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THE ROLE OF L-TYPE Ca2+ CHANNELS IN RELEASE OF ACETYLCHOLINE
FROM MOTOR NERVE TERMINALS FOLLOWING PASSIVE TRANSFER OF
LAMBERT-EATON MYASTHENIC SYNDROME TO MATURE MICE
BY

MICHAEL T. FLINK

A DISSERTATION
Submitted to
Michigan State University
in partial fijlfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY

Department of Pharmacology and Toxicology

2003

ABSTRACT
THE ROLE OF L-TYPE Ca2+ CHANNELS IN RELEASE OF ACETYLCHOLINE
FROM MOTOR NERVE TERMINALS FOLLOWING PASSIVE TRANSFER OF
LAMBERT-EATON MYASTHENIC SYNDROME TO MATURE MICE
BY

MICHAEL T. FLINK

Lambert-Eaton myasthenic syndrome (LEMS) is neuromuscular disorder in which
the release of acetylcholine (ACh) from motor nerves is reduced. LEMS is believed to be due
to circulating antibodies directed against voltage-dependent Ca2+ channels (VDCC) involved
in release of ACh. Passive transfer of LEMS to mature mice by daily injections for 30 days
with plasma from LEMS patients reduces the amplitude of Ca2+ current through P/Q-type
channels and exposes an L-type Ca2+ current not normally found at the motor nerve terminal.
The overall goal of this dissertation was to test the hypothesis that the novel L-type Caz’
current expressed following passive transfer of LEMS to mice participate in release of ACh
from motor nerve temiinals.

Passive transfer of LEMS to mice following plasma injections for 1-30 days reduced
the release of ACh in comparison to controls and induced facilitation of release of ACh during
high frequency nerve-stimulation. Nimodipine, a dihydropyridine L-type Ca2+ channel
antagonist, further reduced the nerve-stimulated release of ACh observed from LEMS, but
not control motor nerve terminals following passive transfer for 20 and 30 days. However,
nimodipine did not affect the nerve-stimulated release of ACh following injection of LEMS
plasma to mice for 1-15 days.

Incubation of motor nerve terminals with the chelator, DM-BAPTA, which rapidly

binds to free Caz' in the cytosol did not affect release of ACh following injection of mice with
control plasma. On the other hand, DM-BAPTA abolished L-type C a” channel involvement
in release of ACh following injection of mice with LEMS plasma. Based upon these findings,
it appears that L-type Ca2+channels involved in release of ACh from LEMS motor nerve
terminals are not tightly coupled with the release apparatus.

The ability of calcium-activated potassium (KCa) channels to induce L-type Caz’
channel involvement in release of ACh from motor nerve terminals was also examined. The
KCa channel antagonist, iberiotoxin, increased the release of ACh from motor nerve terminals
obtained from naive mice only when the membrane was depolarized to potentials more
positive than ~ -42 mV. Furthermore, nimodipine significantly reduced this enhanced release
of ACh induced by iberiotoxin. Thus, it is possible that in LEMS, reduced Caz‘ entry into the
motor nerve terminal may attenuate activation of KCa channels, which increases the extent and
duration of VDCC openings. In turn, this allows L-type Ca2+ channels, which may be located
at sites distant from the release apparatus to become involved in release of ACh.

The studies in this dissertation provide evidence for: 1) L-type Ca2+ channel
involvement in release of ACh from motor nerve terminals occurring only afier prolonged
passive transfer of LEMS to mice, 2) the participation of long-term processes, such as
synthesis and assembly of new channel components for L-type Ca2+ channel involvement in
release of ACh, 3) lack of tight association of L-type Ca2+ channels expressed in LEMS with
the release apparatus, and 4) modulation of L-type Ca” channel involvement in release of

ACh by KCa channels.

For Gussie Zinner

iv

ACKNOWLEDGMENTS

I would first and foremost like to thank my wife, Dana, for her love, support, extreme
patience, and help. I am forever indebted to her encouragement and belief in my abilities to
endure and succeed in the endeavor encompassing this dissertation.

I would also like to thank my advisor, Dr. William Atchison, for his invaluable
guidance. His advice, instruction, support, and friendship have greatly enhanced my abilities
as an independent researcher, thinker, and communicator. I am also grateful for the time and
commitment put forth by my committee members: Drs. James Albers, James Galligan, Peter
Cobbett, and John Goudreau. Their ideas, insight and technical assistance are truly
appreciated. In addition, I am grateful to the members working in laboratories B331, B338
and B307, all of whom have made my graduate experiences quite enjoyable. In particular,
I am appreciative to the fiiendships, training, advice and technical assistance offered by Drs.
Yukun Yuan and Ravindra Hajela.

Finally, I am eternally thankful to my family. Their love, support, and values instilled

in me have made all my successes in life possible.

PREFACE

Some the data contained in this dissertation has been published previously. Chapter Two
appeared as Flink and Atchison (2002) and Chapter Four appeared as Flink and Atchison

(2003)

TABLE OF CONTENTS

LIST OF FIGURES .................................................. xi
LIST OF TABLES .................................................. xv
LIST OF ABBREVIATIONS ......................................... xvi

CHAPTER ONE: INTRODUCTION

A. General Introduction ......................................... 2
B. Neuromuscular transmission ................................... 3
C. Voltage-dependent calcium channels ............................ l3
1. General Description ..................................... 13
2. Functional diversity ..................................... l3
3. Structure of voltage-dependent calcium channels ............... l7
4. Molecular diversity of voltage-dependent calcium channels ....... l9
5. Role of voltage-dependent calcium channel subunits ............ 20
6. Modulation of voltage-dependent calcium channels ............. 23
a. G—protein dependent modulation ..................... 23
b. Kinase-dependent modulation ........................ 24
0. Direct role of Ca2+ in modulation ..................... 26
d. Modulation of Ca2+ channel fimction by proteins involved
in exocytosis .................................... 27
e. Alternative methods involved in modulation ............. 28

D. Voltage-dependent Ca’+ channels involved in neuromuscular

transmission ............................................... 28
E. Ca’+-activated K" channels .................................... 32
1. General information ............................... 32

vii

2. Call activated K" channels and neuromuscular transmission ...... 34

F. Lambert-Eaton Myasthenic Syndrome: General Description ......... 3 5
1. General Clinical Features ................................. 35

2. Physical symptoms ...................................... 36

3. Physical Signs ......................................... 36

4. Electrophysiological characteristics ......................... 38

a. Clinical ......................................... 38

b. Experimental .................................... 40

5. Neuromuscular junction morphology in LEM S ................ 40

G. Lambert-Eaton Myasthenic Syndrome: Autoimmunity .............. 41
1. Clinical and Experiment Findings ........................... 41

2. Antigenic targets ....................................... 44

a. Voltage-dependent calcium channels: general findings ..... 44

b. Voltage-dependent calcium channels: specificity ......... 47

c. Voltage-dependent calcium channels: antigenic sites ...... 55

(1. Additional antigenic targets ......................... 57

3. Antibody etiology ...................................... 59

4. Pathophysiological Mechanisms ............................ 61

a. Electrophysiological features .................... 61

b. Ultrastructural features ............................. 62

c. Radioligand binding ............................... 64

d. Antigenic modulation .............................. 64

e. Direct block of voltage-dependent calcium channels ....... 65

f. Role of complement ............................... 66

H. Voltage-dependent calcium channels and neurological dysfunction . . . . 68

CHAPTER TWO: LAMBERT-EATON MYASTHENIC SYNDROME IN MICE
INDUCES DIHYDROPYRIDINE SENSITIVITY OF SYNAPTIC TRANSMISSION

A. Summary .................................................. 72

viii

B. Introduction ............................................... 73
C. Materials and Methods ....................................... 75
D. Results .................................................... 78
E. Discussion ................................................. 86

CHAPTER THREE: TIME COURSE OF INVOLVEMENT OF L-TYPE Ca2+
CHANNELS IN RELEASE OF ACh DURING PASSIVE TRANSFER OF LEMS TO

MICE

A.
B.
C.
D

E.

Summary ................................................. 1 00
Introduction .............................................. 1 0 1
Materials and Methods ...................................... 104
Results .................................................. 1 07
Discussion ................................................ 1 23

CHAPTER FOUR: IBERIOTOXIN-INDUCED BLOCK OF KC, CHANNELS
INDUCES DIHYDROPYRIDINE SENSITIVITY OF ACh RELEASE FROM
MAMMALIAN MOTOR NERVE TERMINALS

A. Summary ................................................. 140
B. Introduction .............................................. 141
C. Materials and Methods ...................................... 144
D. Results ................................................... 147
E. Discussion ................................................ 159

CHAPTER FIVE: INVOLVEMENT OF P/Q-TYPE Ca2+ CHANNELS IN RELEASE
OF ACh FROM MOTOR NERVE TERMINALS FOLLOWING EXPOSURE TO

LEMS PLASMA
A. Introduction .............................................. 168
B. Summary ................................................. 171

ix

CHAPTER SIX: SUMMARY AND CONCLUSIONS

A. Summary and Conclusions ................................... 173

LIST OF REFERENCES ............................................ 199

Figure 1.1.

Figure 1.2.

Figure 1.3.

Figure 1.4.
Figure 1.5.

Figure 1.6.

Figure 1.7.

Figure 2.1.

Figure 2.2.

Figure 2.3.

Figure 2.4.

Figure 2.5

Figure 2.6.

Figure 2.7.

Figure 2.8.

LIST OF FIGURES

Neuromuscular transmission .................................. 9

Model depicting the role of SNARE proteins in fusion of synaptic vesicle and
nerve terminal membranes .................................. 11

Schematic depiction of the subunit structure of a voltage-dependent Ca2+

channel believed to represent the P, Q, or N subtype ............... 18
G-protein inhibition of voltage-dependent Caz‘ channels ............ 25
EMG recordings of CMAP from control and LEMS patients ........ 42
Single-fiber EMG recordings from two muscle fibers innervated by the same
motor nerve ............................................. 43
Relationship of the alternative and classical complement pathways . . . . 70

Effect of exposure to LEMS plasma for 30 days on quantal content of mouse
hemidiaphragm endplates ................................... 82

Effect of incubation with LEMS plasma for 24 hr on quantal content of mouse
hemidiaphragm endplates ................................... 83

Effect of incubation with LEMS plasma for 2 hr on quantal content of mouse
hemidiaphragm endplates ................................... 84

Effect of exposure to LEMS plasma on quantal content of mouse
hemidiaphragm endplates ................................... 85

Effect of exposure to LEMS plasma for 30 days on facilitation of
neuromuscular transmission ................................. 88

Effect of exposure to LEMS plasma for 24 hr on facilitation of
neuromuscular transmission ................................. 89

Effect of exposure to LEMS plasma for 2 hr on facilitation of neuromuscular
transmission ............................................. 90

Effect of nimodipine on quantal content of mouse hemidiaphragm

xi

Figure

Figure

Flglllt‘

Flair:

Figure 2.9.

Figure 2.10.

Figure 2.11.

Figure 3.1.

Figure 3.2.

Figure 3.3.

Figure 3.4.

Figure 3.5.

Figure 3.6.

Figure 3.7.

Figure 3.8.

Figure 3.9.

Figure 3.10.

Figure 3.11.

preparations exposed to LEMS plasma for 24 hr .................. 91

Effect of nimodipine on quantal content of mouse hemidiaphragm
preparations exposed to LEMS plasma for 2 hr ................... 92

Effect of nimodipine on EPPs from LEMS and control plasma-exposed
neuromuscular junction preparations ........................... 93

Effect of nimodipine on quantal content of mouse hemidiaphragm
preparations exposed to LEMS plasma for 30 days ................ 94

The time course of the effect of plasma from LEMS patient 1 on quantal
content (m) at mice hemidiaphragm preparations. ................ 110

The time course of the effect of plasma from LEMS patient 2 on quantal
content (m) at mice hemidiaphragm preparations. ................ 111

The time course of the effect of plasma from LEMS patient 3 on quantal
content (m) at mice hemidiaphragm preparations. ................ l 12

The time course of the effect of plasma from control patients on MEPP
frequency at mice hemidiaphragm preparations. ................. 114

The time course of the effect of plasma from LEMS patients on MEPP
frequency at mice hemidiaphragm preparations. ................. 115

Effect of LEMS plasma injected for 1 day into mice on facilitation of
transmitter release. ....................................... 116

Effect of LEMS plasma injected for 5 days into mice on facilitation of
transmitter release. ....................................... 117

Effect of LEMS plasma injected for 10 days into mice on facilitation of
transmitter release. ....................................... 118

Effect of LEMS plasma injected for 15 days into mice on facilitation of
transmitter release. ....................................... 119

Effect of LEMS plasma injected for 20 days into mice on facilitation of
transmitter release. ....................................... 120

Effect of LEMS plasma injected for 20 days into mice on facilitation of
transmitter release. ....................................... 121

xii

Figure 3.12.

Figure 3.13.

Figure 3.14.

Figure 3.15.

Figure 3.16.

Figure 3.17.

Figure 3.18.

Figure 3.19.

Figure 3.20.

Figure 3.21.

Figure 4.1.

Figure 4.2.

Figure 4.3.

Figure 4.4.

Effect of nimodipine on facilitation of transmitter release following injection
of LEMS plasma into mice for 20 days. ....................... 122

Effect of nimodipine on quantal content (m) at hemidiaphragm preparations
obtained from mice injected with LEMS plasma for 1 day. ......... 124

Effect of nimodipine on quantal content (m) at hemidiaphragm preparations
obtained from mice injected with LEMS plasma for 5 days. ........ 125

Effect of nimodipine on quantal content (m) at hemidiaphragm preparations
obtained from mice injected with LEMS plasma for 10 days ........ 126

Effect of nimodipine on quantal content (m) at hemidiaphragm preparations
obtained from mice injected with LEMS plasma for 15 days ........ 127

Effect of nimodipine on quantal content (m) at hemidiaphragm preparations
obtained from mice injected with LEMS plasma for 20 days ........ 128

Effect of DM-BAPTA on quantal content (m) at hemidiaphragm preparations
obtained from mice injected with control plasma for 20 days. ....... 130

Effect of DM-BAPTA on quantal content (172) at hemidiaphragm preparations
obtained from mice injected with LEMS plasma for 20 days. ....... 131

Effect of DM-BAPTA on KCl-induced MEPP frequency at hemidiaphragm
preparations obtained from mice injected with control plasma for 20 days
..................................................... 132

Effect of DM-BAPTA on KCl-induced MEPP frequency at hemidiaphragm
preparations obtained from mice injected with LEMS plasma for 20 days

..................................................... 133

Effects of iberiotoxin (IBTx) and nimodipine (nimod) on quantal content of
mouse hemidiaphragm end-plates ............................ 148

Effects of iberiotoxin (IBTx) and nimodipine (nimod) on EPP amplitudes at
mouse hemidiaphragm end-plates ............................ 149

Effects of iberiotoxin (IBTx) and nimodipine (nimod) on resting membrane
potentials from out mouse hemidiaphragm preparations ........... 150

Effects of iberiotoxin (IBTx) and nimodipine (nimod) on MEPP frequency

xiii

 

Figure 4.5.

Figure 4.6.

Figure 4.7.

Figure 4.8.

Figure 4.9.

Figure 4.10.

Figure 4.1 1.

Figure 5.1.

Figure 6.1.

Figure 6.2.

from out mouse hemidiaphragm preparations ................... 151

Effects of iberiotoxin (IBTx) and nimodipine (nimod) on MEPP amplitudes
from out mouse hemidiaphragm preparations ................... 152

Effects of nimodipine (nimod) in the absence of iberiotoxin on quantal content
of mouse hemidiaphragm end-plates .......................... 153

Effects of bay K 8644 (bay K) in the absence of iberiotoxin on quantal content
of mouse hemidiaphragm end-plates .......................... 155

Effects of iberiotoxin (IBTx) and bay K 8644 (bay K) on quantal content of
mouse hemidiaphragm end-plates ............................ 156

Comparative effects of iberiotoxin (IBTx) and nimodipine (nimod) on
amplitudes of MEPPs recorded from muscle preparations in the presence of

varying concentrations of KC] (5-20 mM) ...................... 158

Comparative effects of iberiotoxin (IBTx) and nimodipine (nimod) on MEPP
frequency at mouse hemidiaphragm preparations ................ 160

Effects of nimodipine (nimod) in the absence of iberiotoxin on MEPP
frequency at mouse hemidiaphragm preparations ................ 161

Effect of LEMS plasma on the functional P/Q-type Ca2+ channels involved in
release of ACh from mature mouse motor nerve terminals ......... 170

Hypothetical model depicting the development of fiinctional L-type Ca2+
channels at adult mouse motor nerve terminals .................. 190

Hypothetical model representing the role of KCal channels in LEMS at the
mature motor nerve terminal of mice ......................... 197

xiv

Table 1.1.

Table 1.2.

Table 2.1.

Table 3.1.

Table 4.1.

LIST OF TABLES

Pharmacological and Biophysical Properties of Voltage-Dependent Cai’
Channel Subtypes ........................................... 16

Pharmacological and Biophysical Properties of Ca2'-activated K’ channels 33

Muscle resting membrane potential (RMP), MEPP amplitude, and EPP
amplitude from neuromuscular junction preparations exposed for 2 hr, 24 hr,
or 30 days to control plasma .................................. 81

Muscle resting membrane potentials (RMP) and miniature end-plate potential
(MEPP) amplitudes from preparations exposed to control plasma or plasma
from 3 separate patients with LEMS for 1-20 days ................. 113

Muscle resting membrane potentials (RMP) from control, iberiotoxin, and

iberiotoxin plus nimodipine treated-neuromuscular junction preparations at
various levels of KCl-induced depolarization ..................... 157

XV

.i‘

.1]

Bi

BS

ACh-
AChE-
Aga—IVA—

ANOVA—

BAC-

bay K 8644-

BK-

BSA-

Ca”-

[Cali].-
[Cali]:-
CAMP-

Cng GVIA-
CHAT-
CMAP-

Cme MVIIC-

LIST OF ABBREVIATIONS

acetylcholine
acetylcholinesterase
(o-agatoxin IVA
analysis of variance
action potential
adenosine triphosphate
active zone particles

bovine adrenal chromaffin

1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)pheny1]-3 -

pyridinecarboxylic acid methyl ester

big conductance calcium-activated potassium
bovine serum albumin

calcium

extracellular calcium concentration
intracellular calcium concentration

cyclic adenosine monophosphate
(o-conotoxin GVIA
choline-o-acetyltransferase

compound muscle action potential

(o-conotoxin MVIIC

CREB-
3,4 DAP-
DHP-
DMSO-
DRG-
EA2-
EMG-
EPP-
EtOH-
F (ab)'2,
F(ab)-

FHM-

HEPES-
HLA-
hr-
5-HT-
HVA-

i. v. -

IBTx-

Car/CAMP response element binding protein
3,4 diaminopyridine

dihydropyridine

dimethyl sulfoxide

dorsal root ganglion

episodic ataxia type 2

electromyography

end-plate potential

ethanol

divalent antigen binding fragment of IgG
monovalent antigen binding fragment of IgG
familial hemiplegic migraine

grams

guanosine triphophate

human embryonic kidney
N-Z-hydroxyethylpiperazine-N—2-ethanesulfonic acid
human lymphocyte antigen

hour

5-hydroxytryptamine

high voltage activated

intravenous

iberiotoxin

xvii

msec-

Na’-

immunoglobin

immunoglobin G

intermediate conductance calcium-activated potassium
potassium

calcium-activated potassium
potassium chloride

equilibrium dissociation constant for ligand binding
kilodalton

kilogram

liters

Lambert—Eaton myasthenic syndrome
low voltage activated

quantal content

miniature end-plate potential

mega

milligram

magnesium

magnesium chloride

milliliter

millimolar

millisec

sodium

xviii

NaCl-

NaOH-

NC AM-

nimod—

NM]-

NSF-

o.d.-

PKA-

PKC-

RINm-

SCA6-

SCLC-

SDS-

SCC-

SEM-

SFEMG—

SLE-

SNAP-25-

SNARE-

TEA-

sodium chloride

sodium hydroxide

neuronal cell adhesion molecule
nimodipine

neuromuscular junction
N-ethylmaleimide-sensitive factor

outer diameter

protein kinase A

protein kinase C

rat insulinoma

spinocerebellar ataxia type 6

small cell lung carcinoma
sodium-dodecylsulfate

second

standard error of the mean

single fiber electromyography

small conductance calcium-activated potassium
systemic lupus erythematosus
snaptosome-associated protein of 25 kDa
soluble N-sensitive factor attachment protein receptor
tetraethylammonium

volume

xix

 

VAR/IP-

VDCC-

synaptobrevin

voltage dependent calcium channel

CHAPTER ONE

INTRODUCTION

if?

112

ii;

iii

[iii

Kill”;

111;:

A. General Introduction

Lambert-Eaton myasthenic syndrome (LEMS) is a neurological disorder in which the
nerve-stimulated release of acetylcholine (ACh) from peripheral cholinergic nerves is reduced
(Lambert et a1. , 1961; Lambert and Rooke, 1965; O'Neill and Newsom-Davis, 1988). Patients
with LEMS primarily exhibit skeletal muscle weakness, decreased tendon reflexes, and various
signs of dysautonomia. The pathology underlying LEMS is believed to be due to circulating
antibodies. This idea is based upon the findings that plasma exchange or immunosuppression
transiently alleviates symptoms and signs among patients with LEMS, and the ability to
transfer passively the disease to mice via injection of IgG from patients with LEMS (Lang and
Murray, 1981; Lennon and Fairbanks, 1982; Newsom-Davis and Murray, 1984). Furthermore,
approximately 60% of patients with LEMS have a small cell lung carcinoma (SCLC), which
is believed to initiate the generation of circulating antibodies (Lambert and Rooke, 1965;
O'Neill and Newsom-Davis, 1988).

Although the symptoms and signs of LEMS could represent alterations of post-
synaptic structures similar to those seen in myasthenia gravis, sensitivity of muscle end-plates
to ACh does not appear to be altered in LEMS (Cull-Candy etal., 1980; Lang et al., 1987).
In addition, vesicle size and contents and intraterrninal processes involved in release of ACh
in the motor nerve terminal also are unaltered (Molenaar et al., 1982; Lang and Vincent,
1984). On the other hand, active zone particles, which are believed to represent voltage-
dependent Ca2+ channels (VDCC) are disorganized and fewer in number at motor nerve

terminals from patients with LEMS compared to control terminals. Furthermore,

immunoprecipitation and functional studies have provided additional evidence supporting the
role for circulating antibodies directed against VDCC in patients with LEMS.

Entry of Ca3+ through VDCC into nerve temrinals is a necessary step coupling the
action potential to release of ACh (Katz and Miledi, 1970; Llinas et al. , 1976; Augustine et al. ,
1987). Although multiple VDCC subtypes are known to exist, the specific channel subtype
involved in release of ACh from motor nerve terminals is both species and age-dependent.
During maturation, motor nerve terminals appear to possess multiple VDC C subtypes (Rosato
Siri and Uchitel, 1999; Santafe et al., 2001). However, mature motor nerve terminals
primarily contain one VDCC subtype involved in release of ACh. As such, mature mammalian
motor nerve terminals utilize P/Q- (Katz, 1995; Protti et al. , 1996), whereas amphibians (Sano
et al., 1987) and birds (De Luca et al., 1991) mainly rely on N-type Ca2+ channels to control
release of ACh.

Among the multiple VDCC subtypes, antibodies associated with LEMS preferentially
target the P/Q-type channel (Lennon et al., 1995; Pinto et al., 2002). Thus, based upon the
vital role of VDCC and antibody specificities, the decrease in nerve-stimulated release of AC h
among patients with LEMS appears to be due a decrease in the number of functional VDCC,

particularly those of the P/Q-type.

B. Neuromuscular transmission
The neuromuscular junction represents the synaptic effector junction of the alpha
motor neuron on striated muscle. It is comprised of a specialized cleft or synapse between

motor nerve terminals and a highly defined region of muscle, known as the end-plate. This

specialized space couples release of chemical neurotransmitters from motor nerve terminals
to electrical alterations in the end-plate region, ultimately leading to muscle contraction. The
neuromuscular junction is not just an empty space, but it is composed of a unique form of
basal lamina (Stephens et al., 1985) containing agrin (Gautam et al., 1996), a specialized
enzyme involved in the degradation of the chemical neurotransmitter (Rotundo et al. , 1998),
laminin (Martin et al., 1995) and other proteins involved in mediating the structural and
functional connections between the motor nerve terminal and muscle end-plate region.

The motor nerve terminal contains the chemical neurotransmitter, ACh. It may contain
other neurotransmitters, as well, although the significance of these to normal physiological
fiinction, or distinct pathophysiology is as yet unclear. For example, there is evidence of release
of adenosine from motor nerve terminals (Silinsky, 1984; Redman and Silinsky, 1994, 1995).
ACh is packaged into specialized structures known as synaptic vesicles and is synthesized in
the terminal from acetyl-coenzyme A and choline by the enzymatic reaction involving choline-
O-acetyl-transferase (Browning and Schulman, 1968; Hebb, 1972; Tucek, 1982). Once
synthesized, ACh is stored in synaptic vesicles via an ATPase -dependent transport system
(Breer et al. , 1977; Parsons and Koenigsberger, 1980; Anderson et al. , 1982) whose function
is impaired by the vesicle pump inhibitor vesamicol. Each synaptic vesicle contains
approximately 7,000 -12,000 molecules of ACh (Kuffler and Yoshikami, 1975). Based on
structural and firnctional studies, these synaptic vesicles have been categorized as two distinct
populations--readily releasable and reserve pool (Heinemann et al., 1993; Richards et al.,
2000). The readily releasable pool represents those vesicles that are immediately available for

synaptic transmission, whereas the reserve pool only serves to replenish the readily releasable

pool. Ultrastructural studies have demonstrated that synaptic vesicles representing the readily
releasable pool are located in physical proximity with the nerve temiinal membrane in highly
specialized regions known as active zones (Dreyer er al., 1973; Heuser et al., 1974; Rash er
al., 1974; Heuser er al. , 1979; Ceccarelli et al., 1979; Heuser and Reese, 1981). The active
zones are believed to be sites associated with the process of fast transmitter release (Heuser
et al. , 1974). This assumption is based on the finding that at acutely denervated neuromuscular
junctions, loss of active zones coincides with failure of evoked transmission (Ko, 1981),
exocytosis captured by quick freezing occurs at the active zone (Heuser et al., 1979), and
active zones directly align with densities representing ACh receptors in the end-plate region
of the muscle (Dreyer et al. , 1973; Heuser etal., 1979; Heuser and Reese, 1981; Robitaille et
al,l990)

Movement of vesicles from reserve to readily releasable pools represents a
phenomenon known as “mobilization” and is presumably important in sustaining transmitter
release during periods of intense activity (Delgado er al., 2000; Richards et al. , 2000; Kuromi
and Kidokoro, 2002). The specific processes governing mobilization remain unclear, but the
synaptic vesicle protein, synapsin is thought to play a role in this process by means of a cyclical
series of phosphorylation/ dephosphorylation-dependent interactions with cyto skeletal proteins
such as F -actin (Petrucci and Morrow, 1987; Llinas et al., 1991; Humeau et al., 2001). For
cholinergic synapses, there is evidence that replenishment of the active recycling vesicular pool
occurs primarily from newly-synthesized ACh as opposed to mobilization of pre-filled vesicles
(Collier, 1986). Additionally, at some synapses, there is evidence for “incomplete release” in

a process known euphemistically as “kiss and run” (Ceccarelli et al., 1973; Kraszewski et al. ,

1996; Klingauf er al., 1998; Palfrey and Artalejo, 1998; Fesce and Meldolesi, 1999).

It has been observed using electrophysiological measurements that release of ACh
occurs in defined packets known as quanta (del Castillo and Katz, 1954; Boyd and Martin,
1956; Katz and Miledi, 1967b). As such, each synaptic vesicle is believed to represent a single
quantum of ACh (Heuser and Reese, 1973; Heuser et al., 1979). Various mechanisms can
induce the release of ACh from motor nerves, however, under physiological conditions, release
arises either spontaneously or following action potential -induced depolarization of the nerve
terminal membrane. Spontaneous release of ACh occurs asynchronously and represents single
packets of quanta that cause small depolarizations of the muscle membrane within the end-
plate region (Katz and Miledi, 1963, 1967b). These small fluctuations in the membrane
potential in the end-plate region are known as miniature end-plate potentials (MEPP).
Furthermore, the frequency of asynchronous release of single packets of quanta (MEPP
frequency) increases during KCl-induced depolarization of the nerve terminal membrane. On
the other hand, nerve action potentials induce multiple quanta to be released from motor nerve
terminals in a synchronous manner leading to larger depolarization of the muscle membrane,
which is known as end plate potentials (EPP) (Fatt and Katz, 1951; del Castillo and Katz,
1954; Boyd and Martin, 1956).

Release of ACh from motor nerves (Fig. 1.1) is multi-step process that begins with
generation of an action potential via movement of Na+ and of K+ down their respective
electrochemical gradients and propagation of the action potential along the axon (Hodgkin and
Huxley, 1952). The action potential eventually ceases near the end of the axon, however,

currents spread passively following their initiation by the action potential invade the non-

myelinated nerve terminal and depolarize the membrane (Mallart and Brigant, 1982; Mallart,
1985a). As a result of membrane depolarization, VDCC open, which in turn allows Caz" to
move down its electrochemical gradient from the extracellular environment into the nerve
terminal (Katz and Miledi, 1967a, 1970; Llinas et al., 1981a; Augustine et al., 1987). This
rapid rise in intracellular [Ca2+] occurs in distinct domains at active zones (Llinas el al. , 1992)
and increases the likelihood that synaptic vesicles firse with the membrane to release their
contents into the synaptic cleft (Heuser et al., 1974; Ellisman et al., 1976; Ceccarelli et al.,
1979; Heuser et al., 1979; Harris and Sultan, 1995).

While Ca2+ entry plays a vital role, it is not the only factor required for synaptic vesicle
exocytosis. It has been observed that the delay between the influx of Ca2+ into the nerve
terminal and subsequent changes at the muscle end-plate occur over 60-200 us (Llinas et al.,
1981b; Sabatini and Regehr, 1996), which is faster than most enzymatic processes or chemical
reactions. This observation has led to the supposition that Ca2+ entry induces the completion
of synaptic vesicle and the plasma membrane fusion, a step that was mostly performed prior
to Ca2+ entry into the nerve terminal (Sudhof and Scheller, 2000). This is also supported by
the finding that, under conditions of high osmolarity, synaptic vesicle exocytosis can occur in
the absence of Ca2+ or in Cab-free solutions in the presence of a—latrotoxin (Hubbard et al.,
1968; Rosenmund and Stevens, 1996). As such, Ca2+ most likely acts as key regulator, but
not an absolute requirement for exocytosis.

The steps involved in the regulated release of ACh following Ca” entry into the
terminal remain highly elusive, however, some processes have been suggested. Fusion of

synaptic vesicles with the plasma membrane has been shown to involve the formation of stable

complexes (core complex) of proteins known as SNAREs (Soluble NSF-Attachment Protein
Receptors). SNAREs consist of two groups: nerve terminal, or t-SNAREs (SNAP-25-
{synaptosomal protein of 25 kD} and syntaxin) found on plasma membranes and synaptic
vesicle, or v-SNAREs (synaptobrevin/V AMP) located on vesicle membranes (Trimble and
Scheller, 1988; Oyler etal., 1989; Bennett etal., 1992b; Bennett etal., 1992a). The vital role
these proteins play in synaptic vesicle fiJsion has been demonstrated following exposure of
nerve terminals to CIostridial toxins: botulinum and tetanus (Schiavo et al., 1992b). These
toxins act as proteases to cleave specific proteins of the SNARE complex, ultimately inhibiting
synaptic vesicle exocytosis (Link et al. , 1992; Schiavo et al., 1992a; Blasi et al. , 1993; Schiavo
et al., 1993; Binz et al. , 1994; Schiavo et al., 1994). Furthermore, SNAREs have been shown
to mediate membrane trafficking and secretion in both mammalian and yeast cells (Fig. 1.2.)
(Bennett et al., 1992b; Bennett et al., 1992a). The formation of the core complex is a highly
regulated event that may involve the protein, muncl Sa/nsecl (Hata et al. , 1993; Pevsner et al.,
1994). The rate limiting step in the core complex formation is believed to be the association
of SNAP-25 and syntaxin (Nicholson et al. , 1998), however, this assembly can not take place
until munc18a has dissociated from syntaxin (Pevsner et al. , 1994; Misura et al. , 2000). Once
munc18a dissociates, a highly stable core complex is formed in such a manner that promotes
a condition amenable to synaptic vesicle and plasma membrane fusion (Weber et al., 1998).
Binding of N-ethylmaleimide-sensitive factor (NSF) along with the NSF attachment protein
(oi-SNAP) to this tightly assembled protein structure results in the hydrolysis of ATP and
dissociation of the core complex (Sollner et al., 1993).

Membrane traflicking and release also appear to involve the GTP-binding protein,

 

 

Figure 1.1. Neuromuscular transmission. Stimulation of the nerve leads to initiation and
propagation of an action potential (1). Electrotonic spread of currents generated by the action
potential depolarizes the nerve terminal membrane (2) and induces the opening of voltage-
dependent Ca2+ channels, which permit rapid influx of Ca2+ into the terminal (3). Entry of
Ca2+ initiates release of acetylcholine (ACh) from docked synaptic vesicles (4). Once released
into the synaptic clefi, ACh diffuses across the cleft and binds to receptors located on the end-
plate region of the muscle (5). The effects of bound ACh are rapidly terminated by the
enzymatic processes of AChE (acetylcholinesterase), which cleaves ACh into acetate and
choline (6). Choline is taken back into the cytosol of the nerve terminal by a high affinity
transporter (7). Once inside the terminal, choline is combined with acetleoA by CHAT
(choline-O-acetyltransferase) to reform ACh (8). Free ACh is then taken up into synaptic
vesicles by a specific ATP-dependent transport system (9).

rab3A, which associates with synaptic vesicles (Fischer von Mollard et al., 1990). In the
GTP-bound form, rab3A not only associates with the synaptic vesicle, but with other proteins,
as well (Shirataki et al., 1993; Wang er al., 1997; Cao et al., 1998). The association of these
proteins may act to tether and target the synaptic vesicle to the membrane (Wang et al., 1997;
Orci et al., 1998). During or after exocytosis, GTP bound to rab3A is hydrolyzed to GDP
leading to the removal of rab3A from the synaptic vesicle by guanine nucleotide dissociation
inhibitor (Ullrich et al., 1993), thus limiting the association of the synaptic vesicle with the
plasma membrane. The regulation of synaptic vesicle exocytosis by Ca2+ is thought to involve
the vesicle protein, synaptotagmin (Geppert et al., 1994; Li et al., 1995a; Li et al., 1995b).
Most of the work implicating synaptotagmin as the key Ca2+ sensor is based upon indirect
evidence. Mice deficient in synaptotagmin 1 exhibit decreased Ca21-dependent fast
neurotransmitter release and unchanged Cay-independent spontaneous release of
neurotransmitter (Geppert er al., 1994). Spontaneous (Cf-independent) release of
neurotransmitter, however, is increased in Drosophila possessing synaptotagmin mutants
(DiAntonio and Schwarz, 1994) and decreased in the presence of overexpressed levels of
synaptotagmin in developing spinal neurons (Morimoto et al., 1998). These findings suggest
that synaptotagmin acts as an inhibitor of synaptic vesicle exocytosis, but the effect can be
overridden in the presence of Ca” (DiAntonio and Schwarz, 1994; Morimoto et al., 1998).

Structural analysis of synaptotagmin also has provided evidence for its role as a Ca2+ sensor.
Synaptotagmin possesses two Ca2+ binding regions (C2) on its cytoplasmic side that are

homologous to a region within protein kinase C (Perin et al., 1990; Davletov and Sudhof,

10

’ *\ synatobrevin/V AMP

<20 n; -
, syntaxm

 

 

V

 

 

 

 

O
munc-18/n-sec 1

 

Figure 1.2. Model depicting the role of SNARE proteins in fusion of synaptic vesicle and
nerve terminal membranes. Initiation of synaptic vesicle and nerve terminal membrane firsion
is prevented by munc l8/n-sec 1 binding to syntaxin. Upon dissociation of munc 18/n—sec 1,
syntaxin binds with SNAP-25 on the nerve terminal membrane and forms a highly stable core
complex with synaptobrevin/V AMP. The core complex forces close apposition of the synaptic
vesicle and nerve terminal, leading to membrane firsion and release of vesicle contents.
Binding of NSF and a—SNAP results in hydrolysis of ATP, and dissociation of the core
cf>mplex. The synaptic vesicle then dissociates from the nerve terminal membrane following
binding of munc 18/n—sec 1 to synatxin. Adapted from Sudof and Schellar 2000.

11

1993). It is thought that binding of Ca2+ induces electrostatic and not conformational changes
of synaptotagmin to facilitate association with syntaxin and phospholipids leading to rapid
exocytosis (Shao er al., 1998; Ubach er al., 1998).

Once synaptic vesicles fiise with the nerve terminal membrane, ACh is released and
diffuses across the synaptic clefi where it binds specifically to post-synaptic ACh receptors
localized in the end-plate region of the muscle (Landau, 1978). At physiological resting
membrane potentials, binding of ACh increases the conductance of Na+ and K+ through ACh
receptor-activated cation channels (Takeuchi, 1963), which in turn leads to a localized
membrane depolarization within the end-plate region (Fatt and Katz, 1951; Boyd and Martin,
1956). This change in membrane potential is directly related to the number of ACh receptors
that have opened, and thus the number ACh molecules bound to the ACh receptor.
Electrotonic spread of membrane currents initiated by ACh binding to ACh receptors affect
localized Na” channels on the muscle. At specific levels of depolarization (threshold), voltage-
dependent Nal channels located on the muscle membrane open and Na+ influx rapidly increases
to initiate a muscle action potential. The muscle action potential then activates a cascade of
events that leads to the development of muscle contraction.

Continual activation and re-activation of ACh receptors is promptly terminated by
removal of ACh. This step is accomplished via the action of acetylcholinesterase, an enzyme
which hydrolyzes ACh to acetate and choline (Rotundo et al., 1998; Gaspersic et al., 1999).
Choline is then taken up into the nerve terminal via a specific transporter and reused to form

new ACh (Marchbanks, 1982).

12

C. Voltage-dependent calcium channels

1. General Description

Calcium ions act as second messengers that play vital roles in cellular metabolism,
excitability, contraction, and gene regulation (Augustine et al., 1987; Miller, 1987). The
importance of Ca2+ in cellular firnctions requires precise spatial and temporal control, and as
such various mechanisms exist to control Ca2+ levels within the cell. One such mechanism
includes gating of VDCC, which represent complex heteromeric protein structures that are
sensitive to changes in membrane potential (Miller, 1987). The existence of multiple types of
VDCC, each with distinct biophysical properties, localizations, and densities on cell
membranes, allow precise control of Ca2+ entry into the cell. Based on molecular, biochemical,
pharmacological, and electrophysiological techniques, the following VDC C subtypes have been
identified: T, L, N, P, Q, and R (Table 1.1) (Tsien et al., 1988; Snutch et al., 1990; Zhang et
al., 1993; Randall and Tsien, 1995; Catterall, 2000), referred to now as Cav 1.1-1.4, CaV 2.1-

2.3, and CaV 31-33

2. Functional diversity

The existence of multiple VDCC was initially observed using voltage—clamp studies
with invertebrate egg cells (Hagiwara et al., 1975) in which calcium currents of different
electrophysiological properties were found. Subsequently, two calcium currents of varied
electrophysiological properties also were observed in mammalian neurons (Llinas and Yarom,
1981; Carbone and Lux, 1984) and given the designation of HVA (high voltage activated) or

LVA (low voltage activated), based upon the size of the membrane depolarization required for

13

channel activation. Further classification of HVA and LVA VDCC was made using single
channel and whole cell recordings in chick dorsal root ganglion (DRG) neurons (N owycky er
al. , 1985; Fox et al., 1987a, b). VDCC subtypes of the LVA class were given the designation
of T-type because of their “transient” kinetics and “tiny” conductance, whereas two HV A
class VDCC were designated: L-type, due to their “long lasting” openings and "large"
amplitude current in Ba2+ solutions and N-type, which displayed current properties
intermediate to those of T and L-types ("neither L or T"). T-type Ca2+ channels were shown
to activate at relatively negative potentials, inactive rapidly, and have small single channel
conductance properties. L- and N-type Cazichannels, on the other hand, required much more
positive potentials for activation, inactivated slowly or not at all, and possessed larger single
channel conductances. Although, N-type channels exhibit markedly different
electrophysiological properties than L-type channels in chick DRG neurons, the biophysical
distinction between the two channel subtypes in other model systems is often less clear. Use
of pharmacological agents, however, allows clear identification of these two channel subtypes.
N-, but not L—type channels, are sensitive to firnctional antagonism by the Conus snail toxin,
m-conotoxin GVIA (McCleskey et al., 1987; Tsien et al., 1988; Hillyard et al., 1992;
Grantham et al. , 1994). L-type Ca2+ current, on the other hand is affected by the 3 groups of
organic compounds known as dihydropyridines (DHP), phenylalkylamines and
benzothiazepines (Catterall and Striessnig, 1992; Hofinann et al., 1994). These groups are
typified by the drugs nifedipine, verapamil, and diltiazem, respectively. L-type VDCC are also
antagonized by the peptide toxin, calciseptine, isolated from the black mamba snake,

Dendroaspis poly/epsis poly/epsis. While there have been some reports showing functional

14

antagonism of N-type current by DHPs, this non-selectivity appears to occur in the presence
of high concentrations of antagonists (Diochot er al., 1995). Calciseptine, however, is
selective for L-type VDCC and does not appear to affect entry of Ca2+ through N-type
channels (de Weille et al., 1991; Kuroda er al., 1992; Yasuda et al., 1993) . Currently there
are no specific antagonists for T-type channels, although a number of chemicals antagonize T-
type VDCC (Chuang er al., 1998; Ernst and Kelly, 1998; Martin et al., 2000).

In addition to T, L and N-type channels, three other subtypes of VDCC have been
identified, based on their pharmacological, molecular, and electrophysiological characteristics.
P-type Ca2+ current was initially identified in cerebellum purkinje cells, and were shown to be
highly sensitive to the spider toxin, m-agatoxin IVA (Aga-IVA) (Llinas et al. , 1989; Mintz et
al., 1992). It was later found that when a cloned protein representing the primary subunit
believed to comprise the P-type VDCC was expressed in a cellular model system, the Cal"
current was less sensitive to the effects of Aga—IVA and the inactivation kinetics were different
than originally observed in purkinje cells (Mintz et al. , 1992; Sather et al. , 1993; Zhang et al.,
1993). An Aga-IVA sensitive current also was found in cerebellar granule cells however, this
Ca2+ current exhibited properties resembling those described using cloned P-like channel
proteins and were named Q-type channels (Zhang et al., 1993; Wheeler er al., 1994; Randall
and Tsien, 1995). A sixth VDCC-subtype, aptly named R—type was observed to be resistant
to the actions of the previously described Ca2+ channel antagonists. However, in the presence
of SNX-482, a toxin isolated from the tarantula, Hysterocarates gigas, current through
expressed R-type VDCC channels in mammalian cells or at neurohypophyseal nerve terminals

is reduced (Newcomb et al., 1998). The existence of R-type VDCC cannot, however, be

15

Table 1. 1

Pharmacological and Biophysical

Properties of Voltage-Dependent Ca” Channel Subtypes

 

 

HVA H VA LVA
Caz’ current type L P/Q, N, R T
Structural Ca,.1.1,1.2,1.3,1.4 Cav 2.1, 2.2, 2.3 Cav 3.1, 3.2, 3.3
nomenclature
Previous or, S, C, D, F A, B, E G, H, I
nomenclature

Activation range
Inactivation range

Inactivation

Deactivation rate

Single-channel
conductance

Single-channel
openings

Relative conductance

oo-conotoxin GVIA

Dihydropyridines
co-agatoxin IVA
Calciseptine
SNX-482

Divalent block

Positive to -10 mV Positive to -20 mV

-60to-10mV -120 to -30 mV
Very slow Partial
(r> 500 ms) (I 250-80 ms)
Rapid Slow
25 pS 13 pS
Continual Long burst
reopening
Ba2+ > Caz’ Ba” > Caz"
Resistant Ca,2.2 sensitive
Sensitive Resistant
Resistant Ca,2.l sensitive
Sensitive Resistant
Resistant Ca,,2.3 sensitive
Cd2+ > Ni2+ Cd2+ > Ni”

Positive to -70 mV
-100 to -60 mV

Complete
(1 2:20-50 ms)

Rapid
8 p8

Brief burst,
inactivation

Ba2+ = Ca2+

Resistant

Resistant
Resistant
Resistant
Resistant

Ni2+ > Cd2+

 

Adapted and modified from Hille 2000

16

pro

1811
5 re

and

1 ’. e .
l.”- ii

Q

its.

,9

* lit:

proven based solely by pharmacological antagonism with SNX-482. Newcomb et al. (1998)
have shown that current flow in several rat central neurons was resistant to the actions of

SNX-482.

3. Structure of voltage—dependent calcium channels

Initial biochemical analysis using the transverse tubules of rabbit skeletal muscle
revealed the structure and composition of L-type VDCC. These channels were comprised of
5 separate subunit proteins, each given the designation of al, (125, B, and 7, respectively (Curtis
and Catterall, 1984; Takahashi and Catterall, 1987). It has now been shown that not all
VDCC subtypes consist of 5 subunits. Most HVA C a” channels contain at least four subunits
(01,, (135, [3), however, the association of 7 subunits with non-L type HVA channels is still
unresolved (McEnery et al., 1991; Martin-Moutot et al., 1995b; Liu et al., 1996; Letts et al.,
1998). LVA Ca2+ channels (T-type), on the other hand, are believed to contain only the or,
subunit (Lambert et al., 1998; Leuranguer et al., 1998; Perez-Reyes, 1998).

The structure of the VDCC is shown in Figure 1.3 (See Catterall review, 1998 and
2000). The orl subunit is considered the primary subunit of VDCC and consists of amino acid
sequences organized into four repeated domains (I - IV), with the carboxyl and amino termini
located intracellularly. Each domain is composed of six transmembrane regions (S1 - S6) with
a loop between SS and S6 embedded in the membrane. On the cytoplasmic side of the
membrane, the [3 subunit is found at the amino end of the or1 subunit and contains no
transmembrane spanning regions. The 7 subunit, on the other hand, contains four

transmembrane segments and, in L-type VDCC, is also located in close proximity to the amino

17

EXTRACELLULAR

0L1 0‘2
I II III

 

Lil

 

,3
is:
Q

._.Q; J
{—1

Li ULT

b
”D
i
D
LID
____E, D
[€39
D
95

p—
N
L»
A
LII
ON

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

—— fl

N

DJ

J;

u:
I_J_ :_
_# __ __ a
>__.__

P N

(a) i'
k ‘x

A.

L m
L__g

{L

 

 

INTRACELLULAR

Figure 1.3. Schematic depiction of the subunit structure of a voltage-dependent Ca2+ channel
believed to represent the P, Q, or N subtype. The orl subunit is comprised of 4 domains (I-IV),
each with 6 transmembrane-spanning regions (numbered 1-6). The purely transmembrane-
spanning 6 subunit is linked to the extracelluarly located or2 subunit by a disulfide bond. On
the other hand, the [3 subunit is thought to be located exclusively on the intracellular side of
the membrane. Note that other voltage-dependent Ca2+ channel subtypes may either have an
additional subunit (7), which is not shown in this schematic (L-type found on skeletal muscle)
or be comprised solely of an a, subunit (T-type). Adapted and modified from Catterall 1998.

18

end of the (11 subunit. A disulfide linkage joins the (126 subunits, which are derived from the
same gene (Jay et al. , 1991) and associate with the carboxyl end of the or, subunit. While the
or2 protein contains no transmembrane segments and is located on the extracellular face of the

plasma membrane, the 8 protein is comprised of l transmembrane segment.

4. Molecular diversity of voltage-dependent calcium channels

In conjunction with electrophysiological and biochemical analysis, molecular studies
have elucidated the genes encoding the specific or1 subunit and protein representing each
known VDCC subtype. As such, or, subunits representing the P/Q, N, and, R, channels were
named or1 A, am, and am respectively (Snutch et al., 1990; Dubel et al., 1992; Sather et al.,
1993; Zhang et al., 1993). Although, P- and Q-type channels have different biophysical
properties, it is believed that they are both comprised of same or1A subunit (splice variants of
the same gene); antisense nucleotides directed against the or1A gene or antibodies for the or1A
subunit reduce calcium currents through P- and Q-type channels (Williams et al. , 1992b; Stea
et al. , 1994; Gillard er al. , 1997; Pinto et al. , 1998b). This is further supported by the findings
of Jun er al. (1999), in which mice deficient in the (11’, gene and thus or1A subunit, do not
express P- or Q-type currents. Unlike other VDCC, L- and T- subtypes are compromised of
different a, subunit types depending on the tissue in which the channel was isolated and the
gene encoding the subunit; L-type VDCC were found to contain either the (113,010 or1F or am
subunit (Mikami et al., 1989; Hui et al., 1991; Snutch et al., 1991; Williams etal., 1992a; Hell
et al., 1993), whereas T-type VDCC were comprised of either 0116,01,”, or or" subunits (Perez-

Reyes, 1998). This nomenclature, however, has been revised recently to clarify the structural

19

and functional similarities of each VDCC subtype. This led to grouping of the VDCC into
three families, given the designation of: Ca,.1,2, and 3 (Ertel er al., 2000).

In addition to the multiple or, subunits identified, so far, at least 4 [3, 3 0.26, and distinct
5 7 subunits have also been described. For most of the subtypes (T-type excepted), the
formation of firlly fimctional channels requires the association of specific (11 and accessory
protein subunits, which is highly varied and dependent upon the species and anatomic location
of the channel. To date, however, only a few specific subunit compositions of the different

VDCC have been identified in their native environment.

5. Role of voltage-dependent calcium channel subunits

The or1 subunit confers many of the properties associated with the VDCC, by acting as
the Cal" ion selectivity filter and pore, possessing the channel gating sites, and containing the
voltage sensor and binding sites for many specific pharmacological modulators (Catterall,
2000). The pore of the VDCC is thought to exist in the loop regions between 85- S6
segments for each domain of the orl subunit, and is known as the P-loop. Mutational analysis
has revealed that much of the ion selectivity is detemiined via four glutamic acid sequences in
the P-loop and, as such, loss of these regions is related to decreases in Ca2+ selectivity (Yang
et al., 1993; Yatani et al., 1994; Ellinor et al., 1995). Mutational analysis also has suggested
that the voltage-sensing firnction of the (11 subunit is confined to the S4 segment (Garcia et al. ,
1997). However, rates of channel activation and conductance are influenced via other
segments (Tanabe et al., 1991; Dirksen et al., 1997). Although, HVA channels exhibit

multiple types of inactivation kinetics that differ amongst the different oil subtypes, it has been

20

observed that sequences within the 86 segment and carboxyl terminus are vital for voltage-
dependent inactivation (Wei et al., 1994; Zhang et al., 1994).

Binding sites for pharmacological agents that modulate VDCC mainly reside on the orl
subunit. DHPs have been shown to bind to multiple transmembrane regions of the 01103.1)
subunits (Kalasz et al. , 1993; Kuniyasu et al. , 1998); mutagenesis of the SS segment of domain
III and S6 segment of domain IV dramatically decrease binding of DHP (Peterson et al.,
1997). The presence of Ca2+ ions also is required for binding of DHP to am 3‘ D subunits
(Schneider et al. , 1991). It is believed that Ca” ions keep the al subunit in a conformation that
facilitates DHP binding (Peterson and Catterall, 1995). Other pharmacological agents also have
been observed to affect VDCC specifically by binding to the or, subunit. As such, Ca2+ current
entry through or1A subunits expressed without other auxiliary subunits in model cell systems are
decreased in the presence of (o-Aga IVA and (o- conotoxin (CTx) MVIIC (Sather et al. , 1993;
Zhang et al., 1993). It has been shown using mutational analysis that co-Aga IVA binds to a
site at the carboxyl end of segment S3 in domain IV (Winterfield and Swartz, 2000).

Although expression of or, subunits alone can yield fimctional VDCC, many of the
properties associated with the channel are affected by association with the auxiliary protein
subunits ((125, B, and y) (Perez-Reyes et al. , 1989). Work by Chien et al. (1995) showed that
expression of [32, subunit proteins increase targeting of (11C subunits to cell membranes without
altering alc expression. Other [3 subunit isoforms also target a, subunit proteins to plasma
membranes. As such, B“, 2, 3b, 4 isoforms have been observed to localize expressed or1A
subunit proteins to the plasma membrane of chick osteosarcoma (COS-7) cells, however, or1A

proteins alone or in conjunction with (1,28 proteins are not found at the membrane, but

21

intracellularly (Brice et al., 1997). While the role of the B subunit in targeting oil proteins to
the plasma membrane appears nonspecific, other studies have shown preferential interactions
of the two-subunit proteins. Expression of various isoforms of the B subunit with either or],
am, or am in cells in culture which exhibited membrane polarization with protein sorting
mechanisms analogous to those of neurons, showed differential targeting of the or1 subunit.
This specificity was primarily dependent upon the or] subunit subtype, however, the destination
was also differentially modified based upon the specific B subunit present (Brice and Dolphin,
1999). Thus, calcium currents in cells increase in amplitude following B subunit targeting of
orl proteins to plasma membranes (Singer et al. , 1991; Neely et al. , 1993; Chien et al. , 1995;
Brice et al., 1997; Brice and Dolphin, 1999). Although it is possible that B subunits increase
whole cell Ca2+ currents by increasing expression of functional (1, subunit proteins, studies to
date have shown a lack of effect of B subunits on or, subunit expression (Singer et al., 1991;
Neely et al., 1993; Chien et al., 1995).

Auxiliary subunits also appear to modify the gating kinetics, voltage dependence of
activation and inactivation, and sensitivity of the channel protein to pharmacological agents.
Co-expression of B subunits, facilitates the probability of channel pore openings ofa1A subunit
proteins and, thus increases Ca2+ entry into cells (Neely et a1. , 1993; Shistik et al., 1995). In
addition, voltage-dependent inactivation and activation of orIA subunits is shifted to more
negative potentials and rate of current activation is increased following co-expression of B
subunits with or, subunit proteins (Singer et al., 1991; De Waard and Campbell, 1995).
Conversely, DRG injected with B subunit antisense oligonucleotides, which reduce the

expression B subunit proteins, exhibit a positive shift in the voltage-dependent Ca2+ channel

22

activation (Berrow er al., 1995). Rates of inactivation of channels containing or1A subunits are
increased by co-expression with B subunit isoforms, which occur with differential selectivity
with the following rank order of B 3 > B", = B 4> B2a (fastest to slowest) (Castellano et al. , 1993;
De Waard and Campbell, 1995). DHP- and (o-CTx MVIIC/GVIA binding to onc and am,
subunits, respectively is increased in the presence of B subunit proteins, an effect which is
independent of or, subunit expression (Neely er al., 1993; Nishimura er al., 1993; De Waard
and Campbell, 1995). In addition to B subunits, (125 subunits have also been shown to shift the
voltage—dependence of channel activation and inactivation in the hyperpolarizing direction,
enhance current amplitudes, increase the rate of current activation and inactivation, and alter
the probability of channel openings (Singer et al., 1991; De Waard and Campbell, 1995;
Gumett et al., 1996; Dolphin et al., 1999). While the firnction of 7 subunits in skeletal
muscle is similar to that of other accessory proteins, its role in neurons in vivo is less clear.
However, 7 subunits modulate the voltage-dependence of co-expressed subunits in vitro and
mice with a mutation in a 7 subunit exhibit a unique neurological deficit, known as "Stargazer"
(Letts et al. , 1998). Stargazer mice show prolonged and frequent absence seizures, ataxic gait
and a distinctive head tossing motion that is related to vestibular problems (Felix, 2000). Thus,

7 subunits may play an important role in neurons as well as those observed in skeletal muscle.

6. Modulation of voltage-dependent calcium channels
a. G-protein dependent modulation
Regulation of VDCC involves multiple mechanisms that may act independently or in

concert with one another. One such mechanism involves G-proteins, which have been

23

am
ad

”101

C01;-
31]
Tiles

~\
L‘; 1

observed to modulate VDCC responses to various levels of membrane depolarization (Fig.
1.4.) (Hille, 1994; Dolphin, 1998; Simen and Miller, 1998). The effect of G-proteins on
VDCC kinetics can occur via a direct link between G-protein activation or G-protein coupled
soluble second messengers and VDCC. G-proteins are heteromeric complexes composed of
three subunits designated: a, B, and y (Hille, 1992, 1994). Upon activation of G protein-
coupled receptors, GDP bound to the Ga subunit is exchanged for GTP, which in turn induces
dissociation of GBy from the G-protein complex (Hille, 1992; Hamm, 1998). Voltage-
activation or neurotransmitter binding can induce G-proteins to inhibit directly Ca2+ entry
through N-, R— and P/Q-type channels by shifting the Ca2+ channel from an easily activated to
a more difficult-to activate state (Herlitze et al., 1996; Ikeda, 1996; Qin et al., 1997; Simen
and Miller, 1998; Canti et al., 1999). This inhibition occurs via G137 and not Ga binding to
modulatory sites on VDCC. Characteristics of direct G-protein induced inhibition of N-,
R-, and P/Q-type Ca” channels include: positive shifts in current-voltage relationship, decrease
in whole cell Ca2+ current amplitudes, slowed activation kinetics, and reversal of modulation
by G—proteins during strong depolarization (Herlitze et al., 1996; Ikeda, 1996; Qin et al.,
1997; Simen and Miller, 1998; Canti et al., 1999).

b. Kinase-dependent modulation

L-type Ca2+ channels, on the other hand, do not appear to be affected by direct
coupling to G-protein activation (Bourinet et al., 1996; Meza and Adams, 1998; Bell et al.,
2001). Instead, modulation of L-type channels occurs via G-protein induced second
messenger pathways. Activation of CAMP-dependent protein kinase or phosphinositide 3

kinase pathways following G-protein activation alters cardiac (Reuter et al. , 1983) and smooth

24

transmitter . .
membrane depolarization

 

Figure 1.4. G-protein inhibition of voltage-dependent Ca2+ channels. Hypothetical pathway
depicting direct inhibition of voltage-dependent Ca2+ channels (VDCC) by G—proteins.
Membrane depolarization or transmitter-activation of receptors coupled to G-proteins induces
the exchange of GDP bound to the or subunit of the G-protien for GTP. Binding of GTP, in
turn, causes dissociation of the By subunit, which directly inhibits VDCC by some unknown
mechanism. Adapted from Dolphin, 1998.

25

muscle (Viard er al. , 1999) L-type channel biophysical properties, respectively. As such, direct
phosphorylation plays a key role in the regulation of L-type Ca2+ channels. During periods of
intense stimulation, L-type Ca” channel current at skeletal muscle is greatly enhanced by
phoshorylation of the a and B subunits of the channel by CAMP-dependent protein kinase
(Arreola et al., 1987; Sculptoreanu et al., 1993) (Rotman er al., 1995). Unlike, L-type
channels from skeletal muscle, early investigations observed that phosphorylation does not
change the sensitivity of cardiac muscle L-type channels to voltage, but rather increases Ca”
conductance (Reuter and Scholz, 1977). More recent work has shown that gating kinetics of
cardiac L-type channels is altered following phosphorylation to a state that allows more
channels to open during physiological levels of stimulation (Hess et al., 1984;Yue er al., 1990).
In addition to L-type, P/Q- and N-type Caz" channel activity is also modulated by
phoshorylation. G-protein inhibition of non-L type channels can be relieved by several
mechanisms involving phosphorylation by protein kinase C (Swartz, 1993). Interaction of
P/Q- and N-type channels with the SNARE complex and synaptotagmin is also disrupted
following channel phosphorylation (Yokoyama et al., 1997).
c. Direct role of Ca2+ in modulation
Increased Ca2+ entry also has been shown to alter directly the biophysical properties
of VDCC. L-type channels found in cardiac cells are inhibited following large influxes of Ca2+,
an effect that appears dependent upon calmodulin (Peterson et al. , 1999). It has been shown
that over-expression of calmodulin mutants that are insensitive to Ca” binding prevents Caf-
dependent inactivation of L-type channels (Peterson et al. , 1999). Binding of calmodulin to

a region in the C-terminus of L-type channels is a necessary prerequisite and as such, mutations

26

 

in calmodulin binding regions prevent Ca21-dependent inactivation (Zuhlke and Reuter, 1998).
In contrast, large influxes of Ca2+ can also have a facilitatory effect upon cardiac L-type
channels that is also dependent upon calmodulin (Anderson el al. , 1994). However, in addition
to Ca2+ binding to calmodulin, CazVcalmodulin-regulated protein kinase II (Cam KII) is
required for Cali-dependent facilitation (Xiao et al., 1994; Zuhlke et al., 1999).

In a similar manner, Caz‘ entry through P/Q-type channels has a dual role. Initially
large C az‘ current induces facilitation with a subsequent slower rate of inactivation of P/Q-type
channels via a Caz‘/calmodulin-dependent pathway (Lee et al., 2000). Although, this
mechanism is not well understood, it is believed to occur in a different manner from that
observed with L-type channels (DeMaria et al., 2001).

d. Modulation of Ca2+ channel function by proteins involved in exocytosis

Proteins associated with the core complex of docked vesicles at active zones have been

observed to regulate directly VDCCs. Over expression of the SNARE protein, syntaxin 1A
decreases Cazi- dependent transmitter release (Wu el al. , 1999), whereas reduced expression
0f Syntaxin enhances release (Watanabe et al., 1999). N-, P/Q- and in some instances L-type
Ca” Channel currents have been shown to be inhibited directly by syntaxin (Bezprozvanny et
al., 1995; Wiser et al. , 1996; Wiser er al., 1999). In addition to syntaxin, the SNARE protein,
SN[AP-25 has been also been shown to inhibit P/Q-type Ca2+ channel current (Zhong er al.,
1999). However, in the presence of the Ca2+ binding protein synaptotagmin, inhibition of
VDCC by syntaxin and SNAP-25 is relieved (Wiser et al. , 1997; Wiser et al., 1999; Zhong et
al., 1999). The effect of syntaxin modulation is not the same for each VDCC subtype.

Inhibition of N-type channels by syntaxin can be partially removed by addition of SNAP-25,

27

whereas inhibition of L-type channels by syntaxin is unchanged following addition of SNAP-25
(Wiser et al., 1996; Wiser er al., 1999). It appears, therefore, that complete core complexes
at active zone sites are required for physiological fimction of associated VDCC, which are
regulated via their specific interactions.

e. Alternative methods involved in modulation

As previously described, association of auxiliary subunits modulate many of the
biophysical and pharmacological properties of orl subunits. This may explain differences
observed between P- and Q-type Ca2+ channel subtypes, and as such, may reflect channels
containing identical or1A subunits with different associated auxiliary subunit proteins.
Alternatively, splice variations of or1A subunits could account for dissimilarities of P- and Q-
type channels. Although it is still unclear if variations between these channels represent
splicing or auxiliary subunit differences, both subtypes contain or1A subunits derived from the

same gene (Gillard et al., 1997 ; Pinto et al., 1998a; Jun et al., 1999).

D. Voltage-dependent Caz+ channels involved in neuromuscular transmission
The small size and thus inaccessibility of most motor nerve terminals precludes direct
electrophysiological correlation of presynaptic current carried by specific VDCC subtypes and
concomitant release of ACh. However, recent pharmacological and molecular techniques
have overcome this hindrance and as such, it has been observed that the specific VDCC
subtype(s) involved in release of ACh from motor nerves is highly age- and species-dependent.
Cell bodies of motor neurons from chick embryos initially possess a dominant T-type

Ca2+ current. However, during development, N- and L-type channels assume a major role in

28

controlling Caz’ entry (McCobb et al., 1989). Similarly, L- and N-type channels are found
at developing avian motor terminals and both types are involved in release of ACh (Gray et al. ,
1992)

The involvement of different subtypes of VDCC in Cab-induced ACh release from
motor nerve terminals of rats also displays differential age-dependence (Sugiura and Ko, 1997;
Rosato Siri and Uchitel, 1999; Santafe et al., 2001; Rosato-Siri et al., 2002; Santafe et al.,
2002). During embryonic stages of development, rat motor nerve terminals contain P/Q-, N-,
and L-type channels that all regulate, to some extent, nerve-evoked release of ACh (Sugiura
and Ko, 1997; Rosato Siri and Uchitel, 1999). P/Q- and N-type channels appear to enhance
release, whereas in some experimental conditions, Ca2+ current through L-type channels
actually inhibit release of ACh (Sugiura and Ko, 1997; Rosato Siri and Uchitel, 1999). As
motor nerves of post-natal rats develop, the participation of each VDCC subtype in release of
ACh changes, such that from day 0-4, N- and P/Q- type channels are mainly involved but, by
day 5, N-type channel no longer control release of ACh (Rosato Siri and Uchitel, 1999;
Rosato-Siri et al., 2002). Furthermore, P/Q-type channels appear to be more efficiently
coupled to release of ACh than N-type channels during post-natal days 0-4; this may be due
to differential localization of each channel subtype with the release apparatus (Rosato-Siri er
al., 2002).

Examination using rats of various post-natal days revealed that involvement of multiple
VDCC subtypes with differing effects on ACh release can be explained in part by competition
between poly-innervating motor nerve terminals found at immature end-plates (Santafe et al. ,

2001; Santafe et al., 2002). Entry of Ca2+ through P/Q-, N-, and L-type channels at dually-

29

innervated immature end-plates either enhances or decreases release of ACh, depending upon
the stimulus protocol and the specific terminal activated (Santafe et al., 2001; Santafe er al.,
2002). During maturation one motor nerve will eventually be eliminated, while the other one
remains in contact with the end-plate. However, the roles of VDCC at nerves during synapse
elimination and at those destined to support release of ACh from adult motor nerves are
different. While Ca2+ entry through P/Q-, N-, and L-type channels acts to suppress growth of
axons that eventually will be eliminated, entry of Ca2+ through P/Q-, N-, and L-type channels
induces excitatory processes at immature motor nerves that eventually will mono-innervate
mature end-plates (Santafe et al., 2001; Santafe et al., 2002).

Mature motor nerves appear to possess only one VDCC-subtype principally involved
in release of ACh. Whereas, N-type channels control release of ACh from amphibian (Sano
et al. , 1987) and avian (De Luca et al. , 1991; Gray et al., 1992) mature motor nerve terminals,
P/Q-type channels control release of ACh from adult mammalian motor nerves (Uchitel and
Llinas, 1992;Wessler et al. , 1995; Protti and Uchitel, 1996; Katz et al., 1997). L-type
channels, which are often found co-localized with other VDCC subtypes are not normally
involved in mammalian neuromuscular transmission (Atchison and O'Leary, 1987; Atchison,
1989; Protti et al. , 1996; Katz et al. , 1997), but are involved in release of hormones from the
neurohypophysis from rats (Lemos and Nowycky, 1989), noradrenaline (Owen et al. , 1989)
from adrenal bovine chromaffrn cells, and insulin fi'om human pancreatic B cells (Davalli et al. ,
1996)

In non-physiological settings, however, L-type Ca2* channels can participate in release

of ACh from even adult mammalian motor nerve terminals. Exposure of adult mammalian

3O

motor terminals to a DHP agonist, bay K 8644, which directly modulates the response of L-
type C azi channels to potential changes, enhances release of ACh. This effect can be prevented
by incubation of the preparation with DHP-type antagonists (Atchison and O'Leary, 1987;
Atchison, 1989). More indirect methods have been observed to involve normally silent L-type
Ca2+ channels in release of ACh. Rapid chelation of intracellular Ca2+ using DM-BAPTA
unmasks L-type Ca” current at mouse motor nerve terminals that are not involved in release
of ACh unless protein phosphatases are also inhibited (Urbano and Uchitel, 1999; Urbano et
al., 2001 ). A similar finding was observed at frog motor nerve terminals following an increase
in intracellular levels of protein kinase C (Arenson and Evans, 2001). Alterations in stimulus
conditions induce participation of L—type channels in release of ACh from rat and mouse
motor nerves (Hong and Chang, 1990; Correia-de-Sa et al., 2000a). This effect is thought to
occur through a mechanism involving activation of adenosine A2A receptors (Correia-de-Sa
et al., 2000b). Thus, it appears that mature motor nerve terminals possess L-type Ca”
channels that are normally silent and not involved in release of ACh, but can be unmasked
under certain conditions.

In a similar manner, L-type Ca2+ channels participate in release of ACh during certain
pathological conditions. For example, release of ACh from motor nerves during reinnervation
or following recovery from botulinum toxin-induced poisoning or exposure to antibodies from
patients with amyotrophic lateral sclerosis is sensitive to L-type Ca” channel antagonists (Katz
and Uchitel, 1996; F ratantoni et al., 2000; Santafe and Uchitel, 2000). This effect may well

represent a situation analagous to development of immature motor nerves.

31

E. Caz‘lactivated K“ channels

1. General information

Cay-activated K+ (K0,) channels are found in a variety of tissues, particularly those that
exhibit action potential induced-release of neurotransmitters (Knaus er al., 1996; Anderson
et al., 1988; Vergara er al., 1998). These channels not only appear to be involved in release
of neurotransmitters, but in neuronal firing properties as well. Based upon pharmacological
and biophysical properties, KCa channels can be separated into three families: BK, SK, and IK
channels (Table 1.2.) (Sah, 1996; Vergara et al., 1998). The BK (“big” conductance)
channels, which can be activated by either a rise in cystolic Caz' concentration or membrane
depolarization, are highly selective for K‘, and possess large single channel conductances of
100-400 pS (Marty, 1981). A number of pharmacological antagonists for this channel subtype
exist and include tetraethylammonium, the scorpion toxins iberiotoxin and charybdotoxin, and
the mycotoxins penitrem A and paxilline (Galvez et al., 1990; Knaus er al., 1994; Vergara et
al., 1998). The second type of KCa channel has a small single channel conductance (2-20 pS)
and is thus known as SK (“small” conductance) channels (Blatz and Magleby, 1986). Elevated
intracellular Ca2+ concentrations, but not membrane depolarization, is required for activation
of these channels (Hirschberg er al., 1998). In contrast to BK channels, SK channels are not
sensitive to tetraethylammonium, iberiotoxin, or charybdotoxin. However, current through
SK channels is antagonized by the bee venom apamin, quaternary salts of bicuculline,
tubocurarine, and dequalinium (Blatz and Magleby, 1986; Johnson and Seutin, 1997). On the
other hand, 1-ethyl-2-benzimidazolinone (EBIO) activates the channel by increasing sensitivity

to Ca2+ and thus increasing the probability of channel openings (Olesen et al., 1994).

32

Table 1.2

Pharmacological and Biophysical
Properties of Cay-activated K+ channels

 

 

KC,I subtype BK IK SK
Membrane depolarization Activates No effect No effect
Elevated intracellular [Caz‘] Activates Activates Activates
Single channel conductance 100-400 pS 20-100 pS 2-20 pS
Tetraethylammonium Sensitive No effect No effect
Iberiotoxin Sensitive No effect No effect
Charybdotoxin Sensitive Sensitive No effect
Apamin No effect No effect Sensitive*

 

Adapted from Sah 1996 and Vergara et al. 1998
*Sensitivity is dependent on SK subtype and channel expression system

33

The third channel subtype, IK (“intermediate”) is aptly named because it posses single
channel conductances intermediate (20-100 pS) to those of BK and SK channels (Ishii et al.,
1997b; Logsdon et al. , 1997). In a manner similar to SK channels, 1K channels are insensitive
to voltage changes, but they open in response to elevated cystolic Ca2+ concentration.
Furthermore, they are sensitive to EBIO, charybdotoxin, and clotrimazole, but not to apamin

or iberiotoxin (Ishii et al., 1997a; Logsdon et al., 1997).

2. (332+- activated K+ channels and neuromuscular transmission

Cay-activated Ki" channels have been identified at amphibian and mammalian motor
nerve terminals using pharmacological, electrophysiological, and immunocytochemical staining
techniques. The scorpion venom peptides, Charybdotoxin and iberiotoxin both directly
antagonize Km) current during nerve-stimulation, whereas apamin has no effect. Based on
pharmacological sensitivities, amphibian and mammalian motor nerve terminals appear to
possess BK type KC, channels. The increase in intracellular Ca2+ which activates Km, current
originates from extracellular, but not intracellular sources in mice; as such, block of VDCC by
Mg“, Mn”, or Co2+ prevents activation of Km, current during nerve-stimulation (Mallart,
1985b)

Although the exact role of KC,I channels at motor nerve terminals of mammals and
amphibians is not firlly understood, it is believed that they participate in nerve terminal
membrane repolarization. KC, channels limit the duration of action potentials and Ca2+ entry
at nerve terminals in frog pituitary (Obaid et al. , 1989) and Ca2+ entry and release of ACh from

frog and mouse motor nerve terminals (Mallart, 1985b; Dreyer and Penner, 1987; Anderson

34

er al., 1988; Robitaille and Charlton, 1992; Robitaille et al., 1993; Vatanpour and Harvey,
1995; Xu and Atchison, 1996; Protti and Uchitel, 1997). Furthermore, pharmacological and
immunocytochemical staining techniques have shown that KC, channels are colocalized with
VDCC at the nerve terminal (Robitaille et al., 1993; Protti and Uchitel, 1997). This intimate
localization of these channels most likely ensures a rapid activation of KC, current during Cab

entry that is triggered by an action potential.

F. Lambert-Eaton Myasthenic Syndrome: General Description

1. General Clinical Features

Lambert Eaton myasthenic syndrome occurs in two general populations: those with and
those without detectable neoplasms (Elmqvist and Lambert, 1968; O'Neill and Newsom-Davis,
1988). Among those with neoplasms, small cell lung carcinoma (SCLC) is the most common
type, with approximately 60% of all LEMS patients exhibiting a SCLC (Eaton and Lambert,
195 7; Lambert et al., 1961; Lambert and Rooke, 1965; Lambert and Elmqvist, 1971; O'Neill
and Newsom-Davis, 1988). Evidence of LEMS almost always precedes the diagnosis of
cancer (all types) by 5 months to 3.8 years. On average, the interval between onset of
neuromuscular symptoms and detection of cancer in the majority of patients with LEMS
occurs over 40 years of age. However LEMS has been reported to range from 17 to 79 years
(O'Neill and Newsom-Davis, 1988). Males are more predominately affected than females in
both cancer and non-cancer groups(Elmqvist and Lambert, 1968; O'Neill and Newsom-Davis,

1988).

35

 

2. Physical symptoms

Proximal lower extremity weakness represents the presenting complaint among most
LEMS patients (Lambert et al., 1961 ; Wise and MacDermot, 1962; Lambert and Rooke, 1965;
O'Neill and Newsom-Davis, 1988). A small number of patients, however, present with
generalized weakness, muscle pain and stiffness, autonomic dysfunction, or arm weakness as
their primary symptom. Although arm weakness is often described, it is usually restricted to
the proximal muscles and much less severe compared with that of the lower leg. Autonomic
dysfunction is also a common complaint in approximately 80% of patients with LEMS.
Symptoms include: dry mouth, sexual impotence, and to a lesser extent decreased sweating,
constipation, difficulty with micturition, and blurred vision. (Lambert and Rooke, 1965;
Henriksson et al., 1977; Rubenstein et al., 1979; Khurana and Mayer, 1988; O'Neill and
Newsom-Davis, 1988). Cranial nerves also are affected in LEMS, and patients may exhibit
diplopia and ptosis, dysarthria, dysphagia, and dysphonia. The symptoms related to cranial

nerve involvement, however, are often mild and intermittent.

3. Physical Signs

The most common clinical sign observed in patients with LEMS is that of skeletal
muscle weakness, mainly involving proximal and truncal muscles and most dramatically
affecting the lower limbs (Lambert et al., 1961; Wise and MacDermot, 1962; Lambert and
Rooke, 1965; O'Neill and Newsom—Davis, 1988; Erlington and Newsom-Davis, 1994). During
sustained maximal muscle contraction there is a progressive augmentation in strength followed

by increasing weakness and fatigue. The augmentation occasionally makes it difficult to detect

36

cli

C0.

if

(163

ion

clinical weakness.

Tendon reflexes often are depressed or absent in patients with LEMS. However, after
maximal voluntary muscle contraction, tendon reflexes reappear or augment (Lambert et al.,
1961; Wise and MacDermot, 1962; Lambert and Rooke, 1965). Cranial nerve signs are
common, including ptosis and weakness in neck flexion (O'Neill and Newsom-Davis, 1988).
LEMS patients also exhibit impairments in pupillary light reflex and secretions from lacrimal
and parotid glands (Rubenstein et al., 1979; Heath and Cull, 1988; Khurana and Mayer, 1988;
O'Neill and Newsom-Davis, 1988). Most autonomic abnormalities can be ascribed to defects
of the parasympathetic (cholinergic) system, however two reports have described patients with
sympathetic dysfunction (orthostatic hypotension and increased sensitivity to exogenous
sympathetic agonists) (Mamdani et al., 1985; Khurana and Mayer, 1988). Sympathetic
dysautonomia most likely reflects an effect induced by the underlying neoplasm in the
described patients and not that of LEMS; cardiovascular autonomic dysfunction is commonly
found in patients with bronchial neoplasms/SCLC without LEMS (Park et al., 1972; Gould
et al. , 1986; Maier and Sommers, 1986) and breast cancer patients (Bruera et al. , 1986; Heath
and Cull, 1988). Sensory abnromaltities are not a prominent findings in LEMS (Heath and
Cull, Khurana and Mayer,O'Neil and Newsom-Davis, 1988).

LEMS patients’ signs and symptoms, however, can be dramatically reduced by
pharmacological agents that enhance release of ACh (N ewsom—Davis and Murray, 1984; Oh
et al., 1997; Sanders, 1998; Sanders et al., 2000). Guanidine and 4-aminopyridine, which
prolong the duration of action potentials and enhance Ca2+ entry into the nerve terminal,

enhance AC h release and produce symptomatic relief, albeit frequently accompanied by serious

37

side effects. The actions of a similar compound, 3,4-diaminopyridine (3,4 DAP), on the other
hand, are generally limited to the peripheral nervous system and thus this agent offers
symptomatic relief without serious side effects. Although it may be theoretically possible that
3,4 DAP alters cardiac function by decreasing the rate of ventricular myocyte repolarization,
patients taking 3,4 DAP, 20 mg three times daily for 6 days did not exhibit any changes in
blood pressure or QT interval (which represents the time of ventricular myocyte
repolarization) as measured using EKG (Sanders et al. , 2000) . On the other hand, drugs that
prolong the actions of ACh at motor end-plates by inhibiting acetylcholinesterase alleviate
symptoms in patients with myasthenia gravis, a neuromuscular disorder in which firnctional
ACh receptors on skeletal muscles are reduced, but offer relatively limited benefit in LEMS

patients (Eaton and Lambert, 1957; O'Neill and Newsom-Davis, 1988).

4. Electrophysiological characteristics

a. Clinical

Although neuromuscular disorders (for example, myasthenia gravis) are characterized
by skeletal muscle weakness, the diagnosis of LEMS can be established by electromyography
(EMG). The relevant study utilizes surface electrodes to record compound muscle action
potentials (CMAP) generated from muscle fibers comprising the motor unit (motor nerve +
all muscle fibers innervated by that nerve) during supramaximal depolarization of the nerve
(Eaton and Lambert, 1957). EMG findings associated with LEMS are depicted in Figure 1 .5.
In LEMS, CMAP amplitudes in response to single or low frequency (usually 35 Hz)

stimulation of the motor nerve are depressed, and often can be as low as 10% of normal

38

(Eaton and Lambert, 1957; Elmqvist and Lambert, 1968; Jablecki, 1984; O'Neill and Newsom-
Davis, 1988). During repetitive stimulation at low rates (3 Hz), CMAP also exhibit a
decrementing pattern similar to that seen in myasthenia gravis. High frequency (210 Hz)
stimulation of the motor nerve, on the other hand, induces a progressive increase in the
amplitude of the CMAP. This phenomenon is known as post-activation facilitation (Eaton
and Lambert, 1957; Elmqvist and Lambert, 1968; Jablecki, 1984). Similarly, CMAPs recorded
immediately after maximal voluntary muscle contraction subsequently increase in comparison
to that at rest. It has been postulated that post-activation facilitation reflects Ca2+ build-up in
the nerve terminal that normally does not occur to the same extent as that in patients with
LEMS. Generally, sensory and motor nerve conduction velocities and latencies are
unchanged as are sensory nerve action potential amplitudes in patients with LEMS (Lambert
and Rooke, 1965; Jablecki, 1984; Heath and Cull, O'Neill and Newsom-Davis, 1988). These
findings suggests that mechanisms involved in nerve action potential generation and
conduction are unaffected in LEMS.

Single-fiber EMG (SFEMG), a technique used to measure neuromuscular transmission
in individual end-plates, is abnormal in patients with LEMS (Fig. 1 .6.) (Keesey, 1989; Sanders,
1994). SFEMG is performed during minimal voluntary muscle contractions, and allows
measurements of action potentials from two separate muscle fibers that are innervated by the
same motor nerve. Normally, the time interval between the two action potentials varies from
consecutive nerve discharges, producing neuromuscular "jitter." In some conditions,
generation of an action potential in the second muscle fiber fails; this is known as

neuromuscular "blocking." In LEMS, both jitter and blocking are dramatically increased in

39

comparison to controls and characteristically decrease as the rate of nerve stimulation increases
(Sanders, 1994).

b. Experimental

Intracellular recordings of synaptic transmission from neuromuscular preparations
isolated from patients with LEMS show electrophysiological characteristics consistent with
those found using EMG: EPP amplitudes in response to single depolarizatoin of the motor
nerve are smaller compared to those of control preparations; following high frequency nerve-
stimulation, EPP amplitude increases progressively in size (Elmqvist and Lambert, 1968;
Lambert and Elmqvist, 1971; Cull-Candy et al., 1980). MEPP amplitudes are not
appreciatively changed at LEMS patient tissue preparations nor is their rise or decay times
affected (Elmqvist and Lambert, 1968; Lambert and Elmqvist, 1971 ; Cull-Candy et al. , 1980).
Although spontaneous release of ACh is normally unaltered from LEMS preparations exposed
to buffers containing physiological concentrations of KCl, depolarization of the nerve terminal
membrane by exposure to buffers containing elevated (non-physiological) concentrations of
KCl, which increases the frequency of asynchronous release of ACh in an extracellular
calcium-dependent manner (seen electrophysiologically as MEPPS) is significantly lower than

that observed from controls.

5. Neuromuscular junction morphology in LEMS
Histological analysis of tissue biopsies from LEMS patients does not reveal any gross
abnormalities at motor nerve terminal and end-plate regions when viewed with light

microscopy (Engel and Santa, 1971). Electron microscopic examination of neuromuscular

40

t‘l

junctions from patients with LEMS also reveals normal morphology of the motor nerve
terminals, but alterations in postsynaptic regions. Engel and Santa (1971) found the following
using tissue biopsies obtained from patients with a history of LEMS: mean nerve terminal area
and number of synaptic vesicles were not significantly different from controls, mean vesicle
diameter calculated for each biopsy was also unchanged, but the postsynaptic region was
hypertrophied with increased area and length of the folds and clefts.

Although other investigators have described similar findings at motor nerve terminals,
postsynaptic membrane areas and lengths appeared decreased from patients diagnosed with
LEMS in the reports of Hesselmans et al. (1992) and Tsujihata et al.(1987). This discrepancy
with the findings of Engel and Santa (1971) may reflect differences in the duration of LEMS
in the patients used in the studies. Whereas the patients in the study performed by Engel and
Santa (1971) had a history of LEMS for up to 10 years, the other studies used patients
diagnosed as having LEMS for no longer than 1.5 years prior to their tissue biopsies (Tsujihata

et al., 1987; Hesselmans et al., 1992).

G. Lambert-Eaton Myasthenic Syndrome: Autoimmunity

1. Clinical and Experiment Findings

Early observations that an autoimmune component underlies the pathology in LEMS
were based upon the findings that many patients with defects of neuromuscular transmission
had associated autoimmune disorders (Norris and Panner, 1966; Takamori et al.,
1972;Gutmann et al., 1972). More recent work has shown a higher than normal frequency of

autoimmune disorders, such as vitiligo, pemicious anemia, celiac disease, juvenile-onset

41

Control LEMS
CMAP

pre-exercise
3 Hz stim ulation

 

>

53:35:33“... M %
ll,

50 Hz stim ulation

   

llllllllll .

Figure 1.5. EMG recordings of CMAP from control and LEMS patients. CMAP during
stimulation of the motor nerve at 3 Hz (low frequency) are smaller in amplitude from LEMS
patients in comparison to controls. Following maximal voluntary muscle contractions (post-
exercise), CMAP increase in amplitude in comparison to pre-exercise recordings in LEMS
patients during 3 Hz stimulation of the motor nerve. Prior to exercise, high frequency
stimulation (50 Hz) of the motor nerve leads to the progressive increase in amplitudes of the
CMAP from LEMS, but not control patients. Adapted from Jablecki (1984).

l
l
pre-exercise 1
:il
l

3 sec

42

LEMS Control

 

JUN. j/
_l

1 sec

4-n1\/

Figure 1 .6. Single-fiber EMG recordings from two muscle fibers innervated by the same motor
nerve. Tracings represent superimposed potentials following low frequency single shock
stimulation of a motor nerve from control or LEMS patients. The duration of time between
the two potentials recorded from each muscle fiber innervated by the same motor nerve varies
between superimposed tracings and is known as jitter. Failure in the generation of a potential
in the second muscle fiber is known as blocking. Tracings from LEMS patients exhibit a
higher degree of jitter and blocking in comparison to control patients. Adapted from Sanders

(1994)

43

diabetes and presence of other organ-specific antibodies (Lennon and Fairbanks, 1982; O'N eill
and Newsom-Davis, 1988) in patients with LEMS. Further support for an autoimmune
component was gleaned from the observations that plasma exchange and immunosuppressive
therapy induced marked improvements of symptoms in patients with LEMS with or without
SCLC (Lang and Murray, 1981; Newsom-Davis and Murray, 1984). The autoimmune basis
for LEMS was established by the findings that IgG from patients with LEMS injected into
mice not only duplicated the electrophysiological characteristics observed from EMG
recordings and intracellular electrophysiological recordings of muscle preparations from
patients with LEMS (Lang and Murray, 1981), but ultrastructural studies showed IgG located
at the motor nerve terminals as well (Fukunaga et al., 1983). Passive transfer of LEMS to
mice using IgG in comparison to whole plasma fractions with slightly lower IgG
concentrations showed a greater reduction in release of ACh from motor nerve terminals
exposed to IgG alone (Lang and Murray, 1981). These findings provide strong support for

the hypothesis that LEMS is an autoantibody-mediated disorder.

2. Antigenic targets

a. Voltage-dependent calcium channels: general findings

It was originally proposed that the autoantibody identified in patients with LEMS acts
at the motor nerve terminal and most likely interferes in the utilization of Ca2+ normally
involved in evoked-release of ACh. This proposal was based upon the findings that repetitive
nerve stimulation at high rates or increases in extracellular Ca2+ levels, which both increase the

probability of quantal ACh release improve synaptic transmission (Lambert and Elmqvist,

44

1971 ). Although the specific antigenic target of LEMS antibodies was unclear, entry of C a2 "
through voltage-dependent channels or subsequent intraterminal processes involved in
exocytosis or Ca2+ metabolism represented potential sites of disruption. However, in
conditions of physiological extracellular K+ and zero (or low) Ca2+concentrations from buffer,
the rate of spontaneous release of ACh from LEMS motor nerve terminals was normal (Lang
et al., 1987). This finding suggests that in LEMS, intraterminal processes, which are
independent of extracellular Ca?” levels are unaffected. If this was not the case, the frequency
of occurrence of spontaneous release of ACh would be reduced. In the presence of high
extracellular Caz“ concentrations, nerve-evoked release of ACh also appeared normal,
suggesting that exocytotic mechanisms that occur following Ca2+ entry are also unaffected in
LEMS; elevated extracellular Ca2+ concentrations increase the amount of Ca2+ entering the
terminal during nerve-stimulation. Thus, it appeared based upon these findings that LEMS
antibodies most likely reduce Ca2+ entry into the nerve terminal, presumably by affecting
voltage-dependent C a2+ channels. Experiments involving the cardiac glycoside, ouabain, which
blocks the Nal/K+ exchanger have provided further evidence against an intraterminal target in
LEMS (Wray et al., 1987). Incubation of motor nerve tenninals with ouabain increases the
rate of spontaneous release of ACh via a mechanism independent of Ca2+ entry through VDCC
(Baker and Crawford, 1975). Ouabain-induced release of ACh is not altered following
exposure of motor nerve terminals to LEMS antibodies, thus intraterminal mechanisms are not
affected in LEMS (Lang et al. , 1987; Wray et al. , 1987). Alternatively, the characteristics of
LEMS could be explained by impaired ACh synthesis, storage, and uptake, however, based

upon biochemical and electrophysiological techniques these appear intact (Molenaar et al.,

45

1982; Lang and Vincent, 1984; Meyer et al., 1986).

Other possible targets in LEMS also have been excluded. The observation that release
of ACh is impaired following Ki-induced depolarization of motor nerve terminals from
patients with LEMS, suggests that action potential generation and conduction are not affected
and the antibody effects are limited to nerve terminals. This idea (as described above) has also
been confirmed by direct measurements of conduction velocity and action potentials from
muscle and nerve preparations (Elmqvist and Lambert, 1968; Kim, 1985). Postsynaptic
sensitivity to ACh also appears unaltered and MEPP amplitudes are not appreciably affected
in LEMS (Prior and Newsom—Davis, 1985; Lang et al., 1987). Additional observations
consistent with the idea that LEMS antibodies interfere with Ca2+ entry through VDCC
include the findings that electrical stimulation of motor nerves exposed to LEMS antibodies
exhibit strikingly similar electrophysiological characteristics following exposure to heavy-metal
ions, which are known to block directly Ca2+ channels (Jenkinson, 1957; Silinsky, 1985).

A number of studies have provided more direct support implicating VDCC as the
putative target in LEMS. Incubation of SCLC tissue cultures with IgG from patients with
LEMS inhibits uptake of 45Ca2+ during Ki-induced depolarization (De Aizpurua et al. , 1988).
Furthermore, this inhibition is dependent upon the concentration and time of exposure of the
SCLC to LEMS IgG. Electrophysiological recording of Ca2+ currents from SCLC cells
exposed to LEMS plasma have also corroborated the finding from the K+-induced 45Ca2+
uptake studies; SCLC cells exposed to LEMS IgG for 24 hours reduced the voltage-dependent
Ca2+ current by ~5 8% (Viglione et al. , 1995) in comparison to SCLC cells exposed to control

IgG. In another study, exposure of SCLC cells in culture to LEMS IgG for 24 hours reduced

46

the voltage-dependent Ca2+ entry by up to 70% (Meriney et al. , 1996). Caz’ currents in bovine
adrenal chromaffin cells (BAC) also are reduced following exposure to LEMS IgG. However,
direct injection of Ca?” into the cell, which bypasses the need for VDCC, induced normal
exocytosis (Kim, 1987; Kim and Neher, 1988; Engisch etal., 1999). Similarly, ionomyocin,
which forms pores in membranes specific for Ca2+ entry and also bypasses the need for
VDCC, triggers equal release of ACh from electric ray nerve terminals exposed to LEMS or
control IgG (Satoh et al. , 1998). Although these finding elucidate the antigenic target, they
do not provide direct evidence that VDCC located on mammalian nerve terminals are affected
by LEMS antibodies. However, Ki-induced uptake of 45Ca2+ into rat nerve terminal
preparations (synaptosomes) is reduced following acute exposure to sera from patients with
LEMS (Hewett and Atchison, 1991, 1992a). Along these lines, Ca2+ currents at motor
nerves obtained from mature mice are also reduced following injection of LEMS patient sera
for 30 days (Smith et al., 1995; Xu et al., 1998). Taken together, these observations fiirther
support the hypothesis that LEMS antibodies act on VDCC in multiple systems without
altering intracellular mechanisms involved in exocytosis.

b. Voltage-dependent calcium channels: specificity

Immunoprecipitation assays have been developed which not only extend clinical
findings regarding LEMS, but also offer insight into the specificity of antibodies directed
against VDCC subtypes in LEMS. The various immunoprecipitation techniques all relied on
solubilization of cell membranes, extraction of crude fractions of membrane proteins including
VDCC, purification of VDCC in the presence of radiolabeled anti-VDCC ligand, and

incubation of extracted radiolabeled-ligand-VDCC complexes with LEMS or control patient

47

sera. Following corrections for non-specificity, levels of radioactivity were correlated with
specific antibody binding.

The original description of this assay used the human neuroblastoma cell line, IMR32
(Sher et al., 1989), which exhibits excitability and secretory properties similar to sympathetic
neurons and express N- and L-, and T-type Ca2+ channels (Grassi et al., 1994). VDCC were
partially purfied from IMR32 cells using 125I-(n-conotoxin GVIA('251-to-Cng), a toxin known
to bind N-type channels specifically. Approximately 92% of LEMS patients’ sera bound to
125I-(io-Cng-VDCC complexes. Although there was no significant difference between sera
from LEMS patients with and without SCLC, false positive results were observed in 9% of
control patient sera (included patients with neurological disorders other than LEMS) and 43%
of patients with SCLC without LEMS.

A study performed at the same time but using human derived SCLC or colon
adenocarcinoma tissue cultures, found 52% of LEMS patients had positive antibodies for N-
type VDCC (Lennon and Lambert, 1989). None of the patients with other neurological
diseases (myasthenia gravis, amyotrophic lateral sclerosis, parkinson's disease, and chronic
inflammatory demyelinating peripheral neuropathy) exhibited positive antibodies, whereas 10%
of SCLC patients without LEMS were seropositive. Unlike the study performed by Sher et a1.
(1989), seropositivity was found more frequently among patients with LEMS who possessed
SCLC than in LEMS patients with non— lung tumors or those without any malignancy.

Similar findings from all LEMS patients’ sera tested against VDCC isolated from the
human neuroblastoma cell line, SKN-SH were observed in comparison to the results using

human derived SCLC cultures (Leys et al. , 1989; Leys et al., 1991). Although both SKN-SH

48

and SCLC cells used in this assay posses N-type C az’ channels as determined by 125I-(o-Cng
GVIA binding, SKN-SH cells appear to have a greater number of 125I-(o-Cng GVIA binding
sites. In contrast to earlier findings, a greater percentage of LEMS patients without SCLC
were seropositive compared with LEMS patients with SCLC. The frequency of positive
antibodies was found to be significantly high in sera from patients with other autoimmune
disorders (rheumatoid arthritis and systemic lupus erythematosus (SLE)) (Leys et al. , 1989).
Although it was shown that between patients, antibody titers did not correlate with severity
of the disease, longitudinal studies within individual LEMS patients without SCLC exhibited
an inverse relationship between CMAP amplitudes and antibody titers (Leys et al., 1991).

More recently, immunological tests with greater sensitivity and specificity in detecting
antibodies in LEMS patients have been developed. Instead of extraction of solubilized N-type
VDCC, these newer assays used 125I-co-conotoxin MVIIC (”SI-co-Cme), in concentrations
specific for extracting P/Q-type channels. Lennon et al. (1995) found that 100% of LEMS
patients without lung cancer and 91% with SCLC had high titers of antibodies for 125I-o)-
Cme-VDCC complexes from SCLC and human cerebellar and cerebral membranes.
However, lower antibody titers were found in patients with cancer and no evidence of
neurologic dysfunction or among patients with amyotrophic lateral sclerosis.

Suenaga et al. (1996) have also described similar findings. Although none of the sera
tested from patients with SCLC without LEMS had detectable P/Q-type antibodies, 40% were
seropositive for N-type antibodies. Furthermore, 25% of patients with systemic lupus
erythematuous possessed antibodies against N-, but not P-type channels.

In accordance with the earlier findings, Motomura et al., ( 1997) observed that 92%

49

of LEMS patients were seropositive for P/Q—type channel antibodies, whereas only 33% had
detectable N-type channel antibodies. However, the specificity reported for this assay was
higher than that of earlier reports and as such, all control (including myasthenia gravis,
rheumatoid arthritis and SLE) sera tested were negative for antibodies against P/Q-type
channels. Furthermore, all patient sera with antibodies against N-type channels also possessed
antibodies against P/Q-type channels. Although a comparison of individual LEMS patients
showed that anti-P/Q-type VDCC did not correlate with the severity of the disease,
longitudinal studies within patients receiving therapy exhibited an inverse relationship between
antibody titers and CMAP amplitudes.

Based on the findings using immunoprecipitation assays, antibodies against P/Q- and
N-type VDCC appear to be causally associated with LEMS. Data from the longitudinal
studies further support the role of multiple VDCC subtypes in LEMS. However, antibodies
against P/Q-type channels appear to affect specifically the majority of patients with LEMS,
whereas antibodies to N-type channels appear to have a less prominent and definitive role.

The specific VDCC antibodies involved in LEMS, however, have been better
elucidated using fimctional studies. Early studies found Caz’ current flow through L-, but not
T- type VDCC in undifferentiated mouse neuroblastoma X glioma hybrid cells (N G 108 15)
is reduced following exposure to IgG from LEMS patients in comparison to exposure to
control IgG (Peers et al. , 1990). Similar findings were observed using thyroid C cells exposed
to LEMS IgG; N- and L-, but not T-type Ca2+ currents were inhibited by LEMS IgG (Kim et
al., 1993). In contrast, human neuroblastoma IMR32 cells exposed to antibodies from two

different LEMS patients not only reduced N- and L-, but T-type Ca2+ current as well (Grassi

50

et al., 1994).

Although Grassi et al. (1994 ) observed an apparent nonospecificity of LEMS IgG for
VDCC in IMR32 cells, entry of Ca2+ was reduced to a greater extent through N- in
comparison to L-type channels. Similarly, LEMS IgG had a stronger inhibitory effect on Q-
than L-type Ca2+ channels from rat insulinoma (RINmSF) cells (Magnelli et al. , 1996). These
findings are in direct accordance with the actions of LEMS IgG on SCLC cultures, in which
w-Aga IVA and nicardipine were 38-84% and 18% less effective in reducing Caz’ currents
through P/Q- and L-type channels, respectively (Viglione et al., 1995). Early studies have
suggested that the actions of LEMS IgG at BAC cells occur via downregulation of L-type Ca”
channels (Kim and Neher, 1988; Blandino and Kim, 1993 ), however, several lines of evidence
support the findings that LEMS IgG primarily targets P/Q-type VDCC in BAC cells. These
include the following: ( l) voltage-dependent Ca2+ entry into BAC cells is reduced following
exposure to LEMS sera by approximately 48% (Kim, 1987; Kim and Neher, 1988; Blandino
and Kim, 1993), (2) approximately 40% of the Ca2+ currents in BAC cells are mediated by
P/Q-type channels (Kim, 1998), and (3) P/Q-type Ca2+ currents in BAC cells are reduced
following exposure to LEMS IgG by approximately 80% (Kim, 1998). More compelling
evidence for the specificity of LEMS IgG for P/Q-type Caz’channels has been provided using
human embryonic kidney (HEK) cells expressing transfected cDNA clones encoding human
VDCC subunit proteins (Pinto et al. , 1998a; Pinto et al. , 2002). Voltage-dependent Ca2+ entry
into HEK cells expressing P/Q-type Ca2+ channels (am, (1.26, B.) was inhibited following
exposure to LEMS IgG. On the other hand, current carried through N- ((113, (125, B,,,), L-

(0110.10 (126, B2,“), and R-type channels (am 0196, B4,") expressed in HEK cells was not altered

51

by LEMS IgG. Although some of the patient sera used contained antibodies that
immunoprecipitated N-type Ca2+ channels, Caz’ entry (as determined by whole-cell Baz‘
currents) through N-type Caz" channels transfected into HEK cells was unaltered. This
discrepancy can be explained by the following: transfected N-type channels may not represent
those expressed in vivo due to splice variations or the presence of different subunit
combinations, the density of channels expressed following transfection maybe too high and
N-type channel antibody specificity too low to have an appreciable functional effect, the rate
of N-type channel upregulation may outweigh channel downregulation during LEMS IgG
exposure, and the epitopes on N-type channels may be inaccessible to LEMS IgG when
expressed in HEK cells. A lack of an inhibitory effect of LEMS sera on N-type Caz’ channels
has also been observed using human H146 SCLC cells (Viglione et al., 1995). Similar
findings have also been shown using electric ray organ synaptosomes, which are purely
cholinergic nerve terminals; LEMS IgG inhibited K*-evoked release of ACh from electric ray
organ synaptosomes by reducing P/Q- , but not N-type Ca2+ currents (Satoh et al., 1998).
Other studies also have demonstrated a preferential effect of LEMS IgG for P/Q- over
N-type Ca2+ channels. This specificity of LEMS IgG has been shown using BAC cells
(Engisch et al., 1999). As such, a direct relationship between inhibition of P/Q-, but not N-
type currents in BAC cells and severity of symptoms as measured by CMAP was detected
(Engisch et al., 1999). Furthermore, HVA Ca2+ currents recorded from cultured murine
neonatal motor neurons are reduced to a greater extent than cultured neonatal murine sensory
neurons (Garcia and Beam, 1996; Garcia and Walrond, 1996). The differential effect of

LEMS sera on voltage-dependent Ca2+ entry in rat sensory and motor neurons may be

52

explained by the diversity of VDCC-subtypes on these neurons. Although both these neuron
types from neonatal mammals possess P-, N-, and L-type channels, the predominant VDCC
subtype is believed to be N-, and P-type on neonatal sensory and motor neurons, respectively
(Mintz et al., 1992; Mynlieff and Beam, 1992). Thus, the greater reduction of Ca2+ entry in
motor neurons may be due to the predominance of P/Q-type channels located on these cells
in comparison to sensory neurons that posses N-type channels. These differences may account
for the lack of evidence of sensory nerve dysfunction in patients with LEMS.

Based upon the findings that some LEMS patient sera contain antibodies that reduce
Ca2+ entry through N-type channels and early studies that suggested that autonomic neurons
mainly utilize N-type channels to control catecholamine release (I-Iirning et al., 1988) it was
proposed that dysautonomia in LEMS patients were due to down-regulation of N-type
channels (Lang et al., 1987; Sher et al., 1993; Grassi et al., 1994). However, more recent
findings have shown that transmitter release from the same and different autonomic nerves
relies on Ca2+ entry through P/Q-, N-, R—, and sometimes L-type channels, depending upon the
mode of nerve stimulation (Lemos and Nowycky, 1989; Owen et al. , 1989; Hong and Chang,
1995; Waterman, 1996; 1997). In contrast to earlier suggestions, dysautonomia in LEMS
patients most likely reflect a preferential action on P/Q-type Ca2+ channels at autonomic nerve
terminals. Houzen et al., 1998 found that transmitter release from autonomic nerves
subserved by P/Q-, but not N-type VDCC was reduced following exposure for 6 hours to IgG
from 3 patients with LEMS. Although transmitter release from autonomic nerves obtained
from adult mice injected with 1 of 4 LEMS patient IgG for 8 days attenuated Ca2+ entry

through N-type channels, all 4 LEMS patients IgG affected P/Q-type Ca2+ channels(Waterman

53

 

rial. 1997}
release from
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et al., 1997). In addition, Houzen et al., 1998 found that LEMS IgG reduced transmitter
release from parasympathetic, but not sympathetic nerves. This finding may reflect the VDC C
subtypes subserving transmitter release from parasympathetic and sympathetic nerves used in
their study (N-type channels were involved in transmitter release from both nerve types, but
P/Q-type channels controlled release only from parasymapthetic nerves).

Other direct studies have also shown a preferential effect of LEMS sera on VDCC
subtypes at mammalian motor nerves. Caz‘ currents recorded from motor nerve terminals of
mice injected with LEMS sera for 30 days are reduced in comparison to respective control
recordings (Smith et al., 1995; Xu et al., 1998). Furthermore, Xu et al. (1998) found that
co-Aga IVA was approximately 30% less effective in reducing the amplitude of Caz’ currents
recorded from LEMS motor nerve terminals in comparison to controls and the Ca2+ current
remaining after exposure of LEMS motor nerve terminals to oo-Aga IVA is blocked by
nifedipine. On the other hand, nifedipine did not affect Caz’ currents from control motor nerve
terminals (Xu et al., 1998). These findings suggest that exposure to LEMS sera not only
reduces P/Q-type Caz’ currents, but induces the appearance of a novel L-type Ca” current at
the motor nerve terminal as well. However, acute exposure of rat forebrain synaptosomes to
LEMS sera does not alter the binding characteristics of nitrendipine (Xu et al., 1998), thus
providing evidence that the development of L-type Ca2+ currents requires long-term exposure
to LEMS sera. Similar findings have been observed using cultured mammalian motor neurons
(Garcia and Beam, 1996); while both LVA and HVA currents at cultured mammalian motor
neurons were reduced following exposure to LEMS sera, L-type currents were spared.

Although LEMS antibodies appear to target a heterogeneity of Caz’ channel subtypes,

54

 

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albeit with differing sensitivities, VDCC found at rodent skeletal and cardiac tissue and insect
skeletal muscle are insensitive to the actions of LEMS antibodies. The inability of LEMS
antibodies to affect these channels may be a function of the specific oil subunit comprising the
VDCC on these tissues. In light of these observations, LEMS antibodies do exhibit some
species and anatomic specificity. Furthermore, the preferential action on P/Q-type Ca2+
channels most likely accounts for the observations that neuromuscular transmission, which is
subserved by P/Q-type Ca2+ channels is the primary abnormality in LEMS.

c. Voltage-dependent calcium channels: antigenic sites

The B subunit was found to be antigenic when screened against plasma of LEMS
patients with high titer levels of IgG. However, the B subunit is not considered the actual
target in LEMS based upon several observations. The location of these proteins on the
cytoplasmic side of the membrane makes them normally inaccessible to circulating antibodies
(Catterall, 2000), and injection of B subunit proteins into mice induces high antibody titer
levels against the B subunit without inducing any neurological dysfunctions (Rosenfeld et al.,
1993b; Rosenfeld et al., 1993a; el Far et al., 1995; Verschuuren et al., 1998). The high
frequency and preferential sensitivity of antibodies against P/Q-type Caz” channels in
firnctional and immunoprecipitation assays suggested that antigenic sites reside within the
primary subunit of the channel; namely, the (1M subunit, which contains regions that are
potentially exposed to circulating antibodies. Synthetic peptides representing extracellular S5-
S6 linker segments in each of the four domains of the or1A subunit were used to test
immunoreactivity to LEMS patient sera. It was found that 30% of LEMS patient sera showed

specific antibody binding to the SS-S6 region of Domain IV and 20% of patient sera used was

55

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specific for the 85-86 region of Domain II (Takamori et al., 1997), whereas none of the sera
showed reactivity for Domains I or 111. However, when longer synthetic peptide regions of
85-86 were used, 50% of the LEMS patient sera had antibodies for the SS-S6 regions of
Domain (Iwasa et al. , 2000). This finding implicated the conformational necessity of the SS-
S6 linker region of Domain III to elicit an immunoglobulin response. Immunoreactive linear
epitopes representing regions of the 85-86 linker in Domain II and Domain IV were observed
in greater than 50 % of patient sera tested (Parsons and Kwok, 2002) using a different assay
than that used by (Takamori et al., 1997).

Further support for the 85-86 linker regions of Domains I-IV as potential antigenic
epitopes was also provided by immunization of rats with the synthetic peptides. Rats
immunized with synthetic peptide regions representing S5-S6 linker regions of Domain [1
(40%) and III (60%) induced similar electrophysiological characteristics to those observed in
LEMS (Komai et al., 1999; Takamori et al., 2000b). Although, not every rat developed
neurological deficiencies, these sites may represent potential epitopes involved in eliciting
antibody responses to VDCC in LEMS. One of the problems associated with using synthetic
peptides as antigens is that the conformation of the synthetic peptide may not duplicate that
of the antigen, making its recognition difficult or impossible. The SS-S6 linker regions are all
anchored to transmembrane regions for each domain, and thus synthetic peptides of the linkers
regions may not directly represent the conformation states of these regions. This may explain
any differences that arise between the passive transfer LEMS methodology and that involved
using synthetic peptides. However, it is clear that these epitopes represent potential sites for

VDCC antibody development.

56

(1. Additional antigenic targets

In addition to VDCC, other antigenic targets have been proposed to exist in LEMS.
In particular, the synaptic vesicle protein, synaptotagmin has been implicated. As discussed
earlier, synaptotagmin is believed to act as the calcium sensor and play a vital role in the
docking and fusion of vesicles during exocytosis (Perin et al., 1990; Brose et al., 1992).
Several lines of evidence lend some support to the role of synaptotagmin in LEMS. First, it
was observed that Western blots of partially purified VDCC from rat brains probed with IgG
and plasma from LEMS patients labeled a 58 kDa antigen, which was putatively shown by
partial amino acid sequence to represent synaptotagmin (Leveque et al., 1992). However,
only IgG and plasma from LEMS patients with high antibody titers specific for 125Loo-Cng
GVIA- precipated VDCC labeled the 58-kDa protein on Western blots. Based on similar
molecular weights, it was unclear if the 58-kDa protein actually represented synaptotagmin
or the B subunit that is normally a component of VDCC (Leveque et al., 1992; el Far et al.,
1995). The exclusion of the B subunit was shown by the finding that 6 out of 20 LEM S
patients sera used reacted with synaptotagmin I expressed using E. Coli. (Takamori et al.,
1995; Takamori et al., 2000a). However, only 3 out 6 of these sera reacted specifically with
expressed synaptotagmin alone, whereas the other remaining patient sera exhibited cross-
reactivity to purified Q- and N-type VDCC (Takamori et al., 1995; Takamori et al., 2000a).
In addition, SCLCs, which are believed to express the antigenic sites that initiate the
development of autoantibodies in the majority of LEMS patients, have also been observed to
posses synaptotagmin (David et al. , 1993). Lastly, the EPP quantal content was significantly

reduced for 50% of diaphragm preparations obtained from rats immunized with synthetic

57

synaptotagmin residues, and quantal content increased when these preparations were bathed
in buffers containing elevated Ca2+ concentrations(Takamori et al. , 1994). However, none of
the rats injected with synthetic synaptotagmin were clinically weak or exhibited alterations in
CMAPs. This lack of a pronounced effect may reflect differences between synthetic peptides
as immunogens, which are often only weakly recognized (Takamori et al., 1990; Takamori et
al., 1992) and intact antigens that normally are responsible for initiating LEMS.

However, there are several deficits supporting a role for synaptotagmin as a potential
antigenic target in LEMS. In its native state, the C-terminus of synaptotagmin extends into
the cytoplasm of the nerve terminal, whereas the N-terminus lies within the synaptic vesicle
(Perin et al., 1990) and thus, normally neither component is accessible to the actions of
extracellular LEMS antibodies. During exocytosis, the N—tenninus of synaptotagmin is
exposed to the extracellular surface of the nerve terminal (Shoji-Kasai et al., 1992) and as
such, it has been postulated to represent a potential antigenic site (Leveque et al. , 1992; el Far
et al., 1995; Takamori et al., 1995; Takamori et al., 2000a), however this idea has to yet to
be proven. Additionally, not all investigations have identified synaptotagmin positive
antibodies in LEMS sera; IgG from 14 LEMS patients used to probe Western blots containing
recombinant synaptotagminand human membrane and rat synaptosomal proteins did not label
bands corresponding to synaptotagmin (Hajela and Atchison, 1995). This finding, however,
may reflect a heterogenous specificity among antibodies fiom different patients with LEMS.
Furthermore, immunoprecipitation studies that identified synaptotagmin as a putative binding
site for LEMS antibodies relied upon denaturation of partially purified Ca2+ channels and

therefore, in such a system, epitopes common to both synaptotagmin and VDCC may exist.

58

It has been shown that immunoprecipitation of VDCC with anti-synaptotagmin antibodies was
dependent upon the detergent used to purify partially the VDCC; in the presence of non-
denaturing detergents, anti-synaptotagmin antibodies do not immunoprecipitate VDCC
(Lennon et al. , 1995). It is also possible that LEMS sera contain other specific antibodies that
only recognize proteins associated with VDCC in intact, non-denatured configurations
(Leveque et al., 1992). Furthermore, the specific synaptotagmin congener present in SCLC
cells may not reflect the putative synaptotagmin congener believed to be affected by LEMS
antibodies. Lastly, use of ionomyocin or direct injection of Ca” as means to increase
intracellular Ca2+ concentrations without the need for VDCC overcomes the inhibition of
evoked-transmitter release from synaptosomes (Satoh et al., 1998) or BAC cells (Kim 1988)
exposed to LEMS antibodies. This finding also provides support against synaptotagmin as a
putative target in LEMS. Thus, while there is some evidence consistent with a role for
synaptotagmin in LEMS, the minority of patients’ plasma seropositive for synaptotagmin

antibodies and lack of conclusive evidence suggests that is does not play a major role.

3. Antibody etiology

The involvement of SCLC as a potential etiologic source underlying LEMS was
originally suspected based upon the findings that at least 60% of LEMS patients have a
detectable SCLC and these SCLCs exhibit Caz’ spike electrogenesis in culture (McCann et al.,
1981). This idea was later supported by several other findings: SCLC possess multiple VDCC
subtypes, including L-, N-, and P/Q-type channels that potentially contain antigenic sites

responsible for antibody development in LEMS (McCann et al., 1981; Roberts et al., 1985;

59

De Aizpurua et al., 1988; Pancrazio et al., 1989; Sher et al., 1990; Codignola et al., 1993;
Barry et al. , 1995; Viglione et a1. , 1995; Meriney et al. , 1996); the VDCC- subtypes expressed
on SCLC correlate with the channel subtypes affected in LEMS (De Aizpurua et al., 1988;
Sher et al., 1990; Barry et al., 1995; Viglione et al., 1995); exposure of SCLCs in culture to
antibodies from LEMS patients reduced evoked-Ca” influx (Roberts et al., 1985; (De
Aizpurua et al., 1988; Lang and Newsom-Davis, 1989; Viglione et al., 1995); LEMS
antibodies precipitate VDCC on SCLC (Sher et al., 1990); and finally, clinical improvement
of LEMS follows treatment or removal of SCLC (Chalk et al., 1990; Darnell and DeAngelis,
1993). Thus, the possibility of SCLC as etiologic antigenic source for LEMS appears quite
high. Although the reason VDCC are found on SCLCs is uncertain, it is believed that Ca2+
influx through these channels is necessary for mitosis and antibodies most likely develop
against these VDCC in order to prevent tumor growth (Hafner and Petzelt, 1987).
Approximately 40% of LEMS patients lack a detectable tumor. Thus, the antigenic
etiology is less clear in these cases, however, many patients with LEMS have other
autoimmune disorders or have first-blood relatives with autoimmune diseases (Norris and
Panner, 1966; Gutmann et al., 1972; Takamori et al., 1972; O'Neill and Newsom-Davis,
1988). As such, the incidence of vitiligo, pernicious anemia, celiac disease, juvenile-onset
diabetes mellitus, and thyroid disease is higher than normally expected in patients with LEMS.
In a study performed by Lennon et al. (1982), 48% of LEMS patients without a tumor were
found to have gastric and/or thyroid antibodies. Furthermore, a number of LEMS patients
without a detectable tumor appear to possess specific polymorphisms of genes representing

the different classes of human leukocyte antigens (I-ILA). The human leukocyte antigens

60

(HLA) represent a complex of genes encoding polymorphic molecules (divided into three
classes) found on almost every nucleated cell in the human body and are involved in processing
and presenting foreign antigens to T- cells of the immune system and in the development of
T-cells within the thymus (McCusker and Singal, 1990; Rammensee et al., 1993).
Furthermore, slightly different forms (alleles) of the genes encoding each HLA class exist
between individuals and each allele within a given class is named differently. Based upon
HLA-typing there appears to be a strong association with HLA-B8 in regards to class I, and
a strong association for HLA-DR3 and I-lLA-DRZ among class II with LEMS in patients
without a detectable tumor (Willcox et al., 1985; Parsons et al., 2000; Wirtz et al., 2001).
While the etiology of LEMS antibodies is even more elusive in patients without a detectable

tumor, susceptibility to LEMS is increased in patients possessing specific HLA alleles.

4. Pathophysiological Mechanisms

a. Electrophysiological features

Although Ca2+ entry into BAC cells exposed to LEMS antibodies is reduced, single
Caz’ channel properties, such as activation and inactivation kinetics, ion conductance, and
channel open time are unaltered (Kim and Neher, 1988; Grassi et al., 1994; Magnelli et al.,
1996). This finding suggests that LEMS antibodies react with VDCC in an all or none fashion,
and as such either irreversibly block or induce the elimination of firnctional channels. This
suggestion is further supported by the observations that the relationship between K’-induced
release of ACh and increasing extracellular Ca2+ concentrations is altered at motor nerve

terminals exposed to LEMS antibodies in a manner resembling the actions of an irreversible

61

antagonist (Lang et al. , 1987). Nerve-stimulated release of ACh from LEMS-treated motor
nerve terminals, on the other hand, appears to reflect the actions of a competitive antagonist
(i.e. heavy metals). As such, the effect of LEMS antibodies on reducing maximum release of
ACh can be overcome in the presence of high Caz’ concentrations following nerve-, but not
K"—induced stimulation. This discrepancy can be explained by differences in the methods used
to elicit release of ACh. Unlike high K’ concentrations, electrical depolarization of the nerve
produces much larger simultaneous entry of C az’ into the terminal, thus saturating intraterminal
release mechanisms before saturation of Ca2+ entry through VDCC occurs (Lang eta]. , 1987).
Raising extracellular Ca” concentration, which leads to an increase in the amount of Ca2+
entering the terminal following nerve-stimulation, competes with the saturated release
mechanisms and thus, gives the false appearance that LEMS antibodies act on VDCC in a
manner resembling a competitive antagonist (Lang et al., 1987). Ki-evoked entry of Ca”, on
the other hand does not saturate intraterminal mechanisms and, thus maximum release of AC h
becomes limited by saturation of Caz’ entry instead. Taken together, these observations
support the role of LEMS antibodies as an irreversible antagonist, reducing VDCC in an all
or none fashion.

b. Ultrastructural features

Early investigations examining ultrastructural freeze-fracture analysis of motor nerve
terminals provided compelling evidence that VDCC are eliminated in LEMS. Normally,
ultrastructural analysis of motor nerve terminals reveals the presence of large
intramembraneous structures in the active zone region known as active zone particles (AZPs).

Multiple findings support the idea that VDCC comprise active zone particles. (Llinas et al.,

62

1976; Pumplin et al., 1981; Robitaille et al., 1990). First, the rapid and precise nature of
neuromuscular transmission necessitates a close proximity of Ca2+ channels with synaptic
vesicles ready to be released at active zones. As such, the latency between Cai’ influx and
synaptic vesicle exocytosis is quite small (Llinas et al. , 1976) and exocytosis can be shown via
quick freezing to occur near active zone particles (Heuser et al., 1979). Furthermore,
maximum Caz’ currents in the motor nerve terminal are related to the number of active zone
particles (Pumplin et al. , 1981). More direct support for the association of VDCC and MP5
is provided by the observation that fluorescent staining with w-Cng GVIA, which presumably
represents labeling to VDCC labeled Ca2+ channels at frog motor nerve terminals in regular
patterns corresponding to AZPs, precisely match fluorescent labeled ACh receptors on the
post-synaptic membrane (stained with a—bungarotoxin - a high affinity ligand for the muscle
type nicotinic receptor) in a manner consistent with ultrastructural findings. (Robitaille et al. ,
1990). Furthermore, imaging of fluorescently-labelled Caz’ ions in conjunction with carbon-
fiber electrode techniques to map spatially release of catecholamines from BAC cells reveals
that voltage-dependent C az’ entry occurs in distinct localized regions ("hotspots") and release
of catecholamines mainly occurs within 1.4 pm of these "hotspots." (Robinson et al., 1995).
On the other hand, catecholamine release rarely occured at sites away from these "hotspots. "
These findings provide firrther support for the the close proximity of VDCC and transmitter
release sites.

Tissue biopsies from patients with LEMS displayed a paucity and disorganization of
AZPs and active zones, and clustering of AZPs in comparison to control tissues (Fukunaga,

1982). Similar findings occurred at motor nerve terminals of mice following chronic injection

63

of IgG
proride

511291351

IBIS.
ut-Cglx
same sit.
the plasr
“ng

induce tl

of IgG from patients with LEMS (Fukuoka et al., 1987b). These observations not only
provided one of the first pieces of evidence implicating VDCC as the antigenic target, but also
suggested that loss of VDCC ultimately leads to the pathology of LEMS.

c. Radioligand binding

Other findings have also provided further evidence that VDCC are eventually lost in
LEMS. Incubation of [MR cells for 24 hours with LEMS antibodies decreases binding of 125I-
m-Cng GVIA (Sher er al., 1989). This situation could arise if LEMS antibodies bind to the
same site on the VDCC as 125I-rn-Cng GVIA or if the number of VDCC are eliminated from
the plasma membrane. However, multiple studies have shown that LEMS antibodies and 125I-
w-Cng GVIA bind to discrete sites on VDCC, thus providing evidence that LEMS antibodies
induce the eventual loss of VDCC (as described below).

(I. Antigenic modulation

Although the mechanism involved in reducing functional VDCC is unclear, it has been
proposed that antigenic modulation is a necessary step. Ultrastructural analysis of
neuromuscular junction preparations incubated with LEMS antibodies indicates that a
reduction in the distance between adjacent AZPs initially occurs followed by disorganization
and clustering, eventually leading to loss of AZPs (Engel et al. , 1987 ; Fukuoka et al. , 1987b).
Furthermore, the decrease in distance between AZPs is consistent with the width of binding
regions on the IgG molecule (Fukuoka et al. , 1987a). These findings suggest that modulation
of AZPs, and presumably VDCC, occurs by cross linking by LEMS IgG (Engel et al., 1987;

Fukuoka et al. , 1987a). This idea was tested by exposing motor nerve terminals for 24 hours

to antibodies that were either capable (divalent IgG (F(ab’)2 fragments) or incapable

64

(monovalent IgG (F(ab)) fragments) of cross-linking VDCC (Nagel et al. , 1988; Peers et al.,
1993). Following incubation with LEMS IgG F(ab)2, but not LEMS IgG F(ab) fragments,
AZPs appeared aggregated and depleted (Nagel er al., 1988). In conjunction with this study,
nerve-stimulated release of ACh is also reduced after motor nerve terminals are exposed to
LEMS IgG F (ab)2, but not LEMS IgG F(ab) fragments (Peers et al., 1993). In light of these
results, antigenic cross-linking appears to be a necessary prerequisite for functional loss of
VDCC in LEMS.

e. Direct block of voltage-dependent calcium channels

In addition to a reduction in the number of VDCC, some studies suggest that
irreversible blocking of the channels by LEMS IgG contributes to the reduction in release of
ACh. Binding of LEMS antibodies to VDCC has been reported to occur rapidly (Martin-
Moutot et al., 1995a) and as such, Ki-induced uptake of 45Ca2+ into mammalian nerve terminal
preparations is significantly reduced following incubation with sera from patients with LEMS
for either 3 minutes (Meyer et al., 1986) or 1 hour (Hewett and Atchison, 1991, 1992a).
Similarly, exposure of SCLC or BAC cells in culture for 90 minutes or 30 minutes,
respectively, to LEMS antibodies reduces voltage-dependent Ca2+ entry, which becomes more
pronounced after 24 hours (Johnston et al., 1994) (Kim and Neher, 1988). In contrast,
exposure of mammalian neuromuscular junction preparations in vitro to LEMS antibodies or
sera for 2 hours does not consistently affect nerve-evoked release of ACh (Prior and Newsom-
Davis, 1985; Kim et al., 1988). Similarly, the t1)2 of reduction in release of ACh following
a single i. v. injection of antibodies from one patient with LEMS into mice was observed to

take approximately 4-6 hours (Lambert and Lennon, 1988). It is possible that access to motor

65

 

nerve terminals in such preparations is limited, thus increasing the time required for LEMS
antibodies to have an observable effect. However, in cell cultures and nerve terminal
preparations antibody-induced reduction in Ca” entry occurs too rapidly to be completely
explained by VDCC elimination and thus, loss of function in part is most likely the result of
direct block of the channel, which may be a first step prior to cross linking.
f. Role of complement
In addition to antibody-induced antigenic modulation, other immune components could
be involved in LEMS. Complement has been observed to participate in the pathogenesis of
other neuromuscular disorders. As such, complement components have been found localized
at the end-plate regions of patients with myasthenia gravis and in myasthenia gravis animal
model systems. Thus, it is possible that complement is involved in LEMS as well.
Elimination of foreign bodies is aided by complement, which is comprised of serum
proteins that act sequentially and in concert with one another to form two interconnected
pathways--the classical and alternative pathway (Fig. 1.7.). Although both pathways share
common components, they are activated differently (Goetz, 1987; Loos, 1987; Benjamini et al.,
1996). Whereas the classical pathway is activated by the formation of antibody and antigen
complexes, the alternative pathway is activated by various other substances, such as cobra
venom and some bacteria cell walls and yeast. Many of the protein complexes formed in each
pathway act as enzymes that catalyze the next step and/or aid directly in elimination of the
initiating factor. Most of the complexes are given a number designation preceded by the letter
“.”C Two key protein complexes of complement are C3 and C5. The enzymatic reaction

of C3 by C3 convertase produces C3a and C3b, which are vital components that enhance

66

phagocytosis, immune adherence, and degranulation of mast cells and basophils (Hostetter et
al., 1984). C3b acts a common intermediary between the two complement pathways; it
activates the alternative pathway and leads to its own amplification (Goetz and Muller-
Eberhand, 1971). In addition, C3b also leads to the production of C5 convertase, which
enzymatically cleaves C5 into C5a and C5b. Whereas C5a is important for mast and basophil
cellular degranulation and as an attractant for other immune components, C5b initiates the
formation of the C 5-C9 complex, which possess cytotoxic properties (Porter and Reid, 1978).
The role of complement in LEMS has been investigated using mice either genetically deficient
in C 5 or treated with cobra venom, which leads to the depletion of C3 by continual activation
of the alternative pathway (Prior and Newsom-Davis, 1985; Lambert and Lennon, 1988).
Chronic injection of LEMS antibodies into mice deficient in C5 and C3 and mice with intact
complement systems produced similar impairment of nerve-evoked release of ACh from motor
nerve terminals. Also, muscle biopsies of patients with LEMS examined using immuno-electron
microscopy does not exhibit localized C3 components on the nerve terminal (Engel et al.,
1977; Tsujihata et al., 1987). These findings suggest that complement is not involved in
LEMS. However, reduction in K*-induced 45Ca2+ uptake into mammalian synaptosomal
preparations exposed to LEMS antibodies for 1 hour is dependent upon C3, but not C5
(Hewett and Atchison, 1992b). In addition, factor B was also observed to be required for low
concentrations of LEMS antibodies to impair K’-induced ”Caz’ uptake in these preparations
(Hewett and Atchison, 1992b). Although complement components do not appear to be
involved in LEMS, it is possible that in certain model systems complement enhances the effects

of LEMS antibodies. As such, it has been noted that many of the effects of antibodies on cells

67

are increased in the presence of complement (Benjamini et al., 1996).

H. Voltage-dependent calcium channels and neurological dysfunction

Alterations in functional VDCC have been implicated in not only LEMS, but other
neurological disorders as well. Mutations in the gene encoding the or1A subunit in humans are
associated with episodic ataxia type 2 (EA2), familial hemiplegic migraine (FHM), and
spinocerebellar ataxia type 6 (SCA6) (Ophoffet al., 1996; Zhuchenko et al. , 1997). Although
these disorders result from different mutations of the same gene, the neurologic features
associated with each condition sometimes overlap. EA2 is provoked by stress, exercise, and
fatigue, characterized by ataxia, nystagmus, dysarthria, diplopia and vertigo, and begins in
childhood to late adolescence. This phenotype is the result of frame-shifts and splice-site
mutations that lead to truncated proteins. Mice exhibiting the leaner phenotype have similar
changes in the gene encoding or1A subunits, however, these animals exhibit severe ataxia,
absence seizures, and premature death (Herrup and Wilczynski, 1982; Doyle et al. , 1997; Dove
etal., 1998).

Similarly, FHM presents in childhood, but represents a form of migraine that is
accompanied with an aura and includes hemiparesis, aphasia, or hemianopia. Symptoms and
signs can last a few hours to days, and identical attacks are described in first-degree relative(s).
The disorder is due to missense mutations in the gene encoding the or1A subunit (the
CACNL1A4 gene found on chromosome19p131). Similar mutations also occur in mice,
producing the tottering phenotype (Fletcher et al., 1996; Doyle et al., 1997). Unlike the

phenotype displayed in humans, tottering mice also erdiibit motor and absence seizures, in

68

 

addition to ataxia. Although the tottering phenotype in mice appears analogous to the leaner
phenotype, the characteristics are much less severe. Another mutation in mice at the same gene
locus as the tottering and leaner phenotypes has been observed to cause severe ataxia without
seizues. This phenotype is known as the rolling mouse Nagoya (Mori et al., 2000).

SCA6 usually begins in late adulthood and causes nystagmus, ataxia, dysarthria, and
impaired vibration and position sensation. It is associated isolated with atrophy of the
cerebellum, and it is believed to be due to an expanded CAG repeat in the gene encoding the
onA subunit. In addition to cerebellar changes, absence seizures have been linked to a mutation
in the gene encoding the or,A subunit (Jouvenceau et al., 2001). Alterations in other subunit
proteins comprising the VDCC have also been associated with absence seizures. For example,
reduced expression of B4 subunits in mice leads to the lethargic phenotype, which not only
exhibit instability of gait and convulsions, but absence seizures as well (Burgess et al., 1997)
(McEnery et al. , 1998). Recently, mutations in the B4 subunit have been suggested to underlie
seizures in some human disorders (Escayg et al., 2000). Although it has yet to be
characterized in humans, mice with mutations of the 01252 protein (ducky mutation) exhibit

ataxia and paroxysmal dyskinesia (Barclay et al., 2001).

69

CLASSICAL Antibody-Antigen
PATHWAY l

Clqrs

ALTERNATIVE ' ' -

 

 

PATHWAY
Cm

Cobra venom,
some bacteria —"C3b
cell walls and /
yeaSt +factor B +factor B

I Amplification

C3bB Cij

Jrélctor D +factor D

 

C3bBb

Figure 1.7. Relationship of the alternative and classical complement pathways.
Adapted from Benjamini et al. 1996.

70

CHAPTER TWO

LAMBERT-EATON MYASTHENIC SYNDROME [N MICE INDUCES

DIHY DROPYRIDIN E SENSITIVITY OF SYNAPTIC TRANSMISSION

71

A. Summary

Lambert-Eaton Myasthenic Syndrome (LEMS) is a paraneoplastic disorder in which
autoantibodies apparently target the voltage-gated Caz’ channels that regulate acetylcholine
(ACh) release at motor nerve terminals. P/Q-type Ca2+ channels are primarily involved in ACh
release at mammalian neuromuscularjunctions. Passive transfer of LEMS to mice by repeated
administration of plasma from LEMS patients reduces the amplitude of the perineurial P/Q-type
current, and unmasks a dihydropyridine (DHP)-sensitive L-type Ca2+current at the motor nerve
terminal. The present study sought to detemiine if this DHP-sensitive component contributes
to ACh release. Mice were injected for 30 days with plasma from healthy human controls or
patients with LEMS. For some studies, diaphragms from naive mice were incubated with LEMS
or control human plasma for 2 or 24 hr. End-plate potentials (EPPs) and miniature end-plate
potentials (MEPPS) were recorded from neuromuscular junctions of the hemidiaphragm.
Injection of mice with LEMS plasma caused the characteristic electrophysiological signs of
LEMS: reduced quantal content and facilitation of EPP amplitudes at high frequency
stimulation. Quantal content was also reduced in muscles incubated acutely with LEMS
plasma. Nimodipine, a DHP-type antagonist of L-type Ca2+ channels did not significantly affect
quantal content of muscles incubated for 2 or 24 hr with either control or LEMS plasma or
following chronic injection with control plasma. However, following 30 days injection with
LEMS plasma, nimodipine significantly reduced the remaining quantal content to 57.7 i 3 .3 %
of pre-nimodipine control. Thus, DHP-sensitive Ca2+ channels become involved in synaptic
transmission at the mouse neuromuscular junction after chronic but not acute exposure to

LEMS plasma. However, reductions in quantal release of ACh occur even after very short

72

 

per

6011

but

res;

lie

arc'

fruit

periods of exposure to LEMS plasma. As such, development of L-type Caz’ channel
contribution to ACh release during passive transfer of LEMS appears to occur only after
quantal release is significantly impaired for an extended duration, suggesting that an adaptive

response of the ACh release apparatus occurs in LEMS.

B. Introduction

Lambert-Eaton Myasthenic Syndrome (LEMS) is a paraneoplastic disorder in which
nerve-evoked, Ca2’-dependent release of acetylcholine (ACh) from the presynaptic nerve
terminal is impaired (Elmqvist and Lambert, 1968; Lambert and Elmqvist, 1971). Patients with
LEMS typically exhibit peripheral limb muscle weakness and fatigability, decreased reflexes,
as well as various dysautonomias (Heath et al., 1988; O'Neill et al., 1988; Khurana et al.,
1988). LEMS often is associated with a small cell lung carcinoma (Lennon et al., 1982). The
underlying cause of LEMS is not firlly understood, but the disease is thought to result from
generation of antibodies against the tumor. Passive transfer of LEMS to rodents by chronic
injection of plasma, serum or immunoglobulins faithfirlly duplicates the hallmark
electrophysiological signs of LEMS- namely reduced quantal content, and facilitation of end-
plate potential (EPP) amplitudes during repetitive stimulation (Lang et al., 1983, 1987; Kim,
1985; Prior et al., 1985).
In LEMS, circulating autoantibodies are thought to target the voltage-gated Ca2+
channels involved in release of ACh from the nerve temrinal (Lang et al., 1981, 1983; Lambert
and Lennon, 1988). In several model systems, Ca2+ channel fimction is diminished after

application of LEMS serum or plasma (Kim and Neher, 1988; Hewett and Atchison, 1991 ; Kim

73

int

‘1'
>112“)

(
(I

r" .
. arc

et al., 1993; Garcia et al., 1996; Garcia and Beam, 1996; Pinto et al., 1998). More directly,
in passive transfer models of LEMS, the amplitude of Caz’currents recorded from nerve
terminals of murine neuromuscular preparations is reduced (Smith et al. , 1995; Xu et al., 1998).
Furthermore, active zone particles, which presumably correspond to Ca2+ channels at the nerve
terminal, appear to be disorganized and fewer in number at motor nerve terminals in LEMS
patients (Fukunaga et al., 1982) or LEMS-injected mice compared with control groups
(Fukunaga et al., 1983; Fukuoka et al., 1987). This reduction in the number of Ca2+ channels
at the nerve terminal is likely responsible for the attenuated quantal content seen in LEMS.
Among the multiple types of voltage-gated Ca2+ channels that exist; several including
the L-, N-, and P/Q-types have been shown to control neurotransmitter release; multiple
subtypes often coexist at the same synapse to regulate transmitter release (Turner and Dunlap,
1995). At the mammalian neuromuscular junction the P/Q-type of Ca2+ channel is primarily
involved in release of ACh (Uchitel et al., 1992; Protti et al., 1996; Katz et al., 1996; 1997).
L-type Ca” channels, which participate in release of hormones (Lemos and Nowycky, 1989)
and noradrenaline from chromaflin cells of the adrenal medulla (Owen et al. , 1989), and N-type
channels, which control ACh release at non-mammalian neuromuscular junctions (Sano et al.,
1987), do not appear to be involved normally in the nerve-stimulated release of ACh from
mammalian motor nerve terminals (Atchison and O’Leary, 1987; Atchison, 1989; Uchitel et al.,
1992; Protti et al. , 1996; Katz et al., 1996; 1997). Whereas N-type channels have been shown
to be affected by LEMS serum (Peers et al., 1990; Suenaga et al., 1996), several recent studies
suggest that following exposure to LEMS sera, function of L-type Caz’ channels is spared

(Garcia and Beam, 1996) or unmasked (Smith et al., 1995; Xu et al., 1998). Specifically,

74

ll

0. 1
.qu

injection of mice for 30 days with plasma from LEMS patients attenuates the amplitude of the
P/Q-type Cay current and exposes a DHP-sensitive L-type Caz’ current. Inasmuch as induction
of DHP-sensitive Caz’ current at murine motor nerve terminals might represent an adaptive
response to reduction of function of the normal complement of Ca2+ channels by LEMS
autoimmune attack, the present study was undertaken to determine if this DHP-sensitive
component contributes to the release of ACh from motor nerve terminals in the LEMS passive

transfer model and whether this component develops in tandem with the onset of LEMS.

C. Materials and Methods

Chronic exposure studies. Animal care, handling, and experiments were performed in
accordance with local university (Michigan State University Laboratory Animal Resources) and
national guidelines. Experiments were performed using male ICR mice (20-22 g, Harlan
Sprague-Dawley Laboratories, Madison, WI). Mice were injected once daily intraperitoneally
(11p. ) for 30 days with 1 .5 ml of plasma from patients clinically diagnosed with LEMS or plasma
from healthy (control) patients. Prior to injection with plasma, mice were first injected with 300
mg/kg i.p. of cyclophosphamide to suppress the immune response to exogenous proteins.
After 30 days of plasma injection, animals were sacrificed by decapitation following anesthesia
with 80% CO2 and 20% Oz. The diaphragm muscle with the attached phrenic nerve (Barstad
and Liliheil, 1968) was then removed and prepared for electrophysiological recording.

Acute exposure studies. Naive male ICR mice were sacrificed as described above.
After removal of the diaphragm muscle and the attached phrenic nerve, the tissue was incubated

for 24 hr at room temperature of 23-25°C in Dulbecco’s Minimum Essential Medium with 20%

75

 

517.}:

“but

(v/v) of LEMS or control plasma. The medium was aerated continuously with 95% O2 and 5%
C02. The tissue was washed subsequently in buffered saline solution. Alternatively, the tissue
was incubated for 2 hr at room temperature in buffered saline solution containing 20% of the
appropriate plasma under continual oxygenation (100% 02). After the specified incubation time
the tissue was prepared for electrophysiological recording.
Electrophysiological measurements. Diaphragm preparations were pinned out at
resting tension in a Sylgard-coated chamber and perfirsed continuously with buffered saline
solution at a rate of approximately 1-5 mein. Tissues that were incubated for only 2 hr were
perfiised at a similar rate but contained buffered saline solution with 5% (v/v) plasma. In order
to prevent muscle contraction following stimulation of the phrenic nerve, the diaphragm muscle
was cut approximately 4 mm on either side of the main intramuscular nerve branch (Glavinovic,
1979; Traxinger and Atchison, 1987). This technique does not produce significant changes in
the muscle cable properties (Glavinovic, 1979; Lambert et al., 1981) and permits simultaneous
recordings of end-plate potentials (EPPs) and miniature end-plate potentials (MEPPS) without
the complicating effects of depressing transmitter release with high Mg2+/low Ca2+ solutions or
inducing postjunctional receptor block with d-tubocurarine (Hubbard and Wilson, 1973). All
recordings were made at room temperature of 23-25 °C using conventional intracellular
recording techniques. EPPs and MEPPs were recorded using borosilicate glass microelectrodes
(1.0 mm- o.d., WP Instruments, Sarasota, FL) and having resistance of 5-25 MQ when filled
with 3M KCl. The phrenic nerve was stimulated supramaximally using a suction electrode
attached to a stimulus isolation unit (Grass SIU, Grass Instruments, Quincy, MA) and

stimulator (Grass 888). Recordings were made before and after addition of nimodipine.

76

in

Experiments performed in the presence of nimodipine were done in the dark, in order to prevent
photo-oxidation of this compound. Signals were amplified using either an Axoclamp-Z (Axon
Instruments, Foster City, CA) or WPI 721 (WP Instruments, Sarasota, FL) amplifier and
digitized into a computer for inspection using Axoscope 8.0 (Axon Instruments) software and
analyzed using MiniAnalysis 4.0 software (Synaptosoft, Decatur, GA). EPP and MEPP
amplitudes were standardized to a membrane potential of —50 mV in order to correct for
changes in membrane potential driving force (Katz and Thesleff, 195 7). Recordings from each
preparation were sampled and averaged from at least 5 different endplates before and after
addition of nimodipine, yielding an n value of 1. Quantal content (m) was calculated using the
ratio of the mean amplitude of the corrected EPPs to the mean amplitude of the corrected
MEPPs (Hubbard et al., 1969).
Solutions and Chemicals. The standard buffered saline solution contained (mM): NaCl
137.5, KCl 2.5, MgCl2 1, CaCl2 2, D-glucose 11, HEPES 14, pH adjusted to 7.4 at room
temperature with NaOH and kept under continual oxygenation (100% 02). In order to prevent
depolarization-induced nerve conduction block that can occur when Ki is released from the cut-
muscle fibers, 2. 5 mM KCl was used throughout the experiments (Hubbard and Wilson, 1973;
Glavinovic, 1979; Traxinger and Atchison, 1987). Nimodipine was prepared as a 10 mM stock
solution in 100% ethanol, which was kept at 4° C until use. The final working solution with
nimodipine contained only 0.1% ethanol (v/v). Plasma samples from patients clinically
diagnosed as having LEMS were graciously provided by Drs. Eva L. Feldman and James Albers
(University of Michigan Health System, Ann Arbor, MI, USA), Dr. Andrew Massey (University

of Kentucky Medical Center, Lexington, KY), and Dr. Shin Oh (University of Alabama Medical

77

Center, Birmingham, AL). Plasma was supplied with no identifiers and only patient age and
gender were provided. Plasma was obtained during the course of routine plasma exchange
therapy with the normal informed consent and in accordance with the respective institutions'
human subject committee approval. Control human plasma was donated by healthy volunteers
and obtained as outdated blood donations from the American Red Cross (Lansing, MI).
Nimodipine, cyclophosphamide, and N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
(HEPES) were purchased from Sigma Chemical Co. (St. Louis, MO). All other reagents were
of analytic grade or better.

Statistical Analysis. Differences in quantal content for both acute and chronic
treatment studies in animals receiving LEMS or control plasma were analyzed using a one way
analysis of variance followed by Tukey’s test. P values were set to < 0.05 for all statistical

ICSIS.

D. Results

Effect of LEMS plasma on neuromuscular transmission at low frequency
stimulation. The duration of exposure to control plasma did not affect neuromuscular
transmission from mouse hemidiaphragm preparations. Table 2.1 indicates that there was no
significant difference between the resting membrane potentials, EPP amplitudes, or MEPP
amplitudes recorded from neuromuscular preparations of mice injected chronically with control
plasma or from preparations obtained from naive mice and incubated acutely with control

plasma for 2 or 24 hr (P > 0.05).

78

 

Conversely, Figure 2.] demonstrates that passive transfer of LEMS markedly reduced
evoked release of ACh. Quantal content of EPPs (m) recorded following 0.5 Hz stimulation of
the phrenic nerve from hemidiaphragm preparations obtained from mice injected for 30 days
with LEMS plasma was reduced significantly in comparison to mice injected for the same
duration with control plasma. Similar levels of reduction in m were observed in preparations
from mice injected with plasma from each of three LEMS patients. The composite reduction,
using values obtained from all patients was 45.1 3: 3.1 % of control (Fig. 2.4).

Acute incubation of naive murine neuromuscular preparations with LEMS plasma also
caused a significant reduction in m in comparison to preparations exposed acutely to control
plasma. 2 hr incubation of diaphragm preparations with LEMS plasma reduced m to 61.4 i 5.3
% of control for the composite of three patients (Fig. 2.4). This composite reduction in m for
all three patients was significantly different from that observed in neuromuscular preparations
from mice injected chronically with LEMS plasma as well as from preparations exposed acutely
to control plasma (Fig. 2.4). Plasma from only two of the individual LEMS patients
significantly reduced 111 compared to preparations incubated with control plasma when applied
for 2 hr (Fig. 2.3). Further incubation of naive murine neuromuscular preparations with LEMS
plasma (24 hr) also reduced m to 56.3 :t 5.1 % of 24 hr incubation with control plasma (Fig.
2.4). Plasma from all three of the individual LEMS patients significantly reduced or compared
to preparations incubated with control plasma when applied for 24 hr (Fig. 2.2). However, the
reduction in m following 24 hr incubation with LEMS plasma was not significantly different
from that of neuromuscular junction preparations from mice injected chronically with LEMS

plasma or from naive murine neuromuscular preparations incubated with LEMS plasma for 2

79

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sti.

ele

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hr (Fig. 2.4).

None of the three LEMS exposure paradigms (30 days, 2 hr, and 24 hr) significantly
affected MEPP amplitudes or muscle resting membrane potentials (P> 005- results not shown).

Effect of LEMS plasma on neuromuscular transmission at high frequency
stimulation. In addition to a reduced m at low frequency stimulation, another
electrophysiological characteristic of LEMS is an increase in the amplitude of EPPs, in
comparison to that of the first EPP, evoked during high frequency stimulation of the nerve
(Lang et al., 1983, 1987; Kim, 1985; Prior et al., 1985). 40 Hz stimulation of the phrenic nerve
caused an overall reduction of EPP amplitudes from all three control exposure protocols (Figs.
25, 2.6, and 2.7). On the other hand, LEMS plasma exposure in the 30 day injection, as well
as the 2 hr, and 24 hr incubation paradigms each caused facilitation of the EPP amplitude in
comparison to the initial EPP amplitude (Figs. 2.5, 2.6, and 2.7).

The involvement of DHP-sensitive Ca2+ channels in neuromuscular transmission
from motor nerve terminals treated with LEMS plasma. Normally, DHP-sensitive L-type
Ca” channels are not involved in the nerve-stimulated release of ACh from adult mammalian
motor nerve terminals (Atchison and O’Leary, 1987; Atchison, 1989; Uchitel et al., 1992; Protti
et al., 1996; Katz et al., 1996; 1997). As shown in Figures 2.8, 2.9, and 2.11 the nerve-
stimulated release of ACh recorded from diaphragm preparations of mice injected chronically
with control plasma or from preparations incubated acutely with control plasma was not
sensitive to nimodipine. In addition, quantal content from hemidiaphragm preparations
incubated for 24 or 2 hr with LEMS plasma was not altered in the presence of nimodipine (Figs.

2.8 and 2.9).

80

 

Table 2.1.

Muscle resting membrane potential (RMP), MEPP amplitude, and EPP amplitude from
neuromuscular junction preparations exposed for 2 hr, 24 hr, or 30 days to control plasma

 

Control RMP‘M MEPP Amplitude EPP Amplitude
Treatment (mV) (mV) (mV)
30 days -41.4 i 1.9 0.46 i 0.04 9.9 i 1.2
24 hr -43.7 i 1.7 0.42 i 0.05 9.6 d: 0.84
2 hr -42.9 i 2.1 0.43 i 0.05 9.3 d: 0.92

 

 

 

 

#Values are the mean i SEM of at least 4 preparations of each control exposure.
*Preparations were “cut” to prevent muscle contraction, resulting in depolarized preparations

due to the release of K“‘ from out fibers. Extracellular [K“’] was reduced to 2.5 uM; an
equiosmolar increase in [NaCl] was made.

81

T. a“

mar—i

30 days
100 -

90-
80-
70*

60" * *

 

Quantal Content (% of control plasma)

 

Pt. 1 Pt. 2 Pt. 3

Figure 2.1. Effect of exposure to LEMS plasma for 30 days on quantal content of mouse
hemidiaphragm endplates. Hemidiaphragm preparations were taken from immunosuppressed
mice injected for 30 days with 1.5 ml/day (i.p.). MEPP and EPP amplitudes were recorded
using standard intracellular techniques. EPPs were elicited at a frequency of 0.5 Hz. Quantal
content was determined from each neuromuscular junction preparation using the ratio of the
average EPP amplitude to the average MEPP amplitude. Values are expressed as the
percentage of quantal content fi'om the LEMS plasma-exposed preparations to that of control
plasma-exposed preparations. Each value represents the mean i SEM of at least 5 different
preparations. The asterisk (*) indicates a value significantly different from control (P < 0.05).

82

 

24.hr

 

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Pt.1 Pt.2 Pt.3

Figure 2.2. Effect of incubation with LEMS plasma for 24 hr on quantal content of mouse
herrridiaphragm endplates. Hemidiaphragm preparations were taken from naive mice and
incubated for 24 hr with plasma from one of three LEMS patients (Pt). MEPP and EPP
amplitudes were recorded using standard intracellular techniques. EPPs were elicited at a
frequency of 0.5 Hz. Quantal content was determined from each neuromuscular junction
preparation using the ratio of the average EPP amplitude to the average MEPP amplitude.
Values are expressed as the percentage of quantal content from the LEMS plasma-incubated
preparations to that of control plasma-incubated preparations. Each value represents the mean
:1: SEM of at least 5 different preparations. The asterisk (*) indicates a value significantly
different from control (P < 0.05).

83

2hr

100-

\O
O
l

Quantal Content (% of control plasma)

 

 

Figure 2.3. Effect of incubation with LEMS plasma for 2 hr on quantal content of mouse
hemidiaphragm endplates. Hemidiaphragm preparations were taken from naive mice and
incubated for 2 hr with plasma from one of three LEMS patients (Pt). MEPP and EPP
amplitudes were recorded using standard intracellular techniques. EPPs were elicited at a
frequency of 0.5 Hz. Quantal content was determined from each neuromuscular junction
preparation using the ratio of the average EPP amplitude to the average MEPP amplitude.
Values are expressed as the percentage of quantal content from the LEMS plasma-incubated
preparations to that of control plasma-incubated preparations. Each value represents the mean
i SEM of at least 5 different preparations. The asterisk (*) indicates a value significantly
different from control (P < 0.05).

84

Com bined Patients

 

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2 hr 24 hr 30 days

Figure 2.4. Effect of exposure to LEMS plasma on quantal content of mouse hemidiaphragm
endplates. Hemidiaphragm preparations were taken from immunosuppressed mice injected for
30 days with 1.5 ml/day (i .p.) or from naive mice and incubated for 24 hr or 2 hr with plasma
from one of three LEMS patients. Panel represents the combined patients results for all 3
following 2 hr, 24 hr, and 30 days of exposure to LEMS plasma. MEPP and EPP amplitudes
were recorded using standard intracellular techniques. EPPs were elicited at a frequency of 0. 5
Hz. Quantal content was determined from each neuromuscular junction preparation using the
ratio of the average EPP amplitude to the average MEPP amplitude. Values are expressed as
the percentage of quantal content from the LEMS plasma-exposed preparations to that of
control plasma-exposed preparations. Each value represents the mean i SEM of at least 5
different preparations for each patient plasma. The asterisk (*) indicates a value significantly
different fi'om 30 days treatment (P < 0.05).

85

m.

.. a
V...»
.uC.

However, as shown for sample records in Figure 2.10 and for composite data from all
preparations in Figure 2.1 1 , nerve-stimulated release of ACh from motor nerve terminals of
mice injected chronically with LEMS plasma was sensitive to nimodipine. As shown in F igiure
2. 1 l , or recorded from preparations obtained from mice injected chronically with LEMS plasma
was reduced in the presence of nimodipine to 57. 7 i 3 .3 % of their nimodipine-free ("control")
value. Plasma from all three LEMS patients induced this nimodipine sensitivity of

neuromuscular transmission.

E. Discussion

LEMS is associated with the loss of functional voltage-gated Caz’ channels at motor
nerve terminals and thus the reduction of the nerve-evoked release of ACh. Normally, P/Q-
type, but not L- or N-type Ca2+ channels control the nerve-stimulated release of ACh from adult
mammalian motor nerve terminals (Atchison and O’Leary, 1987; Atchison, 1989; Uchitel et al.,
1992; Protti et al., 1996; Katz et al., 1996; 1997). Passive transfer of LEMS to mice by 30
days of plasma injection attenuates the perineural voltage changes associated with the amplitude
of the P/Q-type C 32+ current and induces the appearance of a DHP-sensitive L-type Ca2+ current
at murine motor nerve terminals (Smith et al., 1995; Xu et al., 1998). The primary objective
of the present study, therefore, was to determine if this novel DHP-sensitive L-type C az’ current
now becomes involved in synaptic transmission from adult mammalian motor nerve terminals
in LEMS.

Nimodipine, a DHP-sensitive L-type Ca2+ channel blocker, had no effect on m from mice

injected chronically for 30 days with control plasma, nor did it affect m in motor nerve terminals

86

incubated acutely for 2 or 24 hr with either LEMS or control plasma. This finding is consistent
with those observed by Xu et al. (1998), in which the binding characteristics of [3H]-
nitrendipine to rat synaptosomal nerve terminal preparations were not affected by acute
application of LEMS IgG. Nimodipine, did however, attenuate m from motor nerve terminals
isolated from mice injected for 30 days with LEMS plasma. Thus, following chronic injection
of mice with LEMS plasma DHP-sensitive L-type Ca” channels become involved in the nerve-
stimulated release of ACh from mammalian nerve terminals.

Although synaptic transmission from neuromuscular preparations isolated from mice and
incubated acutely with LEMS plasma for 2 and 24 hr was not sensitive to nimodipine, m was
reduced in comparison to hemidiaphragm preparations incubated with control plasma. In
addition, facilitation of EPP amplitudes occurred in neuromuscular preparations incubated for
2 or 24 hr with LEMS plasma. The observation that m is reduced after a brief exposure to
LEMS plasma is consistent with the findings of Hewett and Atchison (1991), in which uptake
of 45Ca2+ uptake into rat forebrain synaptosomal preparations during KCl-induced
depolarization is reduced after acute exposure to LEMS IgG. However, this is the first study
that we are aware of in which a consistent reduction in m is observed from neuromuscular
preparations exposed for such a short duration (2 hr) to LEMS plasma. This finding may be
due to the nature of our treatment paradigm, which consisted of a high concentration of plasma
(20%) during the acute incubation phase and the continual presence of a low concentration

(0.5%) of plasma during the recording phase.

87

30 days

 

 

 

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9..
but
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is
754
l 2 3 4

Stimulus number

Figure 2.5 Effect of exposure to LEMS plasma for 30 days on facilitation of neuromuscular
transmission. EPP amplitudes were recorded using standard electrophysiological techniques
from hemidiaphragm preparations taken from mice injected for 30 days with 1.5 ml/day (i.p.)
of LEMS or control plasma. EPPs were elicited by stimulation of the phrenic nerve at 40 Hz.
The symbols, ——0— and ——I— represent LEMS and control plasma-injected groups,
respectively. Values are expressed as the mean ratio fl: SEM of the amplitude of EPPn to that
of the first EPP elicited in the train (n represents the stimulus number). The asterisk (*)
indicates a value significantly different fi'om that of the first EPP (P < 0.05).

88

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l 2 3 4

Stimulus number

Figure 2.6. Effect of exposure to LEMS plasma for 24 hr on facilitation of neuromuscular
transmission. EPP amplitudes were recorded using standard electrophysiological techniques
from hemidiaphragm preparations taken from naive mice and incubated for 24 hr with LEMS
or control plasma. EPPs were elicited by stimulation of the phrenic nerve at 40 Hz. The
symbols, —0— and —I— represent LEMS and control plasma-incubated groups,
respectively. Values are expressed as the mean ratio i SEM of the amplitude of EPPn to that
of the first EPP elicited in the train (n represents the stimulus number). The asterisk (*)
indicates a value significantly different from that of the first EPP (P < 0.05).

89

2hr

 

 

 

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1 2 3 4

Stimulus number

Figure 2.7. Effect of exposure to LEMS plasma for 2 hr on facilitation of neuromuscular
transmission. EPP amplitudes were recorded using standard electrophysiological techniques
fiom hemidiaphragm preparations taken from naive mice and incubated for 2 hr with LEMS
or control plasma. EPPs were elicited by stimulation of the phrenic nerve at 40 Hz. The
symbols, —0— and —I— represent LEMS and control plasma-incubated groups,
respectively. Values are expressed as the mean ratio :t SEM of the amplitude of EPPn to that
of the first EPP elicited in the train (n represents the stimulus number). The asterisk (*)
indicates a value significantly different fiom that of the first EPP (P < 0.05).

90

140_ 24 hr

 

 

 

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Control LEMS Pt. 1 Pt. 2 Pt. 3
Combined

Figure 2.8. Effect of nimodipine on quantal content of mouse hemidiaphragm preparations
exposed to LEMS plasma for 24 hr. Hemidiaphragm preparations were taken from naive mice
and incubated continuously for 24 hr with plasma from one of three LEMS patients (Pt) or
control plasma. EPPs were elicited at a fiequency of 0.5 Hz. Quantal content was determined
fiom each preparation using the ratio of the average EPP amplitude to the average MEPP
amplitude before and after the addition of 10 M nimodipine. Values are expressed as the
percentage of quantal content from preparations after the addition of nimodipine to that of the
same preparation before nimodipine. Each value represents the mean i: SEM of at least 5
different preparations.

91

140- 2 hr

 

 

 

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Control LEMS Ptl Pt. 2 Pt. 3
Combined

Figure 2.9. Effect of nimodipine on quantal content of mouse hemidiaphragm preparations
exposed to LEMS plasma for 2 hr. Hemidiaphragm preparations were taken from naive mice
and incubated continuously for 2 hr with plasma from one of three LEMS patients (Pt. ) or
control plasma. EPPs were elicited at a frequency of 0. 5 Hz. Quantal content was determined
from each preparation using the ratio of the average EPP amplitude to the average MEPP
amplitude before and after the addition of 10 M nimodipine. Values are expressed as the
percentage of quantal content from preparations after the addition of nimodipine to that of the
same preparation before nimodipine. Each value represents the mean d: SEM of at least 5
different preparations.

92

LEMS Control

 

 

A it 1 J
t t it it

 

 

 

>
1 HE J .1
lOms

Figure 2.10. Effect of nimodipine on EPPs from LEMS and control plasma-exposed
neuromuscular junction preparations. EPPs were recorded from A, neuromuscular junction
preparations isolated from mice injected for 30 days, B, neuromuscular preparations isolated
from naive mice and incubated for 24 hr or C, neuromuscular preparations isolated from naive
mice and incubated for 2 hr with LEMS or control plasma. Recordings from the same site are
depicted before (-) and after (+) application of 10 M nimodipine. Each tracing represents the
average of at least 50 EPPs at a stimulation fi'equency of 0.5 Hz recorded from a single
representative preparation.

93

140.1 30 days

 

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Control LEMS Pt. 1 Pt. 2 Pt. 3
Combined

Figure 2.11. Effect of nimodipine on quantal content of mouse hemidiaphragm preparations
exposed to LEMS plasma for 30 days. Hemidiaphragm preparations were taken from mice
injected for 30 days with 1.5 ml/day (i.p.) with control plasma or plasma from one of three
LEMS patients (Pt). EPPs were elicited at a frequency of 0.5 Hz. Quantal content was
determined from each preparation using the ratio of the average EPP amplitude to the average
MEPP amplitude before and after the addition of 10 uM nimodipine. Values are expressed as
the percentage of quantal content from preparations after the addition of nimodipine to that of
the same preparation before nimodipine. Each value represents the mean i SEM of at least 5
different preparations. The asterisk (*) indicates a value significantly different from pre-
nimodipine(P < 0.05).

94

_

 

Chronic passive transfer of LEMS to mice caused the typical clinical
electrophysiological features seen in LEMS patients and reported in other studies: or was
reduced in compari son to control groups and facilitation of the EPP amplitudes occurred at high
frequency stimulation (Lang etal., 1983, 1987; Kim, 1985; Prior etal., 1985). The reduction
in m recorded from neuromuscular preparations obtained from mice injected chronically with
LEMS plasma was significantly greater than that seen in neuromuscular preparations following
2 hr LEMS incubation paradigms. This difference most likely reflects the time course required
to induce LEMS after passive transfer to mice. As shown by Prior et al. (1985), passive
transfer of LEMS to mice by daily injections of LEMS IgG does not induce a maximum
reduction in m until approximately 10 days, and the half-maximal reduction in m occurs after
1.5 days of treatment.

Involvement of the DHP-sensitive L-type channels in synaptic transmission from
mammalian motor nerve terminals following chronic LEMS exposure may reflect recruitment
of normally silent L-type channels which are already present at the terminal. Addition of the
DHP agonist, Bay K 8644 to mammalian neuromuscular preparations increases m (Atchison
and O’Leary, 1987; Atchison, 1989) and L-type channels have been shown to be involved in
the spontaneous release of ACh from mammalian motor nerve terminals in the presence of
physiological concentrations of extracellular KCl (Losavio and Muchnick, 1997). In addition,
pretreatment of mouse neuromuscular preparations with the intracellular Ca2+ buffer, DM-
BAPTA-AM unmasks a nitrendipine-sensitive perineural Ca2+ current (Urbano and Uchitel,
1999). However, this latter study did not find evidence for involvement of this unmasked L-

type channel in ACh release. This discrepancy may be due to the spatial localization of the L-

95

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the l

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befo:

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1K.)
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l'cltit
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the di

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h‘iate

type channels at the nerve terminal. If L-type channels are not in close proximity to the active
zone release machinery (see Robitaille et al., 1990) then most likely the influx of Ca2+ through
the L-type channels would have to occur in a more diffuse manner than that observed with Caz’
channels clustered about the release sites as is postulated to occur normally (Llinas et al.,
1992). DM-BAPTA-AM could therefore, buffer the Ca” entering through L-type channels
before it reached the release apparatus.

One possible explanation for the unmasking of silent DHP-sensitive L-type channels
seen after chronic LEM S-plasma exposure may be a reduction in the Ca2’-activated-potassium
(KO) current at the motor nerve terminal. KC, channels are believed to be located in close
proximity to the Ca?” channels involved in transmitter release from motor nerve terminals
(Robitaille and Charlton, 1992; Robitaille et al., 1993; Xu and Atchison, 1996; Protti and
Uchitel, 1997). These KCa channels respond to Caz’ influx through Ca2+ channels localized at
active zones (N -type in frog and presumably, P/Q-type in mice and rats) and may normally limit
the duration and extent to which the nerve terminal remains depolarised in response to an action
potential. Loss of Ca2+ channels due to LEMS plasma exposure may reduce activation of these
Kc. channels, thus prolonging the duration and extent of nerve terminal depolarization. This
enhanced nerve terminal depolarization could allow silent L-type channels, which may be
located at a site distant from the ACh release machinery to remain open long enough to become
involved in synaptic transmission (Smith et al., 1995). The DHP-sensitivity seen after chronic,
but not acute LEMS exposure could reflect the difference in the extent to which or is reduced
among the different experimental paradigms. Unlike 2 hr LEMS incubation, chronic injection

of mice with LEMS plasma may reduce Ca2+ influx at the nerve terminal enough to decrease

96

the KCa current, which in turn could unmask silent L-type channels. Although, there was no
significant difference in the reduction of in between chronic injection of mice with LEMS
plasma and 24 hr LEMS incubation, this finding may be misleading. It is likely that the
predominant Caz' channel involved in transmitter release, presumably the P/Q-type in our
preparations, may be lost to a greater extent in the chronically exposed LEMS mice than
following 24 hr LEMS incubation because m in the 30 day injection regimen was reduced by
~40-45 % by nimodipine, whereas at 24 hr, 0% of the quantal release was nimodipine sensitive.
Thus, the apparent similar reduction in m may occur because L-type Ca2+ channels now
comprise a significant portion of the nerve-evoked release of ACh in the chronically exposed
LEMS preparations. The possible distinct location of L-type Ca” channels from the normal
release sites may preclude involvement of these Ca2+ channels in activation of KCa channels.
Thus, at the motor nerve temrinals of mice injected chronically with LEMS plasma, activation
of KC, channels may be attenuated in comparison to motor nerve temrinals exposed to LEMS
plasma for only 24 hr.

Alternatively, the ability of chronic LEMS exposure to induce the involvement of L-type
channels in synaptic transmission may reflect more than simple unmasking of channels already
present. This is supported by the inability of acute LEMS-plasma incubation to induce DHP-
sensitivity of transmitter release despite reducing m. For example, processes that take a longer
time, such as synthesis of new mRNA and then new channels, may be required. Along these
lines, L-type channels become involved in transmitter release at newly forming or regenerating
mammalian neuromuscular preparations (Katz et al., 1996; Sugiura and Ko, 1997; Rosato Siri

and Uchitel, 1999; Santafe et al., 2000, 2001). However, morphological data do not support

97

the notion that LEMS treatment induces degeneration or sprouting of new nerve terminals
(Fukunaga et al., 1982, 1983; Fukuoka et al., 1987).

Finally, various studies support the idea that the fast nature of the transmitter release
process at motor nerve terminals necessitates a close association between voltage-gated Ca2+
channels and transmitter release sites (Robitaille et al. , 1990; Sugiura et al., 1995; Seagar and
Takahashi, 1998). Perhaps relocalization of L-type channels to active zones may be needed
before they become involved in transmitter release (Polo-Parada et al., 2001).

Involvement of L-type Ca2+ channels in transmitter release from adult mammalian motor
nerve temrinals may act as a compensatory mechanism in LEMS to overcome the chronic loss
of functional P/Q-type Ca2+ channels. Indirect evidence suggests that a similar phenomenon
occurs in humans with LEMS. Respiratory failure in a patient with LEMS has been shown to
be associated with the administration of verapamil (Krendel and Hopkins, 1986). In addition,
the signs of LEMS developed in a patient with ischemic heart disease who received the L-type
antagonist, diltiazem and disappeared as the patient’s serum levels of diltiazem decreased (Ueno
and Hara, 1992). Thus, it is possible that in patients with LEMS L-type Caz’ channels also
become involved in synaptic transmission at the neuromuscular junction. As such, these
patients might benefit from therapies directed specifically at facilitating current flow through
L—type Ca2+ channels.

In conclusion, following passive transfer of LEMS by chronic injection, the ACh
secretory process assumes a considerable component of L-type Ca2+ channel sensitivity. While
the mechanism underlying this adaptive change is unclear, it may reflect a compensatory process

to sustain transmission in the face of an autoimmune attack at the nerve terminal.

98

CHAPTER THREE

TIME COURSE OF INVOLVEMENT OF L-TYPE Ca2+ CHANNELS IN RELEASE

OF ACh DURING PASSIVE TRANSFER OF LEMS TO MICE

99

A. Summary

Lambert-Eaton myasthenic syndrome (LEMS) is a cholinergic nerve disorder, in which
evoked-release of ACh is diminished. Antibodies directed against voltage-dependent calcium
channels (VDCC) normally involved in ACh release are believed to underlie the pathology of
the disease and as such, P/Q-type Ca2+ channels, the primary subtype involved in ACh from
adult mammalian motor nerve terminals, represent the major antigenic target in LEMS. Chronic
injection of sera from patients with LEMS for 30 days into mice not only reduces the amplitude
of the P/Q-type current, but unmasks a L-type current that is involved in release of ACh from
the motor nerve terminal. The present study sought to further elucidate the time course of
development of L-type Ca” channel involvement in release of ACh. Quantal content from
neuromuscular junction preparations obtained from mice injected with plasma for 1-20 days
from LEMS patients 1 and 2 was significantly reduced in comparison to control plasma.
However, plasma from LEMS patient 3 did not significantly affect quantal content until day 10.
Facilitation of end-plate potential (EPP) amplitudes during high frequency stimulation of the
nerve occurred from preparations exposed to plasma from patients 1 and 2 at all days tested,
but not from controls. In accordance with the effect of plasma from patient 3 on quantal
content, facilitation of EPP amplitudes was not observed at day 1 or 5, but occurred by day 10.
Nimodipine, a dihydropyridine antagonist, did not affect quantal content from preparations
taken from mice injected with control or LEMS patient 3 plasma for 1-20 days or LEMS
patients 1 and 2 for 1-15 days. However, the remaining quantal content and facilitation of EPP
amplitudes from preparations obtained from mice injected for 20 days with plasma from

patients 1 and 2, or just patient 2, respectively were significantly reduced in the presence of

100

nimodipine. In addition, incubation of preparations exposed to plasma from patient 1 for 20
days with DM-BAPTA-AM prevented the involvement of L-type Ca2+ channels in release of
ACh, whereas DM-BAPTA-AM did not affect ACh release from control preparations.
Although reductions in quantal content can occur in a short-time following exposure to LEMS
plasma, it appears to take longer for L-type Ca2+ channels to become involved in release.
Whatever the exact mechanism that is involved, it appears as though L-type Ca2+ involved in
release of ACh following exposure to LEMS plasma are not located in close proximity to the

normal release apparatus.

B. Introduction

Lambert-Eaton myasthenic syndrome (LEMS) is neuromuscular disorder in which
evoked-release of acetylcholine (ACh) from nerve temiinals is impaired, causing skeletal muscle
weakness, decreased tendon reflexes, and autonomic dysfirnction (Elmqvi st and Lambert, 1968;
Lambert and Elmqvist, 1971; Heath and Cull, 1988; Khurana and Mayer, 1988; O'Neill and
Newsom-Davis, 1988). Several lines of evidence have shown that the defect in
neurotransmission is due to circulating antibodies; as such, plasma exchange and use of
immunosuppressant medications transiently alleviates many of the symptoms and signs
associated with LEMS (Lang and Murray, 1981; Lennon and Fairbanks, 1982; Newsom—Davis
and Murray, 1984; O'Neill and Newsom-Davis, 1988). Also, injection of IgG from patients
with LEMS into mice replicates the electrophysiological characteristics of the disease--namely,

reduced quantal content and facilitation following high frequency nerve stimulation (Lambert

101

and Elmqvist, 1971; Lang et al., 1983; Kim, 1985; Prior and Newsom—Davis, 1985; Lambert
and Lennon, 1988).

The putative target of the antibody associated with LEMS is believed to be the VDCC,
normally involved in release of ACh from nerve terminals (Lang and Murray, 1981; Lambert
and Lennon, 1988). Ultrastructural studies have shown that active zone particles, which
presumably correspond to VDCC are disorganized and fewer in number at nerve terminals
exposed to IgG from patients with LEMS compared with nerve terminals exposed to IgG from
control patients (Fukunaga, 1982; Fukuoka et al., 1987). While single VDCC kinetics and
amplitude are unaffected, entry of Caz’ through VDCC into various cell types treated with
LEMS IgG is reduced in comparison to cells treated with control IgG (Kim and Neher, 1988;
Kim et al., 1993; Garcia and Beam, 1996). More direct evidence has shown that Caz’ currents
at neuromuscular junctions treated with LEMS sera is reduced in comparison to control
treatments (Hewett and Atchison, 1991; Smith and Atchison, 1995; Xu and Atchison, 1998).
Storage and synthesis of ACh and postsynaptic structures, however, are unaffected at
neuromuscular junctions exposed to LEMS sera (Lang and Vincent, 1984). Taken together,
the effect of LEMS IgG on reducing nerve-evoked release of ACh appears to occur by
decreasing the number of functional VDCC at the nerve terminal.

Entry of Ca2 * through VDCC into nerve terminals is a necessary step coupling the action
potential to release of transmitter (Llinas et al., 1976; Augustine et al., 1987). Based on
pharmacological, biophysical, and molecular characteristics, various VDCC have been
identified: P, Q, L, N, T, and R (Nowycky et al., 1985; Tsien et al., 1991; Zhang et al., 1993;

Randall and Tsien, 1995). During development, motor nerve terminals have been shown to

102

possess multiple VDCC subtypes involved in ACh release, some of which become less
important in the later stages of development (Sugiura and Ko, 1997; Rosato-Siri and Uchitel,
1999; Santafe et al., 2001; Rosato-Siri et al., 2002). Upon maturation, release of ACh from
motor nerves primarily involve only one VDCC subtype. As such, P/Q-type VDCC are the
main subtype involved in ACh release at mature mammalian motor nerve terminals (Uchitel and
Llinas, 1992; Katz, 1995; Protti and Uchitel, 1996). In contrast, release of ACh from motor
nerves of amphibians and birds is dependent on Caz’ entry through N—type VDCC (Sano et al.,
1987; De Luca et al., 1991; Gray et al., 1992). L-type VDCC, which are often found
colocalized on the same nerve terminal with other VDCC subtypes, can participate in the
release of catecholamines and hormones (Lemos and Nowycky, 1989; Owen et al., 1989;
Turner et al., 1993).

A smaller percentage of patients with LEMS have been shown to possess antibodies
directed against multiple VDCC subtypes, however, over 90% of patients have antibodies

specifically for the P/Q subtype (Suenaga et al., 1996). In corroboration with these findings
many studies have shown a direct action of LEMS IgG on P/Q channels while sparing or
unmasking L-type VDCC (Smith and Atchison, 1995; Garcia and Beam, 1996; Xu and
Atchison, 1998). Specifically, chronic treatment of mice with plasma from LEMS patients
reduces the amplitude of P/Q-type Caz’ currents and exposes a DHP-sensitive L-type Caz’
current, which becomes involved in ACh release at mouse motor nerve terminals (Smith and
Atchison, 1995; Xu and Atchison, 1998; Flink and Atchison, 2002; Giovannini et al., 2002).
While development of L-type channels may represent a compensatory means to overcome loss

of fimctional VDCC at the nerve terminal, the present study sought to elucidate further the

103

time t

LEM!

C. .11

wet
guidel
labor:
from p
blite u
immun
inditai.
0: and

isolatec

time course of development of L-type VDCC involved in ACh following passive transfer of

LEMS to mice.

C. Materials and Methods

Passive transfer of LEMS. All experiments involving handling and use of animals
were done in accordance with local (Michigan State University Animal Resources) and national
guidelines. Male ICR mice weighing 20-22g were obtained from Harlan Sprague-Dawley
Laboratories (Madison, WI) and given daily intraperitoneal (i .p.) injections of 1.5 ml of plasma
from patients clinically diagnosed with LEMS or healthy volunteer controls for 1 to 30 days.
Mice were initially injected (i.p.) with cyclophosphamide (300 mg/kg) in order to prevent an
immune response to exogenous plasma proteins. Following the specified number of days as
indicated in the results section, animals were anesthetized via inhalation of 80% CO2 and 20%
O2 and sacrificed by decapitation. The diaphragm and attached phrenic nerves were then
isolated as described by Barstad and Lilleheil (1968).

BAPTA loading. Dimethyl BAPTA (1,2-Bis(2-amino-5-methylphenoxy) ethane-
N,N,N’,N’-tetraacetic acid tetrakis (acetoxymethyl)ester) was loaded into mouse motor nerve
terminals using a modification of procedures described by Urbano and Uchitel (1999). Uncut
hemidiaphragm preparations were incubated at room temperature of 23-25°C (v/v) under
continual oxygenation (100%02) in Ca2'—free physiological saline containing either 25 uM DM-
BAPTA-AM or 0.0125 % DMSO (control vehicle). Following 2 hr incubation, the tissue was
washed in Cali-free buffer for 10 min and then washed in normal buffer solution for 30 min.

Electrophysiology. Neuromuscular transmission was examined using isolated phrenic
nerve hemidiaphragm preparations pinned out at resting tension in a Sylgard-coated chamber

and perfused at a rate of 1-5 ml/min with physiological saline containing (in mM): NaCl 137. 5,

104

KC12.5, MgCl2 1, CaC122, D-glucose 11, HEPES 14, pH adjusted to 7.4 at room temperature
of 23-25°C with NaOH and under continual oxygenation (100% 02). The muscle was cut
approximately 4 mm on either side of the main intramuscular nerve branch in order to prevent
muscle contraction following stimulation of the phrenic nerve (Glavinovic, 1979; Traxinger and
Atchison, 1987). This technique allows simultaneous recordings of end-plate potentials (EPPs)
and miniature end-plate potentials (MEPPS) without significant changes in the muscle cable
properties (Hubbard and Wilson, 1973) or synaptic transmission. The cut hemidiaphragm
preparation was perfused with cold oxygenated physiological saline solution for 30 min; ionic
gradients that may have been disrupted by cold temperatures were then restored by perfusing
the tissue in oxygenated buffer maintained at room temperature of 23-25°C for an additional
3 0 min. All experiments were performed using conventional intracellular recording techniques.
EPPs and MEPPs were recorded using borosilicate glass microelectrodes (1 .0 mm- o.d., FHC,
Bowdoinham, ME, USA) and having resistance of 5-20 MQ when filled with 3M KCl. The
phrenic nerve was stimulated supramaximally using a suction electrode attached to a stimulus
isolation unit (Grass SIU, Grass Instruments, Quincy, MA) and stimulator (Grass 888). Signals
were amplified using a WPI 721 (WP Instruments, Sarasota, FL, USA) amplifier and digitized
into a computer for inspection using Axoscope 8.0 (Axon Instruments, Foster City, CA, USA)
software and analyzed using MiniAnalysis 5.0 software (Synaptosoft, Decatur, GA, USA).
Quantal content of end-plate potentials. Recordings from each preparation were
sampled and averaged from at least 5 different endplates from the same preparation to
determine the mean amplitude of the EPPs and MEPPs before and after addition of nimodipine,
yielding an n value of 1. Recordings were rejected if the 10-90 % EPP rise time was greater
than 1 ms. EPPs with amplitudes 2 10 mV were corrected for nonlinear summation

(McLachlan and Martin, 1981) using the formula V6,,“r = V/[(1-.8*V)/E], where V is the

105

CO
lllt

1in

usir
11.35
days

19315

uncorrected EPP amplitude, E is the resting membrane potential, and Va," is the corrected EPP
amplitude. Averaged EPP and MEPP amplitudes were first standardized to a membrane
potential of —50 mV using the formula, Vdf = V/E *-50 in order to correct for changes in
membrane potential driving force (Katz and Thesleff, 1957), where V,If represents the corrected
EPP amplitudes due to driving force and V represents the uncorrected EPP amplitude prior to
correction for nonlinear summation. Quantal content was calculated using the ratio of the
mean amplitude of the corrected EPPs to the mean amplitude of the corrected MEPPs
(Hubbard et al., 1969).

Statistics. Statistical significance between the various treatment groups was analyzed
using a one way analysis of variance followed by Tukey’s test. A two way analysis of variance
was used to examine statistical significance between MEPP frequencies of various treatment
days from control and LEMS-treated preparations. P values were set to < 0.05 for all statistical
tests.

Facilitation of end-plate potential amplitudes. EPP amplitudes were recorded from
LEMS- and control-muscle preparations during 40 Hz stimulation of the phrenic nerve. The
effect of exposure of mouse muscle preparations to LEMS or control plasma on the induction
of facilitation of EPP amplitudes was determined by first normalizing the EPPn amplitude to that
first EPP amplitude (EPPn/EPPI), where n = the stimulus number. Before averaging across
experiments, the average normalized EPP ratio for the 2'“l to 20th stimulus from one tissue
preparation was then calculated to yield a Facilitation ratio (Average (EPPn/EPP1)2_22). The
effect of nimodipine was also examined on the EPP amplitudes during 40 Hz stimulation of the
phrenic nerve and calculated by taking the ratio of the Facilitation ratio after nimodipine to the
Facilitation ratio before nimodipine. This value was denoted Inhibition of facilitation ratio to

represent the effects of nimodipine EPP amplitudes during high frequency nerve-stimulation.

106

graci
Systr

Lexi

the r.
C om
Only
lulur
.111,

than
Dbl-l
Ietrak

fill (it

Chemicals. Plasma samples from patients clinically diagnosed as having LEMS were
graciously provided by Drs. Eva L. F eldman and James Albers (University of Michigan Health
System, Ann Arbor, MI, USA), Dr. Andrew Massey (University of Kentucky Medical Center,
Lexington, KY, USA), and Dr. Shin Oh (University of Alabama Medical Center, Bimringham,
AL, USA). Plasma was obtained during the course of routine plasma exchange therapy with
the normal informed consent, in accordance with the respective institutions' Human Subjects
Committee or Institutional Review Board approval and was supplied without any identifiers.
Only patient age and gender were provided Control human plasma was donated by healthy
volunteers and obtained as outdated blood donations from the American Red Cross (Lansing,
MI, USA). Nimodipine, cyclophosphamide, and N-2-hydroxyethylpiperazine-N-2-
ethanesulfonic acid (HEPES) were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
DM-BAPTA-AM (1,2-Bis(2-amino-5-methylphenoxy)ethane-N,N,N’,N’-tetraacetic acid
tetrakis(acetoxymethyl)ester) was obtained from Molecular probes Inc (Eugene, OR, USA).

All other reagents were of analytical grade or better.

D. Results

Effect of passive transfer of LEMS to mice on release of ACh from motor nerves
at low frequency stimulation. Mice were injected for 1- 20 days with plasma either from
three separate patients (designated “Patient 1, 2, or 3") with LEMS or control patients. Passive
transfer of LEMS to mice with plasma from patient 1 and 2 significantly reduced release of
ACh following stimulation of the phrenic nerve at 0.5 Hz in comparison to control plasma
treatments for 1 - 20 days (Figs. 3.1, 3.2). On the other hand, quantal release of ACh from
motor nerves obtained from mice injected with plasma from patient 3 only became significantly

different in comparison to control plasma after 10 days (Fig. 3.3) of treatment. Resting

107

membrane potentials and MEPP amplitudes were not significantly different between any of the
patient plasma or control treatment groups (Table 3.1). In addition, spontaneous release of
ACh was not affected by injection of LEMS or control plasma at any day tested (Figs. 3.4, 3 .5).

Effect of passive transfer of LEMS on high frequency nerve-stimulated release of
ACh from motor nerves. In addition to reduced quantal release of ACh from motor nerves
exposed to LEMS plasma, another electrophysiological characteristic of LEMS is enhanced
facilitation of ACh release following high frequency nerve-stimulation. Treatment of mice with
plasma from LEMS patients 1 and 2 significantly increased EPP amplitudes following 40 Hz
stimulation of the phrenic nerve to a greater extent than did control plasma. The average ratios
of amplitudes of EPPZ,20 to EPP1 recorded from muscle preparations taken fiom mice injected
with plasma from patients 1 and 2 for 1- 20 days were significantly greater when compared to
preparations treated with control plasma (Figs. 3.6-3.11). On the other hand, treatment of
mice with plasma from patient 3 did not significantly affect averaged ratios of amplitudes of
EPPZ,20 to EPPl in comparison to control plasma preparations until after 10 days of treatment
(Figs. 36-39, 3.11).

Development of Dihydropyridine sensitivity of release of ACh from motor nerves
exposed to LEMS plasma. Nimodipine, a DHP-type L-type antagonist, significantly reduced
the evoked-release of ACh following low frequency (0.5 Hz) stimulation of the phrenic nerve
from preparations taken from mice treated with plasma from patients 1 or 2 for 20 days in
comparison to pre-nimodipine treatment (Fig. 3.17). However, nimodipine was ineffective at
reducing release of ACh from preparations taken from mice injected with plasma from patients
1 or 2 for 1 - 15 days (Figs. 3. 13-3. 16) or from preparations taken from mice injected with
control plasma for 1 - 20 days(Figs. 3.13-3.17). Plasma from patient 3 did not produce DHP-

sensitive L-type channel involvement in ACh at any day tested (Figs. 3. 13-3. 17). Furthermore,

108

nimodipine had no effect on spontaneous ACh release (measured as changes in MEPP
frequency) from preparations exposed to either LEMS or control plasma for 1-20 days (Figs.
3.4, 3.5).

The involvement of L-type Caz" channels in ACh release following high frequency
nerve-stimulation was also examined from preparations exposed to plasma from patient 1 for
20 days. Nimodipine, significantly reduced the averaged amplitude ratios of EPP}20 to EPPl
from preparations exposed to patient 1 plasma, but not control plasma for 20 days in
comparison to averaged amplitude ratios of EPP}20 to EPPl before nimodipine treatment (Fig.
312).

Effect of DM-BAPTA loading into motor nerve terminals following passive
transfer of LEMS. The proximity of L-type Ca2+ channels to the transmitter release apparatus
was examined using the rapid chelator of Ca2+, DM-BAPTA. Adler et al. (1991) observed a
reduction in evoked-release of ACh from squid nerve terminal following incubation with
BAPTA, suggesting that the location of VDCC involved in glutamate release {tour this terminal
is not tightly coupled with the release apparatus. However, VDCC at motor nerve terminals
of mice exhibit a very tight coupling with the transmitter release apparatus (Urbano and Uchitel,
1999). For these experiments, muscle preparations obtained from mice injected with control
plasma for 20 days were loaded with 25 uM DM-BAPTA-AM or DM-BAPTA control vehicle
(.0125%, v/v DMSO). Following low frequency stimulation of the phrenic nerve, release of
ACh was unchanged from control preparations loaded with DM-BAPTA in comparison to
control preparations loaded with DMSO (Fig. 3.18). Thus, the complement of VDCC
involved in release of ACh from mouse motor nerves appears tightly coupled with the normal

release process. Furthermore, DM-BAPTA control (DMSO) had no effect on exposing L-type

109

Patient 1
80-

 

 

 

A
E "
m
is: ‘t.
— \
Q1 60‘ -- ‘\
—-1 9: \
O \\
I: \
E ‘s
c \
U s‘
\e 40- x;
SJ * ‘~‘~~-
§ 7% “um-I
3':
is
20 ' a .

0 5 1'0 15 20
Day

Figure 3.1. The time course of the effect of plasma from LEMS patient 1 on quantal content
(m) at mice hemidiaphragm preparations. Hemidiaphragm preparations were obtained from
mice injected with 1.5 m1/day(i.p.) of plasma from LEMS patient 1 for the number of days
shown. Quantal content was determined from each neuromuscular junction preparation using
the ratio of the average EPP amplitude to the average MEPP amplitude. Values are expressed
as the percentage of quantal content from the LEMS plasma-treated preparations to that of
control plasma-treated preparations. Each value represents the mean i SEM of at least 5
different preparations. The asterisk (*) indicates a value significantly different from control
plasma (P < 0.05).

110

Ili/

8 OTPatlent 2

60- xx}

m (% control plasma)
3. 1
Hit
l—I-l

 

20 - . - .
0 5 10 15 20

Day

 

Figure 3.2. The time course of the effect of plasma from LEMS patient 2 on quantal content
(or) at mice hemidiaphragm preparations. Hemidiaphragm preparations were obtained from
mice injected with 1.5 ml/day (i.p.) of plasma from LEMS patient 2 for the number of days
shown. Quantal content was determined from each neuromuscular junction preparation using
the ratio of the average EPP amplitude to the average MEPP amplitude. Values are expressed
as the percentage of quantal content from the LEMS plasma-treated preparations to that of
control plasma-treated preparations. Each value represents the mean :t SEM of at least 5
different preparations. The asterisk (*) indicates a value significantly different from control
plasma (P < 0.05).

111

Patient 3

60‘ a: \‘i
4:

404

m (% control plasma)

a?
3!.

 

20 . . . . . .
O 5 10 15 20 25 30
Day

 

Figure 3.3. The time course of the effect of plasma from LEMS patient 3 on quantal content
(m) at mice hemidiaphragm preparations. Hemidiaphragm preparations were obtained from
mice injected with 1.5 ml/day (i.p.) of plasma from LEMS patient 3 for the number of days
shown. Quantal content was determined from each neuromuscular junction preparation using
the ratio of the average EPP amplitude to the average MEPP amplitude. Values are expressed
as the percentage of quantal content from the LEMS plasma-treated preparations to that of
control plasma-treated preparations. Each value represents the mean i SEM of at least 5
different preparations. The asterisk (*) indicates a value significantly different from control
plasma (P < 0.05).

112

Table 3.]

Muscle resting membrane potentials (RMP)” and miniature end-plate potential (MEPP)
amplitudes from preparations exposed to control plasma or plasma from 3 separate patients

with LEMS for 1-20 days

 

 

 

 

 

 

 

 

 

 

 

Days Control":A Patient 1“ Patient 2“ Patient 3'“
1 -38.3 3:27 -4l.6i: 1.1 -40.3:t1.4 -41.5:t2.7
0.738 :1: 0.052 0.662 i .027 0.786 3: .047 0.694 :1: .057

5 -38.1 :1: 1.8 -41.6 :t 1.9 -39.4 i 2.6 -38.7 :l:1.7
0.785 :1: 0.036 0.677 i .023 0.695 :1: .060 0.672 3: .024

10 -38.1:t 7.2 -39.5 $1.3 40.9 :1: 3.6 -39.5 :E 1.1
0.698 :t 0.039 0.725 i .046 0.720 :1: .031 0.720 :t .021

15 -39.0 :t 2.9 -40.9 21:19 -41.5 d: 1.3 -41.2 :1: 2.7
0.711 :1: 0.037 0.746 i .037 0.707 :1: .023 0.680 d: .033

20 -38.2 i 2.7 -38.6 i 3.4 -40.5 d: 1.5 -39.3 :1: 2.2
0.794 :f: 0.064 0.694 :t .044 0.700 :1: .038 0.687 d: .021

 

”Preparations were cut to prevent muscle contractions resulting in release of K from the
fibers and depolarization of the membrane. Values are the mean :1: SEM of at least four

preparations for each treatment group.

R" denotes measurements of RMP (mV) and MEPP amplitude (mV), respectively.

113

 

1,0 , Control plasma - Dre-nimodipine
[Illinunodqnne

0.8-

0.6-

MEPP frequency (Hz)

 

 

 

 

 

 

 

 

 

10 15 20
Days

F1811 Fe 3 .4. The time course of the effect of plasma from control patients on MEPP frequency
at mice hemidiaphragm preparations. MEPP frequency was determined fiom hemidiaphragm
preparations taken fi'om mice injected with 1 .5 ml/day (i. p.) of plasma from control patients for
the time indicated. Recordings from each preparations were made in the presence or absence
( p r e‘ nimodipine”) of 10 uM nimodipine. Each value represents the mean i SEM of at least

5 d“ I erent preparations.

114

q LEMS plasma

 

 

 

 

 

 

 

 

 

 

1.0
- pre-nimodipine
nimodipine
0.8 ‘
A
i 0.6 -
>3
0
L".
<1)
:3
8
9b 0.4 7
Q.‘
E
0.2 r
0.0

 

10 15
Days

Figure 3.5. The time course of the effect of plasma from LEMS patients on MEPP frequency
at mice hemidiaphragm preparations. MEPP frequency was determined from hemidiaphragm
Preparations taken from mice injected with 1.5 ml/day (i. p.) of plasma fi'om LEMS patients for
the time indicated. Recordings from each preparations were made in the presence or absence
(“Pie—nimodipine”) of 10 uM nimodipine. Each value represents the mean 2: SEM of at least
5 different preparations.

115

Da 1
1.2- y

Facilitation ratio

 

 

Control Pt. 1 Pt. 2 Pt. 3

Figure 3.6. Effect of LEMS plasma injected for 1 day into mice on facilitation of transmitter
release. EPP amplitudes were recorded from hemidiaphragm preparations taken from mice
injected with 1.5 ml/day (i.p.) of plasma from control or LEMS patients (Pt.) for 1 day. Values
are expressed as the mean i SEM of the Facilitation ratio (see Materials and Methods). The
asterisk (*) indicates a value significantly different from control plasma (P < 0.05).

116

 

Day 5

0.9-

Facilitation ratio
0
(P

0.34

 

 

0.0
Control Pt. 1 Pt. 2 Pt. 3

Figure 3.7. Effect of LEMS plasma injected for 5 days into mice on facilitation of transmitter
release. EPP amplitudes were recorded from hemidiaphragm preparations taken from mice
injected with 1 .5 ml/day ( i.p.) of plasma from control or LEMS patients (Pt.) for 5 days. Values
are expressed as the mean i SEM of the Facilitation ratio (see Materials and Methods) The
asterisk (*) indicates a value significantly different from control plasma (P < 0.05).

117

p”fl‘fi

III II. - I “ Hill

Day 10

Facilitation ratio

 

 

Control Pt. 1 Pt. 2 Pt. 3

Figure 3.8. Effect of LEMS plasma injected for 10 days into mice on facilitation of transmitter
release. EPP amplitudes were recorded from hemidiaphragm preparations taken from mice
injected with 1.5 ml/day (i.p.) of plasma from control or LEMS patients (Pt.) for 10 days.
Values are expressed as the mean 3: SEM of the Facilitation ratio (see Materials and Methods).
The asterisk (*) indicates a value significantly different from control plasma (P < 0.05).

118

“re 1

ii

inj
Ct
331

0 Day 15

Facilitation ratio

 

 

Control Pt. 1 Pt. 2 Pt. 3

Figure 3.9. Effect of LEMS plasma injected for 15 days into mice on facilitation of transmitter
release. EPP amplitudes were recorded from hemidiaphragm preparations taken from mice
injected with 1.5 ml/day (i.p.) of plasma from control or LEMS patients 15 days. Values are
expressed as the mean i SEM of the Facilitation ratio (see Materials and Methods). The
asterisk (*) indicates a value significantly different from control plasma (P < 0.05).

119

 

2.0-

 

, rt 1.
.. i ii

1 .Oi'i‘ii;

it it ~r— ,

iffifiifei

EPPn/EPPI

i\
Fifi;

 

 

0.0 . . . . . . 4
1 4 7 10 13 16 19 22
Stimulus number

Figure 3. 10. Effect of LEMS plasma injected for 20 days into mice on facilitation of transmitter
release. EPP amplitudes were recorded during 40 Hz stimulation of the phrenic nerve from
hemidiaphragm preparations taken from mice injected with 1.5 ml/day (i.p.) of plasma from
control or LEMS patient 1 for 20 days. Graph representing the mean d: SEM of the normalized
EPP plotted against a train of 20 stimuli recorded from LEMS patient 1 (—A—) or control (—I-—)
plasma-exposed hemidiaphragm preparations.

120

Facilitation ratio

 

 

Control Pt.1 Pt.2 Pt. 3

Figure 3. 1 1 . Effect of LEMS plasma injected for 20 days into mice on facilitation of transmitter
release. EPP amplitudes were recorded during 40 Hz stimulation of the phrenic nerve from
hemidiaphragm preparations taken from mice injected with 1.5 ml/day (i.p.) of plasma from
control or LEMS patients (Pt) for 20 days. Graph representing the mean 3: SEM of the
Facilitation Ratio (see Material and Methods). The asterisk (*) indicates a value significantly
different from control plasma (P < 0.05).

121

Inhibtion of facilitation
ratio (%)
Ox
;

 

 

 

Control Pt. 1

Figure 3.12. Effect of nimodipine on facilitation of transmitter release following injection of
LEMS plasma into mice for 20 days. EPP amplitudes were recorded during 40 Hz stimulation
of the phrenic nerve from hemidiaphragm preparations taken from mice injected with 1 .5 ml/day
(i.p.) of plasma from control or LEMS patients (Pt) for 20 days. Graph representing the mean
i SEM of the Inhibition of facilitation ratio (see Materials and Methods). The asterisk (*)
indicates a value significantly different from pre-nimodipine preparations (P < 0.05).

122

Caz‘ channels involved in release of ACh from control motor nerves (Fig. 3.18). In contrast,
release of ACh at nerve-muscle preparations obtained from mice treated with plasma from
patient 1 for 20 days and exposed to DMSO alone was sensitive to block by nimodipine,
whereas preparations treated the same way but loaded with DM-BAPTA no longer exhibited
DHP-sensitivity (Fig.3.19). It appears therefore, that L-type channels that are involved in
release of ACh from motor nerves exposed to LEMS plasma for 20 days are not in close
proximity to the normal transmitter release sites.

In order to ensure that the terminals were effectively loaded with DM-BAPTA, MEPP
frequency was examined using muscle preparations bathed in physiological saline containing
12.5 mM KCl. MEPP frequency was significantly lower from preparations treated with control
plasma and patient 1 plasma loaded with DM-BAPTA when compared to preparations treated
identically with plasma (control plasma or patient 1) but not DM-BAPTA (exposed to DM-

BAPTA control; DMSO), respectively (Figs. 3.20, 3.21).

E. Discussion

Immature motor nerve terminals contain multiple subtypes of VDCC involved in release
of ACh, some of which become less important during maturation (Sugiura and Ko, 1997;
Rosato—Sin and Uchitel, 1999; Santafe et al., 2001; Rosato-Siri et al., 2002). For instance,
mature mammalian motor nerves contain one primary subtype of VDCC involved in ACh
release; the specific mammalian subtype is species-dependent (Uchitel and Llinas, 1992; Katz,
1995; Protti and Uchitel, 1996). P/Q—, but not N— or L— type VDCC are involved in release
of ACh from mature mammalian motor nerves (Sano et al., 1987; De Luca et al., 1991; Gray
etal., 1992; Uchitel and Llinas, 1992; Katz, 1995; Protti and Uchitel, 1996). Chronic exposure

of adult mammalian motor nerve terminals to sera from patients with LEMS reduces P/Q-type

123

 

Da 1
150- y

”a 125*
.E
.E‘
'g 100-
.§
:1
EL” 75-
2..
cf, sol
E

25‘

 

 

Control Pt.1 Pt. 2 Pt. 3

Figure 3.13. Effect of nimodipine on quantal content (m) at hemidiaphragm preparations
obtained from mice injected with LEMS plasma for 1 day. Hemidiaphragm preparations were
obtained taken from mice injected with plasma from control or LEMS patients (PI) for 1 day.
Quantal content was determined from each preparation using the ratio of the average EPP
amplitude to the average MEPP amplitude before and after the addition of 10 uM nimodipine.
Values are expressed as the percentage of quantal content from preparations after the addition
of nimodipine to that of the same preparation before nimodipine (“pre—nimodipine”). Each
value represents the mean i SEM of at least 5 different preparations.

124

)

1pine

m (% pre—nimod

 

 

Control Pt.1 Pt.2 Pt.3

Figure 3.14. Effect of nimodipine on quantal content (In) at hemidiaphragm preparations
obtained from mice injected with LEMS plasma for 5 days. Hemidiaphragm preparations were
obtained taken from mice injected with plasma from control or LEMS patients (Pt) for 5 days.
Quantal content was determined from each preparation using the ratio of the average EPP
amplitude to the average MEPP amplitude before and after the addition of 10 uM nimodipine.
Values are expressed as the percentage of quantal content from preparations after the addition
Of nimodipine to that of the same preparation before nimodipine (“pre-nimodipine”). Each
value represents the mean i SEM of at least 5 different preparations.

125

Day 10
120-

)

rpine

m (% pre—nimod

 

 

Control Pt.1 Pt.2 Pt. 3

Figure 3.15. Effect of nimodipine on quantal content (m) at hemidiaphragm preparations
obtained from mice injected with LEMS plasma for 10 days. Hemidiaphragm preparations were
obtained taken from mice injected with plasma from control or LEMS patients (Pt) for 10 days.
Quantal content was determined from each preparation using the ratio of the average EPP
amplitude to the average MEPP amplitude before and alter the addition of 10 11M nimodipine.
Values are expressed as the percentage of quantal content from preparations after the addition
of nimodipine to that of the same preparation before nimodipine (“pre-nimodipine”). Each
value represents the mean i SEM of at least 5 different preparations.

126

 

Day 15
120-

1pine)

d

-n1m0

m (% pre

 

 

Control Pt. 1 Pt. 2 Pt. 3

Figure 3.16. Effect of nimodipine on quantal content (m) at hemidiaphragm preparations
obtained from mice injected with LEMS plasma for 1 5 days. Hemidiaphragm preparations were
obtained taken from mice injected with plasma from control or LEMS patients (Pt) for 15 days.
Quantal content was determined from each preparation using the ratio of the average EPP
amplitude to the average MEPP amplitude before and after the addition of IOuM nimodipine.
Values are expressed as the percentage of quantal content from preparations after the addition
of nimodipine to that of the same preparation before nimodipine (“pre-nimodipine”). Each
value represents the mean 1 SEM of at least 5 different preparations.

127

 

150 20 days

3 125-
.E
.9
"g 100
.E
=1
9 75-
Q.
G
e, 50
S

25-

 

 

Control Pt.l Pt.2 Pt.3

Figure 3.17. Effect of nimodipine on quantal content (m) at hemidiaphragm preparations
obtained from mice injected with LEMS plasma for 20 days. Hemidiaphragm preparations were
obtained taken from mice injected with plasma from control or LEMS patients (Pt) for 20 days.
Quantal content was determined from each preparation using the ratio of the average EPP
amplitude to the average MEPP amplitude before and after the addition of 10 uM nimodipine.
Values are expressed as the percentage of quantal content from preparations after the addition
of nimodipine to that of the same preparation before nimodipine (“pre-nimodipine”). Each
value represents the mean i SEM of at least 5 different preparations. The asterisk (*) indicates
a value significantly different from pre-nimodipine preparations (P < 0.05).

128

Caz" currents and unmasks L-type Caz‘ currents involved in ACh release terminals (Smith and
Atchison, 1995; Xu and Atchison, 1998; Flink and Atchison, 2002;Giovannini et al., 2002).
The primary objective of this study was to examine further the time course of development of
L-type VDCC involved in ACh from motor nerves following passive transfer of LEMS to mice.

Nimodipine, a dihydropyridine-sensitive L-type Ca2+ channel antagonist, significantly
reduced low frequency stimulated-release of ACh from motor nerve terminals from mice
injected for 20 days with plasma from 2 out 3 patients with LEMS in comparison to mice
injected with control patient plasma for 20 days. However, passive transfer of LEMS to mice
with plasma from 3 separate patients for 1, 5, 10, and 15 days did not induce DHP-sensitive L-
type Caz" channel involvement in evoked-release of ACh from motor nerves. These findings
corroborate earlier work, which showed that L-type channel involvement in ACh release from
motor nerves exposed to LEMS plasma occurs only after prolonged exposure (Flink and
Atchison, 2002; Giovannini et al., 2002). Although, MEPP frequency was unaffected by
nimodipine at control and LEM S-treated preparations at all days tested, high-frequency nerve-
evoked release from motor nerves exposed to LEMS patient plasma was reduced in the
presence of nimodipine. Thus, it appears that strong depolarization of the nerve terminal is
necessary in order to involve L-type Ca2+ channels in release of ACh following passive transfer
of LEMS to mice by injections for 20 days. This findings correspond to those of earlier reports
describing the strong voltage-dependence necessary for activation of L-type channels (Miller,
1987).

Passive transfer of LEMS to mice produced electrophysioligical characteristics similar
to those of earlier reports. Evoked-release of ACh from motor nerves following exposure of
mice with plasma from 2 out 3 three LEMS patients for 1 and 5 days and from all 3 patients

after 10, 15 and 20 days was reduced significantly in comparison to the corresponding controls.

129

Fw -'fl"u
.

 

Control
125-

100-

75-

m
(% pre-nimodipine)

 

 

DMSO BAPTA

Figure 3.18. Effect of DM-BAPTA on quantal content (m) at hemidiaphragm preparations
obtained from mice injected with control plasma for 20 days. Hemidiaphragm preparations
obtained from mice injected with plasma from control patients for 20 days were incubated with
either 25 uM DM-BAPTA-AM or .0125% DMSO (BAPTA vehicle) for 2 hr. Quantal content
was determined from each preparation using the ratio of the average EPP amplitude to the
average MEPP amplitude before and after the addition of IOuM nimodipine. Values are
expressed as the percentage of quantal content from preparations after the addition of
nimodipine to that of the same preparation before nimodipine (“pre-nimodipine”). Each value
represents the mean i SEM of at least 4 different preparations.

130

Patient 1
150

  
 
 
 

é

m
(% pre-nimodipine)
$

 

DMSO BAPTA

Figure 3.19. Effect of DM-BAPTA on quantal content (m) at hemidiaphragm preparations
obtained from mice injected with LEMS plasma for 20 days. Hemidiaphragm preparations
obtained from mice injected with plasma from LEMS “patient 1" for 20 days were incubated
with either 25 11M DM-BAPTA-AM or .0125% DMSO (BAPTA vehicle) for 2 hr. Quantal
content was determined from each preparation using the ratio of the average EPP amplitude to
the average MEPP amplitude before and after the addition of IOuM nimodipine. Values are
expressed as the percentage of quantal content from preparations after the addition of
nimodipine to that of the same preparation before nimodipine (“pre-nimodipine”). Each value
represents the mean i SEM of at least 4 different preparations. The asterisk (*) indicates a
value significantly different from pre-nimodipine preparations (P < 0.05).

131

 

Control

20'
16‘

12‘

MEPP frequency (Hz)

 

 

 

DMSO DM-BAPTA

Figure 3.20. Effect of DM-BAPTA on KCl-induced MEPP frequency at hemidiaphragm
preparations obtained from mice injected with control plasma for 20 days. Hemidiaphragm
preparations obtained from mice injected with plasma from control patients for 20 days were
incubated with either 25 11M DM-BAPTA-AM or .0125% DMSO (BAPTA vehicle) for 2 hr
and subsequently exposed 12.5 KCl in the buffer. Each value represents the mean i SEM of
at least 4 different preparations. The asterisk (*) indicates a value significantly different from
DMSO preparations (P < 0.05).

132

 

Fiv
lite
Pie;
inn
and
31 1e
Dbl:

Patient 1

MEPP frequency (Hz)

 

   

 

 

DM SO DM-BAPTA

Figure 3.21. Effect of DM-BAPTA on KCl-induced MEPP frequency at hemidiaphragm
preparations obtained from mice injected with LEMS plasma for 20 days. Hemidiaphragm
preparations obtained from mice injected with plasma from LEMS “patient 1" for 20 days were
incubated with either 25 uM DM-BAPTA—AM or .0125% DMSO (BAPTA vehicle) for 2 hr
and subsequently exposed 12.5 KCl in the buffer. Each value represents the mean i SEM of
at least 4 different preparations. The asterisk (*) indicates a value significantly different from
DMSO preparations (P < 0.05).

133

However, facilitation of ACh release, which is normally seen at motor nerves exposed to LEMS
IgG was only observed after injection of plasma from 2 of the 3 LEMS patients at all days
tested. For the third LEMS patient’s plasma, facilitation of ACh release did not occur until
low frequency nerve- stimulated release of ACh was already significantly reduced (day 10). This
finding suggests that facilitation of ACh release following high fi'equency nerve-stimulation
requires a significant reduction in the entry of Ca2+ through VDCC into the nerve terminal.

Development of L-type Ca” channel involvement in release of ACh following passive
transfer of LEMS may depend on the extent to which the number of functional P/Q-type VDCC
is reduced at the motor nerve terminal. Evoked-release of ACh from motor nerve terminals
following treatment with LEMS plasma for 1 day was significantly greater than that seen after
20 days when L-type channel involvement became obvious. Although, reduction in ACh release
appears unchanged at days 10, 15, and 20, loss of P/Q-type VDCC at motor nerve terminals
most likely is greatest following the 20 day treatment regimen. L-type Ca2+ channels may
therefore, assume the role of lost P/Q-type VDCC following the 20 day regimen. In turn, the
extent of quantal content reduction appears the same at days 20, 15, and 10. This is firrther
supported by the findings of Giovonnini et al.(2002), which showed that following passive
transfer of LEMS for 9 days, release of ACh was sensitive to an L-type Ca2+ antagonist only
during further blockade of P/Q-type VDCC with (n-Aga-IVA. However, when evoked-ACh
release was examined without significant blockade of P/Q-type channels, L-type Ca2+ channels
did not appear involved in release of ACh.

While involvement of L-type Ca2+ channels in release of ACh following passive transfer
of LEMS is dependent on the extent of P/Q-type VDCC loss, other mechanisms must be
involved as well. This can be explained by the following: inhibition of P/Q-dependent Cai’

currents by the fimnel web spider toxin, w-Aga-IVA at motor nerve terminals exposed to

134

I6

llit.

Unn
Pres
but

ripe
LEM
LEM
”ban

“01 th.

control patient sera for 30 days does not unmask L-type Ca2+ currents (Xu and Atchison, 1998);
L-type Ca2+ channels do not appear to be involved in ACh release following the 10 or 15 day
treatment regimens with LEMS plasma, even though the extent of nerve evoked-release of AC h
appears significantly different from that observed following 1 or 5 days of treatment; motor
nerve terminals exposed to plasma from patient 3 do not involve L-type VDCC in nerve
evoked-release of ACh even after significant reduction in ACh release occurs.

It is possible that involvement of L—type Ca” channels in ACh release following passive
transfer of LEMS is due to unmasking of normally silent channels already present on or near
the motor nerve terminal. This possibility is supported by the findings that pretreatment of
mammalian motor nerve terminals with bay k 8644, a DHP-type agonist, increases evoked
release of ACh, which can be firrther reduced by addition of the DHP-sensitive antagonist
nimodipine (Atchison and O'Leary, 1987; Atchison, 1989). Block of adenosine (A2) receptors
at rat motor nerve terminals also exposes L-type Ca2+ channel involvement in ACh release
(Correia-de-Sa et al. , 2000a, b). Furthermore, inhibition of protein phosphatases in conjunction
with rapid chelation of Ca2+ using BAPTA in the mammalian motor nerve terminal also
unmasks L—type Ca2+ channels involved in ACh release (Urbano et al., 2001). However, in the
presence of rapid chelation (BAPTA-treatment) alone, L-type Ca2+ current becomes apparent,
but is not involved in ACh release (Urbano and Uchitel, 1999). Interestingly, in our
experiments DM-BAPTA abolished L-type Ca2+ channel involvement in ACh release from
LEMS-treated motor nerve terminals. Thus, if inhibition of protein phosphatases occurs at
LEMS-treated motor nerve terminals, then incubation with DM-BAPTA should have either
enhanced or not affected L-type Ca2+ channel involvement in ACh release, however this was

not the case.

135

It appears that development of L-type Ca” channels involvement in ACh from motor
nerves is multifaceted. The time course describing the development of DHP-sensitivity implies
that processes, which take a long time are required, such as denovo- mRN A synthesis and Ca2+
channel protein translation and assembly. For instance, following botulism toxin-induced
poisoning, reinnervating or recovering motor nerves express L-type Ca2+ channel involvement
in ACh release (Katz et al., 1996; Santafe et al., 2000). However, the motor nerve terminal
does not appear to be damaged in LEMS (Fukunaga, 1982; Fukuoka et al., 1987; Tsujihata et
al., 1987). So this involvement presumably doesn’t result from reinnervation. Alternatively, L-
type channel development may occur rapidly, but not be present at a location in which their
involvement in ACh release is possible, perhaps during exposure LEMS IgG these channels
relocate along the terminal to a point at which they can become involved in ACh release.

Loading of motor nerve terminals with the rapid onset Ca2+ chelator, DM-BAPTA did
not affect evoked-release of ACh in solutions containing low concentrations of KCl . This
suggests that the coupling between the VDCC normally involved in ACh from adult mammalian
motor nerves is tightly coupled with the release machinery. Although this release corroborates
the results of Urbano and Uchitel (1999), it is in contrast to that result reported by Adler et al
(1992). However, this discrepancy most likely reflects the differences in species (squid stellate
ganglion) used in their study, for which coupling of VDCC and release may not be as tight as
that seen at adult mammalian motor nerve terminals. In the presence of DM-BAPTA, L-type
Ca2+ channels do not appear involved in ACh release from motor nerve terminals exposed to
LEMS plasma, suggesting that these channels are located at a site distinct from that of the
normal complement of VDCC involved in ACh release. Thus, DM-BAPTA most likely binds
C a2 i that enters the terminal through L-type channels before the Ca2+ reaches ACh release sites.

It is believed that VDCC normally involved in ACh release are clustered near active zone

136

release sites (Llinas et al., 1992). If L-type channels are located at a distinct site, then the influx
of Ca2+ through the L-type channels would likely occur in a more diffuse manner than what is
thought to occur normally. This possibility is further supported by the findings that L-type Ca2+
channels are not involved in spontaneous release of ACh. At resting membrane potentials, the
number of open L-type Ca2+ VDCC that allow entry of Ca2+ into the terminal may too small to
induce-spontaneous release of ACh. (unstimulated MEPP frequency) Depolarization of the
nerve terminal, on the other hand, would increase the probability of L-type channel openings
and thus allow enough Ca2+ to entry the terminal be involved in ACh release.

The lack of development of L-type Ca2+ channel involvement in release of ACh
following passive transfer with plasma from patient 3 is puzzling, but this observation may
reflect differences in the specificity of the antibodies found in different patients with LEMS.
Recent work has shown that over 90% of patients have antibodies directed against the P/Q-type
VDCC (Suenaga et al., 1996). Functional analysis has firrther shown that IgG from patients
with LEMS reduces Ca2+ uptake into cells containing P/Q-, but not N-, L-, or R VDCC (Pinto,
1998; Pinto et al., 2002). The specificity of LEMS IgG for P/Q-type VDCC, which are the
major VDCC involved in ACh release from adult mammalian motor nerves, may explain why
the primary clinical sign in LEMS is muscle weakness. However, antibodies directed against
non- P/Q-type VDCC have also been detected in plasma from patients with LEMS (Lennon and
Lambert, 1989; Sher et al., 1989, El Far et al., 1995). As such, incubation of neuroblastoma
IMR32 (Grassi et al., 1994) or neuroblastoma X glioma hybrid (Peers et al., 1990) cells with
LEMS IgG has been shown to decrease entry of Ca2+ through N-type and L-type VDCC. The
presence of antibodies against multiple subtypes of VDCC may explain why the development

of L-type C a2 * channels was not observed following passive transfer of LEMS with plasma from

137

patient 3. Plasma from this patient may also contain antibodies specific for not only P/Q-, but
L-type Ca2+ channels as well.

While one may question L-type Caz" channel involvement in ACh release from motor
nerves of patients with LEMS, it has been shown that addition of L-type Ca” channel
antagonists worsens or unmasks symptoms in LEMS patients (Krendal and Hopkins 1986,
Ueno et al. 1992). Taken together with our findings, L-type Ca2+ channels assume an important
role in the release of ACh in LEMS and thus may act as a compensatory mechanism to maintain

a given level of transmitter release following loss of P/Q-type VDCC.

138

CHAPTER FOUR

IBERIOTOXIN-INDUCED BLOCK OF Kc. CHANNELS REVEALS
DIHYDROPYRIDINE SENSITIVITY OF ACh RELEASE FROM MAMMALIAN

MOTOR NERVE TERMINALS

139

A. Summary

The role which Ca2‘-activated-K' (K0,) channels play in regulating acetylcholine (ACh)
release was examined at mouse motor nerve terminals. In particular, the ability of the
antagonist iberiotoxin to recruit normally silent L-type Caz’ channels to participate in nerve-
evoked release was examined using conventional intracellular electrophysiological techniques.
Incubation of cut hemidiaphragm preparations with 10 11M nimodipine, a dihydropyridine
(DHP) L-type C az‘ channel antagonist, had no significant effect on quantal content of end-plate
potentials following nerve-stimulation in comparison to untreated (control) preparations.
However, 1 11M bay K 8644, a DHP L-type Caz’ channel agonist enhanced quantal content to
134.7 i 3.5 % of control. Iberiotoxin (150 nM) increased quantal content to 177.5 i 9.9 % of
control, whereas iberiotoxin in the presence of nimodipine increased quantal content to only
145.7 2: 10.4 % of control. Coapplication of 1 uM bay K 8644 with iberiotoxin did not
significantly increase quantal content firrther than did treatment with iberiotoxin alone. The
effects of iberiotoxin and nimodipine alone or in combination on MEPP frequency in response
to KCl-induced depolarization were also examined using uncut hemi-diaphragm preparations.
Nimodipine alone had no effect on MEPP frequency from preparations incubated in
physiological saline containing 5-20 mM KCl. Moreover, iberiotoxin alone or combined with
nimodipine also had no effect on MEPP frequency in physiological salines containing 5-15 mM
KCl. However, when the [KCl] was raised to 20 mM, iberiotoxin significantly increased MEPP
frequency to 125.6% of iberiotoxin-free values; combined treatment with both nimodipine and
iberiotoxin prevented this increase in MEPP frequency (102.2% of iberiotoxin-free values).

Thus, loss of functional KC, channels can unmask normally silent L-type Ca2+ channels to

140

participate in quantal release of ACh from motor nerve terminals particularly under conditions

of intense nerve terminal depolarization.

B. Introduction

Release of acetylcholine (ACh) from motor nerves is a highly controlled process that
requires entry of Ca” through voltage-dependent Ca2+ channels into the nerve terminal
(Augustine et al., 198 7; Katz et al., 1997). Multiple Ca2+ channel subtypes are known to exist;
thus far pharmacological, electrophysiological, and molecular characteristics of L-, T-, N-, P-,
Q-, and R- type channels have been described (Tsien et al., 1988; Zhang et al., 1993; Catterall,
1998; Newcomb et al., 1998). Often more than one Ca2+ channel subtype coexists at the same
nerve terminal to control transmitter release (Lemos and Nowycky, 1989; Turner et al., 1993;
Elhamdani et al., 1998). However, the specific Caz’ channel phenotype primarily involved in
ACh release is both species- (Sano et al., 1987; De Luca et al., 1991; Uchitel et al., 1992;
Protti et al., 1996; Katz et al., 1997) and age-dependent (Sugiura and Ko, 1997; Rosato Siri
and Uchitel, 1999; Santafe et al. , 2001 ). Release of ACh from mature mammalian motor nerves
relies primarily on entry of Ca?” through P/Q- (Uchitel et al., 1992; Protti et al., 1996; Katz et
a1 ., 199 7), but not N-type Ca2+ channels, which are found on motor nerves of amphibians (Sano
et al., 1987) and birds (De Luca et al., 1991). Developing mammalian motor nerves on the
other hand, possess multiple Ca2+ channel subtypes involved in the release of ACh, some of
which become less important during maturation (Sugiura and Ko, 1997; Rosato Siri and

Uchitel, 1999; Santafe et al., 2001).

141

 

til
del

Cu:

L-type Ca2+ channels, which can be found colocalized with other Ca2+ channel
phenotypes, participate in release of noradrenaline from chromaffin cells of the adrenal medulla
(Owen et al., 1989) and in the release of oxytocin and vasopressin (Lemos and Nowycky,
1989). Pharmacological evidence has implicated a potential role of normally silent L-type Ca2+
channels in release of ACh from mature mammalian motor nerves. As such, bay K 8644, a
dihydropyridine-sensitive L-type Ca” channel agonist, but not nimodipine, a dihydropyridine-
sensitive L-type Caz’ channel antagonist alters the release of ACh from mammalian motor
nerves (Atchison and O’Leary, 1987; Atchison, 1989). Inhibition of protein phosphatases in
conjunction with rapid chelation of intracellular Ca2+ in the motor nerve terminal also exposes
a L-type Ca2+ channel component involved in release of ACh from mammalian motor nerves
(Urbano et al., 2001 ). Moreover, during certain pathological conditions, L-type Ca2+ channels
can also participate in release of ACh as well (Katz et al., 1996; F ratantoni et al., 2000; Santafe
et al., 2000; F link and Atchison, 2002; Giovannini et al., 2002).

The control of ACh release from motor nerves requires not only precise and rapid
opening of Ca2+ channels at the nerve terminal, but also mechanisms to close these channels as
well. One such mechanism involves the opening of K+ channels, which return the depolarized
membrane to resting potential and thus alters the open state of the voltage—dependent Ca2+
channels (Llinas et al., 1981 ; Augustine, 1990). Three types of K+ currents have been identified
at mammalian motor nerve terminals: a slow and fast voltage-dependent K*-current and a Ca2+-
dependent-K+ current (IKCa) (Mallart, 1985; Tabti et al., 1989). The voltage-dependent K+
currents are both sensitive to 3,4 diaminopyridine (DAP), whereas tetraethylammonium (TEA)

affects only the fast component. 1K0, on the other hand is activated in the presence of Ca2+ and

142

sensitive to TEA, but not to 3,4 DAP. IKC, can also be distinguished phannacologically from
the voltage-dependent K+ current in the presence of various scorpion toxins (Tabti et al., 1989;
Vatanpour and Harvey, 1995). Although the precise role which KCa channels play at the nerve
terminal is unclear, they most likely participate in attenuating Ca2+ -dependent transmitter
release by contributing to repolarization of the nerve terminal and thus altering the open state
of voltage-dependent Caz’ channels (Mallart, 1985; Robitaille and Charlton, 1992). Evidence
exists that show KCa channels and voltage-dependent Ca2+ channels are not only colocalized at
motor nerve terminals, but influence one another as well (Robitaille and Charlton, 1992;
Robitaille et al., 1993; Xu and Atchison, 1996; Protti and Uchitel, 1997).

It has been recently reported that passive transfer of Lambert-Eaton myasthenic
syndrome (LEMS), a neuromuscular disorder, which causes fimctional loss of P/Q-type Ca2+
channels and thus a decrease in the depolarization-induced entry of Ca2+ into the motor nerve
terminal (Lambert and Elmqvist, 1971; Fukunaga et al., 1983; Hewett and Atchison, 1991)
appears to involve L-type channels in ACh release (Flink and Atchison, 2002, Giovannini et al.,
2002). LEMS however, has not been shown to damage the nerve terminal nor cause sprouting
of newly-formed terminals (Fukunaga et al., 1983; Tsujihata et al., 1987). In LEMS, reduced
entry of Ca2+ into the nerve terminal following membrane depolarization could attenuate
activation of KCal channels. This could, in turn, slow repolarization of the motor nerve terminal
and thus increase the probability that colocalized or spatially-removed Ca2+ channels become
involved in ACh release. For example, a component of transmitter release from sympathetic
and motor nerves has been shown to depend upon Ca2+ entry through L-type channels under

conditions of intense stimulation or following inhibition of voltage-dependent K+ currents

143

(Hong and Chang, 1990; Somogyi et al., 1997; Correia-de-Sa et al., 2000a,b). The present
study was designed, to determine if loss of firnctional KCal channels unmasks normally silent L-
type Caz’ channels involved in release of ACh from mammalian motor nerves. To accomplish
this, iberiotoxin, a specific antagonist for KC,I channels, was used to block KCal channels and the
resulting sensitivity of release of ACh to DHP-type antagonists such as nimodipine was tested

at murine motor nerve terminals.

C. Materials and Methods

Electrophysiology. Experiments were performed using male ICR mice (20-22 g,
Harlan Sprague-Dawley Laboratories, Madison, WI) in accordance with local university
(Michigan State University Laboratory Animal Resources) and national guidelines. Animals
were sacrificed by decapitation following anesthesia with 80% CO2 and 20% Oz. The
diaphragm muscle with the attached phrenic nerve was then removed (Barstad and Lilleheil,
1968) and pinned out at resting tension in a Sylgard-coated chamber. The tissue was perfused
continuously with buffered saline solution containing (mM): NaCl 137.5, KCl 2.5, MgCl2 l,
CaCl2 2, D-glucose 11, HEPES 14, pH adjusted to 7.4 at room temperature of 23-25°C with
NaOH and kept under continual oxygenation (100% 02) at a rate of approximately 1-5 ml/min.
The diaphragm muscle was transected into the two hemidiaphragms, and one hemidiaphragm
was then cut approximately 4 mm on either side of the main intramuscular nerve branch in order
to prevent muscle contraction following stimulation of the phrenic nerve (Glavinovic, 1979;
Atchison, 1989). This technique does not produce significant changes in the muscle cable

properties (Glavinovic, 1979). Depolarization-induced nerve conduction block that occurs

144

when K’ is released fi'om the cut-muscle fibers was prevented by the use of buffered saline
solution containing 2.5 mM KCl throughout the experiments (Glavinovic, 1979; Atchison,
1989). Only one hemidiaphragm preparation per mouse was used for any given experiment.

Intact muscle preparations were used in experiments examining the effect of iberiotoxin
on MEPP frequency in the presence of varying concentrations of KCl (5 - 20 mM). Osmolarity
was adjusted for changes in concentrations of KCl with an equiosmolar change in the
concentration of NaCl. All experiments were performed at room temperature of 23-25°C using
conventional intracellular recording techniques. EPPs (end-plate potentials) and MEPPs
(miniature end-plate potentials) were recorded using borosilicate glass microelectrodes (1.0
mm- o.d., WP Instruments, Sarasota, FL) and having resistance of 5-15 MQ when filled with
3M KCl. The phrenic nerve was stimulated supramaximally at a frequency of 0.5 Hz using a
suction electrode attached to a stimulus isolation unit (Grass SIU, Grass Instruments, Quincy,
MA) and stimulator (Grass S88). Signals were amplified using a WPI 721 (WP Instruments,
Sarasota, FL) amplifier and digitized into a computer for inspection using Axoscope 8.0 (Axon
Instruments, Foster City, CA) software and analyzed using MiniAnalysis 5.0 software
(Synaptosoft, Decatur, GA).

Data analysis and statistics. Control recordings were first made from untreated
muscle preparations and subsequent recordings were made from the same preparation following
incubation with the relevant drug treatment for the time indicted in the figure legends.
Recordings from at least 5 different end-plates were used to determine the mean amplitude of
the EPPs (average of 10 recordings per endplate) and MEPPs for each drug treatment from the

same neuromuscular junction preparation, yielding an n value of 1. Averaged EPP and MEPP

145

amplitudes were first standardized to a membrane potential of —50 mV in order to correct for
changes in membrane potential driving force (Katz and Thesleff, 1957). EPPs were then
corrected for nonlinear summation (McLachlan and Martin, 1981) using the formula V0,," =
V/((1-.8*V)/E), where V is the uncorrected EPP amplitude, E is the resting membrane
potential, and V“,1T is the corrected EPP amplitude. Quantal content was calculated using the
ratio of the mean amplitude of the corrected EPPs to the mean amplitude of the corrected
MEPPs (Hubbard et al., 1969). Statistical significance between the various treatment groups
was analyzed using a one way analysis of variance followed by Tukey’s test. A two way
analysis of variance was used to compare the effect of drug treatments on MEPP amplitudes
in the presence of varying concentrations of KCl. P values were set to < 0.05 for all statistical
tests.

Drugs and chemicals. Nimodipine, S-(—)bayK8644, and N-2-hydroxyethylpiperazine-
N-2-ethanesulfonic acid (HEPES) were purchased from Sigma Chemical Co. (St. Louis, MO).
Iberiotoxin was obtained from Alomone labs (Jerusalem, Israel). All other reagents were of
analytic grade or better. Nimodipine and S-(-) bay K 8644 were prepared as a 20 mM and 10
mM stock solution, respectively in 100% ethanol, which was kept at 4° C until use. The final
working buffered saline solution with nimodipine and S-(-) bay K 8644 contained only 0.05%
and 0.01% ethanol (v/v), respectively. Control experiments contained an equivalent
concentration of the respective vehicle. Experiments performed in the presence of nimodipine
or S-(—) bay K 8644 were done in the dark, in order to prevent photo-oxidation of these
compounds. Iberiotoxin was prepared as a stock solution in deionized water containing 0.01%

bovine serum albumin (w/v), and was used within a two-week period. Before incubation with

146

iberiotoxin, 0.01% bovine serum albumin was added to the buffered saline solution in order to

prevent non-specific binding of toxin to the chamber, tubing, and glassware.

D. Results

Effects of KC, and L-type Ca2+ channels on neuromuscular transmission.
Incubation of cut neuromuscular preparations with iberiotoxin increased quantal content of
nerve- evoked release of ACh as shown in sample records (Fig. 4.2) and composite data (Fig.
4. 1) to 177. 5 i 9.9 % of control values. Further treatment of neuromuscular preparations with
nimodipine, a dihydropyridine L-type Ca2+ channel antagonist, in the presence of iberiotoxin
reduced the enhancement of quantal content observed with iberiotoxin alone to 145.7 1- 10.4
% of control (Fig. 4.1). The final ethanol concentration (0.05%) used in physiological saline
containing nimodipine and iberiotoxin had no significant effect upon the release of ACh, MEPP
amplitudes or frequency, and muscle resting membrane potentials in comparison to iberiotoxin
alone (data not shown). Similarly, iberiotoxin and nimodipine had no effect on resting
membrane potentials (Fig. 4.3), MEPP frequency (Fig. 4.4), or MEPP amplitudes (Fig. 4.5)
recorded from the cut preparations. Incubation of preparations with iberiotoxin vehicle (0.01%
bovine serum albumin) alone or with nimodipine also had no effect on quantal content in
comparison to untreated controls (Fig. 4.6).

Although L-type Ca2+ channels do not normally participate in nerve-evoked release of
ACh from adult mammalian motor nerve terminals, use of the dihydropyridine L-type Ca”
agonist, bay K 8644 has been shown to enhance ACh release from rat motor nerve terminals

(Atchison and O’Leary, 1987; Atchison, 1989). Addition of bay K 8644 to cut neuromuscular

147

200‘ 3':
A
3 180‘
I:
= 160‘
e
O 140‘
\°
3 120-
E
3 100‘
8 80‘
C)
E 60-
§ 40—
5
O’ 20‘
0-

 

 

IBTx IBTx + nimod

Figure 4. 1. Effects of iberiotoxin (IBTx) and nimodipine (nimod) on quantal content of mouse
hemidiaphragm end—plates. Recordings were first made in the absence of any drug treatments
(control), then subsequently made after 1 hr incubation with 150 nM iberiotoxin and then
following firrther incubation with 10 uM nimodipine for 25 min in the presence of iberiotoxin.
IVIEPP and EPP amplitudes were recorded using standard intracellular techniques. EPPs were
elicited at a frequency of 0.5 Hz. Quantal content was determined from each hemidiaphragm
preparation using the ratio of the average EPP amplitude to the average MEPP amplitude.
Values are expressed as the percentage of quantal content from drug-treated preparations to
that of control preparations. Each value represents the mean i SEM. of at least 4 different
preparations. The asterisk (*) and double dagger (1) indicate a value significantly different from
control and from control and iberiotoxin alone, respectively (P < 0.05).

148

control IBTx IBTx + nimod

 

 

 

 

 

l 1 l J3

4ms

Figure 4.2. Effects of iberiotoxin (IBTx) and nimodipine (nimod) on EPP amplitudes at mouse
hemidiaphragm end-plates. Each tracing represents the average of 10 EPPs at a stimulation
frequency of 0. 5 Hz recorded from a single end-plate before (control), after the addition of 150
nM iberiotoxin, and following incubation with both iberiotoxin (150 nM) and nimodipine (10
11M). Each representative tracing was recorded from a different end-plate.

149

-40-

-50-

-60 -

Resting membrane potential (mV)

-70 -

 

 

control IBTx IBTx + nimod

Figure 4.3. Effects of iberiotoxin (IBTx) and nimodipine (nimod) on resting membrane
potentials from cut mouse hemidiaphragm preparations. Recordings were made in the absence
of any drug treatments (control), following 1 hr incubation with 150 nM iberiotoxin alone, and
following further incubation with IOuM nimodipine and 150 nM iberiotoxin for an additional
25 min. Each value represents the mean :t SEM. of at least 4 different preparations.

150

0.7-

MEPP frequency (Hz)

 

 

control IBTx IBTx + nimod

Figure 4.4. Effects of iberiotoxin (IBTx) and nimodipine (nimod) on MEPP frequency from cut
mouse hemidiaphragm preparations. Recordings were made in the absence of any drug
treatments (control), following 1 hr incubation with 150 nM iberiotoxin alone, and following
firrther incubation with IOuM nimodipine and 150 nM iberiotoxin for an additional 25 min.
Each value represents the mean i SEM. of at least 4 different preparations.

151

MEPP amplitude (mV)

 

 

control IBTx IBTx + nimod

Figure 4.5. Effects of iberiotoxin (IBTx) and nimodipine (nimod) on MEPP amplitudes fiom
cut mouse hemidiaphragm preparations. Recordings were made in the absence of any drug
treatments (control), following 1 hr incubation with 150 nM iberiotoxin alone, and following
further incubation with IOuM nimodipine and 150 nM iberiotoxin for an additional 25 min.
Each value represents the mean i S.E.M. of at least 4 different preparations.

152

120-

100-

00
O
1

 

 

Quantal content (% control)
0\
0

vehicle vehicle + nimod

Figure 4.6. Effects of nimodipine (nimod) in the absence of iberiotoxin on quantal content of
mouse hemidiaphragm end-plates. Recordings were first made in the absence of any drug
treatments (control), then subsequently made after 1 hr incubation with iberiotoxin vehicle (0.01
% BSA) alone, and then following further incubation with 10 11M nimodipine for 25 min in the
presence of iberiotoxin vehicle. MEPP and EPP amplitudes were recorded using standard
intracellular techniques. EPPs were elicited at a frequency of 0.5 Hz. Quantal content was
determined from each neuromuscular junction preparation using the ratio of the average EPP
amplitude to the average MEPP amplitude. Values are expressed as the percentage of quantal
content from drug-treated preparations to that of control preparations. Each value represents
the mean 2+: S.E.M. of at least 4 different preparations.

153

preparations enhanced quantal content to 134.7 at 3.5 % of control (Fig. 4.7). Treatment of cut
preparations with bay K 8644 in conjunction with iberiotoxin did not cause a significant
difference from the effect of iberiotoxin alone (Fig. 4.8).

Effects of KC, and L-type Ca2+ channels on MEPP frequency. The effect of
iberiotoxin and nimodipine on changes in MEPP frequency from motor nerve terminals was
examined in the presence of varying concentrations of KCl. Resting membrane potentials
recorded from uncut muscle preparations in the absence of any drug treatments (control),
following incubation with iberiotoxin alone or with nimodipine were not significantly different
from each other (Table 4.1). Although MEPP amplitudes (Fig. 4.9) appeared to increase
slightly in the presence of nimodipine as KCl concentrations was increased, comparisons of
results for all treatment groups within and between different KCl concentrations (5 -20 mM)
revealed no significant differences. MEPP frequency, on the other hand, increased in
conjunction with the increase in KC] concentration (Figs. 4.10, 4.11). Treatment of
preparations with iberiotoxin alone or plus nimodipine had no significant effect on MEPP
frequency in comparison to one another or to control treatment in physiological saline
containing either 5,10, or 15 mM KCl (Fig. 4.10). However, when the physiological saline
contained 20 mM KCl, iberiotoxin significantly increased MEPP frequency to approximately
125.6% of control treatment (Fig. 4.10). Further addition of nimodipine to preparations in the
presence of iberiotoxin significantly reduced the enhancement of MEPP fi'equency observed
with iberiotoxin alone approximately 102.2 % of control in the presence of 20 mM KCl (Fig.
4. 10). However, MEPP frequency was not significantly different in the presence of nimodipine

and iberiotoxin when compared with control treatment. Furthermore, MEPP frequency was

154

r. .. N
9?" a: 3.3 3

Quantal content (% control)
8

 

 

 

O
;

vehicle vehicle +bay K

Figure 4.7. Effects of bay K 8644 (bay K) in the absence of iberiotoxin on quantal content of
mouse hemidiaphragm end-plates. Recordings were first made in the absence of any drug
treatments (control), then subsequently made after 1 hr incubation with iberiotoxin vehicle
(0.01% BSA) and then following fiirther incubation with 1 uM bay K 8644 for 25 min in the
presence of iberiotoxin vehicle. MEPP and EPP amplitudes were recorded using standard
intracellular techniques. EPPs were elicited at a frequency of 0.5 Hz. Quantal content was
determined from each neuromuscular junction preparation using the ratio of the average EPP
amplitude to the average MEPP amplitude. Values are expressed as the percentage of quantal
content fi'om drug-treated preparations to that of control preparations. Each value represents
the mean 25 SEM. of at least 4 different preparations. The asterisk (*) indicates a value
significantly different from either control or iberiotoxin vehicle (P < 0.05).

155

Quantal Content (% control)

 

 

IBTX IBTx+bayK

Figure 4.8. Effects of iberiotoxin (IBTx) and bay K 8644 (bay K) on quantal content of mouse
hemidiaphragm end-plates. Recordings were first made in the absence of any drug treatments
(control), then subsequently made after 1 hr incubation with 150 nM iberiotoxin and then
following further incubation with 1 uM bay K 8644 for 25 min in the presence of iberiotoxin.
MEPP and EPP amplitudes were recorded using standard intracellular techniques. EPPs were
elicited at a frequency of 0.5 Hz. Quantal content was determined from each neuromuscular
junction preparation using the ratio of the average EPP amplitude to the average MEPP
amplitude. Values are expressed as the percentage of quantal content from drug—treated
preparations to that of control preparations. Each value represents the mean i SEM. of at
least 4 different preparations. The asterisk (*) indicates a value significantly different from
control (P < 0.05).

156

Table 4.1

Muscle resting membrane potentials (RMP) from control, iberiotoxin, and iberiotoxin plus
nimodipine treated-neuromuscular junction preparations at various levels of KCl-induced

 

 

depolarization
[KCI] Control Iberiotoxin Iberiotoxin +
(mM) (mV) (mV) nimodipine
(mV)
5 -70.1 :1: 2.4A -71.3 :t 2.0 -70.5 at 2.0
10 -60.2 i 2.0 -60.9 :t 3.1 -60.0 :t 2.6
15 -53.3:tZ.1 -51.9i1.8 -51.7i1.0
20 -45.8 d: 1.3 -42.0 :t1.7 -43.7 3:1.9

 

A Each value represents the mean RMP i S.E.M. from at least 4 preparations for each
’KCl concentration.

157

1.0 _ l I control

 

 

 

 

 

 

                

  
 

 

 

 

 

 

-IBTx
WBTX+nimod
0.8‘
9
E
00.6“ 7 y
“C 7 ¢ ¢
3 a a it
'2- iii?
0.4“ / / / /
«t a a a a
e d d d d
e a a 22
a 2 2a
802‘ a a a 2
a a a a
a r a a
a r / /
/// /
0.0-—/ /‘ fig

5 10 15 20
KC] concentration (mM)

Figure 4.9. Comparative effects of iberiotoxin (IBTx) and nimodipine (nimod) on amplitudes
of MEPPs recorded from muscle preparations in the presence of varying concentrations of KCl
(5-20 mM). MEPP amplitudes were measured using standard intracellular techniques fi'om
untreated (control) preparations, following 1 hr incubation with 150 nM iberiotoxin, and then
subsequently after 25 min incubation with 10 uM nimodipine in the presence of 150 nM
iberiotoxin. Each value represents the mean i S.E.M.. of at least 4 different preparations.

158

not significantly affected in the presence of nimodipine vehicle (0.05% ethanol) and iberiotoxin
in comparison to iberiotoxin alone in physiological salines containing 20 mM KCl (data not
shown). Incubation of uncut preparations with iberiotoxin vehicle alone or in the presence of
nimodipine had no significant effect on MEPP frequency in comparison to control treatment in

physiological saline containing 5,10, 15, or 20 mM KCl (Fig. 4.11).

E. Discussion

Normally, P/Q-, but not L-type channels are primarily involved in the nerve-evoked
release of ACh from adult mammalian motor nerve terminals (Uchitel et al., 1992; Protti et al.,
1996; Katz et al., 1997). L-type Ca2+ channels can, however, participate in ACh release from
motor nerves during development (Sugiura and K0, 1997; Rosato Siri and Uchitel, 1999;
Santafe et al., 2001) and in the presence of certain pathological conditions (Katz et al., 1996;
Fratantoni etal., 2000; Santafe et al., 2000; Flink and Atchison, 2002, Giovannini et al., 2002).
In one such pathological condition, Lambert-Eaton myasthenic syndrome (LEMS),
autoantibodies not only reduce the entry of Ca” into the motor nerve terminal (Lambert and
Elmqvist, 1971; Fukunaga et al., 1983; Hewett and Atchison, 1991), but also induce L-type
Ca” channels to participate in ACh release (Flink and Atchison, 2002; Giovannini et al., 2002).

Other conditions exist in which L-type channels can participate in ACh release as well.
In the presence of specific pharmacological agents, normally silent L-type Ca2+ channels on or
near the motor nerve terminal become involved in ACh release (Atchison, 1989; Hong and
Chang, 1990; Urbano et al., 2001). Taken together, it is therefore possible that in LEMS,

involvement of L-type Ca2+ channels in release of ACh from motor nerve terminals, may in

159

150‘ —-— control *
-- 0-- IBTx l
125 d --------A IBTx + nimod .’

t—l

O

O
l

\I
LI]
1

()1
O
1

N
(I!
1

 

MEPP frequency (Hz)

I

O
L

 

 

5 1'0 1'5 20
KCI concentration (mM)

Figure 4.10. Comparative effects of iberiotoxin (IBTx) and nimodipine (nimod) on MEPP
frequency at mouse hemidiaphragm preparations. Recordings were made from preparations
before (control), after perfirsion with iberiotoxin (150 nM) for 1 hr, and following perfusion
with both nimodipine (10 11M) and iberiotoxin (150 nM) for an additional 25 min in buffers
containing either 5-20 mM KCI. An equiosmolar substitution was made in the NaCl
concentration to compensate for the varying KCl concentrations in the buffer. Each value
represents the mean i: S.E.M. of at least 4 different preparations. The asterisk (*) and double
dagger (1) indicates a value significantly different from either control or iberiotoxin alone,
respectively (P < 0.05).

160

140 a
- I - control

-O-- vehicle

120‘ -------A vehicle + niI‘nOCl

 

A

N

E 1004

V

5‘ 80-

5::

8

U, 60-

8

4—4 404

Q—r

9% 20-
0...

 

 

5 1'0 1'5 20
KC] concentration (mM)

Figure 4.1]. Effects of nimodipine (nimod) in the absence of iberiotoxin on MEPP frequency
at mouse hemidiaphragm preparations. Recordings were made from preparations before
(control), after perfusion with iberiotoxin vehicle (0.01 % BSA) for 1 hr, and following
perfusion with both nimodipine (10 nM) and iberiotoxin vehicle (0.01 % BSA) for an additional
25 min in buffers containing either 20, 15, 10, or 5 mM KCl. An equiosmolar substitution was
made in the NaCl concentration to compensate for the varying KCl concentrations in the buffer.
Each value represents the mean i S.E.M. of at least 4 different preparations.

I61

part be due to unmasking of silent channels.

One possible reason for involvement of the L-type Caz’ channels in release of ACh from
motor nerves during LEMS may involve a corresponding reduction in Kc. currents at the nerve
terminal. KC, channels are activated in the presence of Ca2+ and colocalized at active zone
regions with Cali channels normally involved in release of ACh (P/Q- type at mammalian and
N-type at amphibian motor nerve terminals) (Robitaille and Charlton, 1992; Robitaille et al.,
1993; Xu and Atchison, 1996; Protti and Uchitel, 1997). Although the exact role of KCa
channels is unknown, they are believed to aid in the repolarization of the nerve terminal
following the incoming action potential and thus, limit the duration over which Ca2+ channels
remain involved in transmitter release (Mallart, 1985; Robitaille and Charlton, 1992). In
LEMS, reduced entry of Ca?" into the nerve terminal may attenuate activation of colocalized
KC, channels and in turn alter repolarization of the membrane. This prolonged depolarization
of the nerve terminal may lead to changes in gating kinetics of L-type Caz’ channels to the
extent that they now become involved in ACh release. In fact, intense nerve terminal
depolarization has been shown to alter the phenotype of Ca2+ channels involved in ACh release
from motor (Hong and Chang, 1990; Correia-de-Sa et al., 2000a,b) and sympathetic nerves
(Somogyi et al., 1997). The present study was designed, to examined if loss of functional Kc.
channels at motor nerve terminals could expose silent L-type Ca2+ channels involved in ACh
release.

Incubation of muscle preparations with iberiotoxin, a KCal channel antagonist,
significantly increased quantal content in comparison to untreated terminals. This finding is

consistent with those of earlier reports (Robitaille and Charlton, 1992; Robitaille et al., 1993;

162

Vatanpour and Harvey, 1995), and those in which KC, channel block affects perineurial Cai’
currents recorded at motor nerve terminals (Xu and Atchison, 1996). Thus, functional
antagonism of KC, channels influences Ca2+ current flow and in turn enhances the release of
ACh.

Further, treatment of muscle preparations with nimodipine, a dihydropyridine-sensitive
L-type C az‘ channel antagonist in the presence of iberiotoxin reduced the enhanced ACh release
caused initially by iberiotoxin alone. Therefore, it appears that normally silent L-type Ca” can
participate in ACh release from motor nerve terminals lacking firnctional KC,l channels. These
findings are consistent with those reported by Hong and Chang (1990), in which L-type Caz’
channels are involved in ACh release from mammalian motor nerve terminals pretreated with
3,4 diaminopyridine (DAP), which blocks voltage-dependent K+ channels. Interestingly, block
of L-type C a2+ channels reduces the increased duration, but not the enhanced amplitude of EPPs
caused by 3,4- DAP. Unlike 3,4-DAP, iberiotoxin had no effect up the duration of EPPs, but
only affects the amplitude of EPPs. The reason for this difference is unclear, but may reflect
functional and local differences between voltage-dependent-K’ channels and KC, channels.
Block of K, channels, which are colocalized with voltage-gated Ca2+ channels, enhances
transmitter release faster than that observed with block of voltage-gated K+ channels
(Vatanpour and Harvey, 1995). Also, voltage-dependent K*, but not KCa channels, cause
repetitive transmitter release following a single stimulation (Hong and Chang, 1990; Vatanpour
and Harvey, 1995), which may influence the gating of L-type channels. The intimate
colocalization of KC, with Ca2+ channels involved normally in release may allow KC, to have a

faster and more direct effect upon these Ca2+ channels than that observed with loss of voltage-

163

gated K’ channels alone. My findings and those reported by Hong and Chang (1990) are
however, in contrast with those reported by Giovannini et al. (2002), where enhanced ACh
from mouse motor nerve terminals in the presence of the voltage-dependent K” channel
antagonist, 4-aminopyridine was unaffected by L-type Ca2+ channel antagonists. This
discrepancy most likely reflects differences in experimental protocols.

The effect of iberiotoxin on MEPP frequency was also examined. Addition of5,10,15,
or 20 mM KCl to the buffer caused a corresponding depolarization of the endplate resting
membrane potential and presumably of the nerve terminal as well. MEPP frequency increased
significantly as the resting membrane potential became more depolarized. This was unaffected
by iberiotoxin and/or nimodipine in buffer containing 5, 10, or 15 mM KCl. However, in the
presence of 20 mM KCl, addition of iberiotoxin significantly further increased MEPP
frequency, whereas further addition of nimodipine in the presence of iberiotoxin attenuated the
enhanced release caused by iberiotoxin alone. The amplitude of MEPPs was unaltered in the
presence of iberiotoxin at all concentrations of KCl tested. Thus, the effect of iberiotoxin on
ACh release appears to occur only when the membrane is depolarized by KC] concentrations
greater than 15 mM. This is consistent with the result on evoked release in which the
membrane potential is markedly depolarized from rest. It is also consistent with the biophysical
properties of L-type Caz’ channels which require strong depolarization from rest to induce
opening.

The exact mechanism involved in unmasking silent L-type Caz’ channels during block
of KCal channels is unclear, and may be multifaceted. KC, channels may exert a direct effect by

altering localized membrane potentials and thus, prevent opening of L-type Ca2+ channels,

164

which require strong depolarization for activation (Miller, 1987; Tsien et al., 1988; Zhang et
al., 1993). As such, recruitment of silent L-type Ca2+ channels involved in ACh release is
evident during prolonged or high frequency stimulation of motor (Correia-de-Sa et al., 2000b;
Correia-de-Sa et al., 2000a) and sympathetic nerves (Somogyi et al., 1997) or during block of
voltage—dependent K‘ channels (Hong and Chang, 1990). Also, L-type Ca2+ channels, which
may be located at a site distinct from active zone regions, may require prolonged periods of
openings in order to allow diffusion of Ca” through these channels to reach the release
machinery (Miller, 1987; Elhamdani et al., 1998).

Alternatively, activation of L-type Ca2+ channels in the presence of iberiotoxin may
involve more indirect mechanisms. Enhanced release of ACh from mammalian motor nerves
by or, adrenergic-receptor activation is abolished following functional antagonism of L-type
channels (Wessler et al., 1990). Intense and prolonged depolarization has been implicated to
enhance Cai’ currents by increasing phosphorylation of L-type Ca” channels (Sculptoreanu et
al., 1995). In the presence of muscarinic receptor-dependent activation of protein kinase C,
L-type Caz’ channel activity at adult rat major pelvic ganglia is increased. Furthermore, it has
been postulated that at mammalian motor nerve terminals, L-type Ca2+ channels are in close
proximity to A2", adenosine receptors and activation of A2,, adenosine receptors during
prolonged depolarization most likely unmasks L-type Ca2+ channels via activation of protien
kinases (Correia-de-Sa et al., 2000a). More direct evidence also supports the role of protein
kinases in activating L-type Ca2+ channels involved in ACh release from mature mammalian
motor nerves (Urbano et al., 2001).

The ability of bay K 8644, a DHP-sensitive L-type channel agonist to enhance ACh

165

release in comparison to control treatments, fithher supports the notion that silent L-type
channels exist at mammalian motor nerve terminals and corroborates previous reports (Atchison
and O’Leary, 1997; Atchison, 1989). However, incubation of motor nerve terminals with bay
K 8644 in the presence of iberiotoxin did not alter the release of ACh in comparison to
iberiotoxin alone. Thus, if iberiotoxin simply unmasked silent L-type channels then addition of
bay K 8644, which increases the duration of L-type Cazichannel openings (Nowycky et al.,
1985), should have further increased ACh release. It is possible that mechanisms involved in
L-type channel activation or L-type channel involvement with ACh release become saturated
in the presence of iberiotoxin and thus, bay K 8644 has no additional effect. Alternatively,
iberiotoxin and bay K 8644 may act through similar mechanisms that reach a maximum state
of activation in the presence of either drug alone. Although, iberiotoxin does not bind to L-type
Ca3+ channels, loss of KCa channels in the presence of iberiotoxin may activate secondary
pathways that act in a manner similar to that of bay K 8644 directly. The precise reason for the
inability of bay K 8644 to enhance the iberiotoxin-induced release of ACh is still elusive,
however.

In conclusion, loss of firnctional KCa channels, which presumably delays nerve terminal
membrane repolarization, activates normally silent L-type Ca2+ channels involved in ACh release
at adult mammalian motor nerves. The role which these silent L-type Ca2+ channels play at the
adult mammalian motor nerve terminal remains unclear, but may offer a means to maintain a
certain level of ACh release during periods of intense nerve stimulation or in certain
pathological conditions, in which involvement of Ca” channels normally responsible for ACh

release is impaired.

166

CHAPTER FIVE

INVOLVEMENT OF P/Q-TYPE Ca2+ CHANNELS IN RELEASE OF
ACETYLCHOLINE FROM MOTOR NERVE TERMINALS FOLLOWING

EXPOSURE TO LEMS PLASMA

167

A. Introduction

Voltage—dependent Ca2+ channels (VDCC) serve a vital role in neuronal activity and as
such, couple the action potential to release of transmitters from the nerve terminal (Augustine
et al., 1987; Katz et al., 1997). Multiple types VDCC have been identified based upon
electrophysiological, molecular, and pharmacological techniques and thus, far include the
following subtypes: T, L, N, R, P, and Q (Tsien et al., 1988; Zhang et al., 1993; Catterall,
1998; Newcomb et al., 1998). Although the exact role of each VDCC subtype throughout the
nervous system is still unclear, those channels involved in release of ACh from motor nerve
terminals have been identified. However, the specific VDCC subtype coupled to release of ACh
from motor nerve terminals vary based upon species (Sano et al., 1987; De Luca et al., 1991;
Uchitel et al., 1992; Protti et al., 1996; Katz et al., 1997) and age (Sugiura and Ko, 1997;
Rosato Siri and Uchitel, 1999; Santafe et al., 2001). During development, release of ACh from
immature motor nerve terminals is controlled by Ca2+ entry through multiple subtypes of
VDCC; immature motor nerve terminals from mice and rats utilize P/Q-, N-, and L-type Caz’
channels to affect release of ACh (Sugiura and Ko, 1997; Rosato Siri and Uchitel, 1999;
Santafe et al., 2001). On the other hand, mature motor nerves mainly possess one VDCC
subtype involved in release of ACh. Based on pharmacological sensitivities to agatoxin IVA,
a specific antagonist for P/Q-type Ca2+ channels and to m-conotoxin GVIA, a specific
antagonist for N-type Ca2+ channels, the VDCC subtypes involved in release of ACh at mature
motor nerve terminals have been identified. As such, P/Q- and N- Ca2+ channel subtypes are
involved in release of ACh from mature mammalian (Uchitel et al., 1992; Protti et al., 1996;

Katz et al., 1997) and amphibian motor nerve terminals (Sano et al., 1987), respectively.

168

In Lambert-Eaton myasthenic syndrome (LEMS), a neurological disorder affecting
release of ACh, antibodies down-regulate multiple subtypes of VDCC. However, the P/Q-type
C az“ channel appears to be affected preferentially (Lennon et al. , 1995; Garcia and Beam, 1996;
Houzen et al., 1998 Pinto et al., 1998a; Engisch et al., 1999; Pinto et al., 2002). Based upon
the VDCC subtype controlling release of ACh from mature mammalian motor nerve terminals
and antibody specificities in LEMS, it is clear why neuromuscular weakness is the most
common symptom reported by LEMS patients. Although direct access to the mammalian
motor nerve terminal is precluded by its small size, indirect electrophysiological recordings have
been used to determine the effect of passive transfer of LEMS to mice on the P/Q-type Ca2+
currents at the motor nerve terminal. Overall, Caz’ current amplitudes are reduced from motor
nerve terminals of mice exposed to LEMS plasma in comparison to those at motor nerve
terminals exposed to plasma from disease-free individuals. However, the actions of agatoxin
IVA, which normally antagonize the majority of P/Q-type Ca2+ currents are reduced at LEMS
motor nerve terminals in comparison to controls (Xu et al., 1988); agatoxin IVA reduces the
amplitude of the P/Q-type Ca2+ currents at control motor nerve terminals by approximately
90%, whereas agatoxin IVA only attenuates the P/Q-type Ca2+ current amplitude by
approximately 45% from LEMS motor nerve terminals. Thus, it appears that passive transfer
of LEMS to mice reduces the number of functional P/Q-type Ca2+ channels. Furthermore, the
percent Caz‘ current remaining following passive transfer of LEMS is greater than that observed

at controls, indicating the possible expression of a Ca2+ current not normally observed.

169

Patient #1

3100—
>

_

.E

S 80—
H

N

00

‘1‘

Q)

a 60- :s
3

V

’E _
8 40
=

O

U

3 20—
g 4:
5

O’

 

 

LEMS control

Figure 5.1. Effect of LEMS plasma on the functional P/Q-type Ca2+ channels involved in
release of ACh from mature mouse motor nerve terminals. Diaphragm neuromuscular
preparations were obtained from mice (20-22g) following injection ofLEMS or control plasma
for 30 days. Prior to injection with plasma, mice were first injected with 300 mg/kg
cyclophosphamide in order to suppress the immune response to foreign proteins. Muscles
were cut on either side of the main intramuscular nerve branch to prevent contractions that
occur during nerve-stimulation. EPPs and MEPPs amplitudes were determined using
conventional intracellular recording techniques. EPPs were elicited during 0.5 Hz stimulation
of the phrenic nerve. Quantal content was determined using the ratio of the average EPP
amplitude to the average MEPP amplitude. Values are expressed as the percentage of quantal
content from plasma-exposed preparations after addition of 150 nM agatoxin IVA to those
before addition of agatoxin IVA. The asterisks (*) indicates a value significantly different from
pre-agatoxin IVA treated preparations (P < 0.5 Hz).

170

B. Summary

The effect of passive transfer of LEMS plasma to mice on the number of functional P/Q-
type Cal" channels involved in release of ACh from the motor nerve terminal was examined
(Fig. 5.1). Following injection of control plasma into mice for 30 days, agatoxin IVA reduced
the nerve-stimulated release of ACh to approximately 90%. On the other hand, agatoxin IVA
only inhibited the nerve-stimulated release of ACh by approximately 50% following passive
transfer of LEMS to mice by daily injections for 30 days. These findings are consistent with
those reported by Xu et al. (1998), in which passive transfer of LEMS to mice following 30
days of injections had a similar effect on the P/Q-type Ca2+ current at the motor nerve terminal.
Furthermore, the ability of agatoxin IVA to reduce almost completely the nerve-stimulated
release of ACh from control preparations also corroborates the findings reported elsewhere,
which have shown that P/Q-type Ca” channels are the primary subtype of VDCC involved in
release of ACh from mature mammalian motor nerve terminals. The effect of agatoxin IVA
on release of ACh following injection of mice with LEMS plasma for 30 days in comparison
to controls has two important implications: 1) the number of firnctional P/Q-type Ca2+ channels
involved in nerve-stimulated release of ACh is reduced in LEMS and 2) the component of
nerve-stimulated release of ACh unaffected by agatoxin IVA may reflect Ca2+ entry through

a VDCC subtype not normally found at the mature mammalian motor nerve terminal.

171

CHAPTER SIX

SUMMARY AND CONCLUSIONS

172

Summary and Conclusions

The overall aim of this dissertation was to examine the role of L-type Ca” channels in
release of ACh from motor nerves following passive transfer of LEMS to mature mice.
Although antibodies from patients with LEMS have been observed to down-regulate a variety
of functional voltage-dependent Caz’ channel subtypes, the P/Q-type channel appears to be the
primary target (Lennon er al., 1995; Garcia and Beam, 1996; Houzen et al., 1998 Pinto et al.,
1998a; Engisch et al., 1999; Pinto et al., 2002). Furthermore, among the multiple VDCC
subtypes known to exist, the P/Q-type channel appears to be the predominant channel subtype
involved in release of ACh from mature mammalian motor nerve terminals (Uchitel et al., 1992;
Protti et al., 1996; Katz et al., 1997). Based on these findings, the paucity of nerve-stimulated
release of ACh and resulting skeletal muscle weakness that ensues in patients with LEMS is
therefore not surprising.

Passive transfer of LEM S to mature mice also reduces nerve-stimulated release of AC h
stimulation (Lambert and Elmqvist, 1971; Lang et al., 1983; Kim, 1985; Prior and Newsom-
Davis, 1985; Lambert and Lennon, 1988). Along these lines, LEMS antibodies injected into
mature mice for 30 days attenuate P/Q-type Ca2+ current at motor nerve terminals (Xu et al.,
1998) However, L-type Caz’ current, which is not normally found at mature mouse motor
nerve terminals is present following passive transfer of LEMS for 30 days (Xu et al., 1998).
Not only is this finding in contrast with the known effects of LEMS antibodies, but represents
expression of a Caz’ current not normally observed.

It is possible that this novel L-type Ca2+ current may participate in ACh release and/or

complex signaling pathways that have as of yet, no clearly identified fimction. However, based

173

on the effects of LEMS antibodies, it seems likely that this novel current is involved in release
of ACh in order to compensate for the reduction in P/Q-type channels. Furthermore, during
maturation of some mammalian motor nerves, not only P/Q-, but L-type Ca2+ channels
participate in release of ACh as well (Sugiura and Ko, 1997; Rosato Siri and Uchitel, 1999;
Santafe et al., 2001 ; Rosato-Siri et al. , 2002; Santafe et al., 2002). At birth, developing muscle
fibers are innervated by multiple motor nerves, which through a process of activity-dependent
synaptic elimination, decrease until only one nerve is eventually associated with a single end-
plate (Rosato Siri and Uchitel, 1999; Santafe et al. , 2000; Santafe et al. , 2002). It appears that
the function of multiple subtypes of VDCC found at motor nerve terminals differs depending
on the eventual role of the motor nerve in synaptic transmission in mature mammals; motor
nerves destined to become involved in release of ACh in mature mammals possess multiple
VDCC subtypes, including P/Q- and L-types that are both involved in release of ACh
throughout development. On the other hand, P/Q- and L-type Ca2+ channels located on motor
nerve terminals destined for elimination are believed to be involved in activation of cytoplasmic
proteases that aid in neuronal death and only weakly participate in release of ACh (Rosato Siri
and Uchitel, 1999; Santafe et al., 2001; Santafe et al., 2002). Thus, motor nerves that release
the largest amount of ACh during development survive, whereas those nerves that release
minimal ACh eventually are eliminated. Although the role of multiple VDCC subtypes on
surviving motor nerve terminals during development is unclear, it is believed they act to keep
Ca2+ entry and thus, ACh release, at high levels until expression of P/Q-type Ca2+ channels
becomes maximal (Rosato Siri and Uchitel, 1999; Santafe et al., 2001; Santafe et al., 2002).

Based on these findings, L-type Ca2+ current observed after passive transfer of LEMS to

174

mature mice most likely reflects actions of multiple subtypes of VDCC on motor nerve
terminals destined to survive development. Thus, it was my contention that this novel L-type
Ca3+ current at motor nerve terminals of mice with LEMS may compensate for lost P/Q-type
Ca2+ channels and become involved in release of ACh.

Following passive transfer of LEMS to mice by 30 days of daily injection, the spider
toxin, m-Aga IVA, at concentrations used to inhibit both P- and Q-type Ca2+ channels, reduced
the nerve stimulated-release of ACh from motor nerves to a lesser extent than that observed
when applied to control preparations; m-Aga IVA inhibited the nerve-stimulated release of
ACh by approximately 90% at neuromuscular junction (NMJ) preparations obtained from
mature mice following injection of control plasma for 30 days, whereas w-Aga IVA only
reduced release of AC h by approximately 50% following injection of LEMS plasma into mature
mice for 30 days. This finding is consistent with the effects of passive transfer of LEMS to
mature mice on the P/Q-type Ca2+ currents (Xu and Atchison, 1998). The inability of w-Aga
IVA to reduce quantal content at LEMS NMJ preparations to the same extent as those
observed from controls, suggests that LEMS plasma attenuates the number of fimctional P/Q-
type channels at the motor nerve terminal of mature mice. Furthermore, the remaining quantal
content observed after the addition of co-Aga IVA to LEMS NMJ preparations indicates a
possible role of another VDCC subtype involved in release of ACh. This finding lends support
to my contention that L-type Ca” currents observed at motor nerve terminals following
injection of LEMS plasma for 30 days into mature mice is involved in release of ACh.

However, in order to determine clearly the role of L-type Ca2+ current in release of ACh

at LEMS motor nerves, the pharmacological sensitivity to the dihydropyridine L-type Caz’

175

channel antagonist, nimodipine was examined. As such, nimodipine reduced the nerve-
stimulated release of ACh at NMJ preparations obtained from mature mice injected with LEMS
plasma for 30 days, but did not affect release of ACh at NMJ preparations obtained from mice
injected with control plasma for a similar duration. These findings support the idea that L-type
Caz’ current at motor nerve terminals in LEMS effect release of ACh.

This involvement of L-type Caz’ channels in release of ACh from LEMS motor nerve
terminals may represent an unmasking of silent L-type channels already present on the motor
nerve terminal. Although L-type Ca2+ current is not normally observed at mature mouse motor
nerve terminals, various mechanisms exist to unmask current through these channels, which can
participate in release of ACh. L-type Ca2+ channels become involved in ACh release from
mature mammalian motor nerve terminals following incubation with the dihydropyridine
agonist, bay K 8644 by increasing the duration of channel openings (Atchison and O'Leary,
1987; Atchison, 1989) or during increased protein phosphorylation in conjunction with
intracellular Ca” chelation (Urbano et al., 2001).

Based on these observations, it seemed plausible that involvement of L-type Ca2+
channels in release of ACh following exposure of motor nerves to LEMS plasma results from
unmasking of normally silent channels. While this possibility can not be rejected, experiments
contained in this dissertation provide some evidence that L-type Ca” channel involvement in
release of ACh from LEMS motor nerves is due to more than merely unmasking of silent
channels. For instance, L-type Caz’ channels did not become involved in release of ACh from
motor nerves exposed to LEMS plasma for 2 or 24 hours. This finding could potentially be due

to the differences in the extent of reduction of quantal content between 30 days injection and

176

2 or 24 hour exposures. Injection of mature mice for 30 days with plasma from patients with
LEMS not only significantly reduced the nerve-stimulated release of ACh, but induced
facilitation in the release of ACh during high frequency stimulation of the motor nerve as well.
These findings were not observed using control preparations and are consistent with those
reported in other model systems and those observed from EMG recordings of patients with
LEMS (Eaton and Lambert, 1957; Elmqvist and Lambert, 1968; Jablecki, 1984; O'Neill and
Newsom-Davis, 1988). Similarly, exposure of NMJ obtained fi'om mature mice to LEMS
plasma for 2 and 24 hours also induced the electrophysiological characteristics of LEMS.
However, under the experimental conditions used to examine the effect of LEMS plasma on
motor nerve terminals exposed for 30 days and 2 and 24 hours, differences in the extent of
reduction of nerve-stimulated release of ACh were only significantly different between 2 hours
and 30 day treatment regimens. The lack of significance between the other treatment groups
may reflect the manner in which the effect of LEMS plasma on ACh release was measured. It
is possible that injection of mice with LEMS plasma for 30 days reduces the number of
functional P/Q-type Ca2+ channels involved in ACh release to a significantly greater extent
compared to exposure for 24 hours to LEMS plasma. However, expression of L-type Ca2+
current following 30 days, but not 24 hours of LEMS exposure somewhat compensates for the
reduced number of P/Q-type Ca2+ channels and thus, differences in nerve-stimulated release of
ACh appear similar. It is possible therefore, that significant differences of LEMS antibodies
on the motor nerve terminal are obscured among the different treatment groups. Nevertheless,
2 and 24 hour exposure of motor nerve terminals to LEMS plasma induces the overall reduction

in nerve-stimulated ACh release to an appreciable extent without involving L-type Caz’ channels

177

in ACh release. Thus, the involvement of L-type Caz’ channels in release of ACh from motor
nerve terminals of mature mice following injection of LEMS plasma for 30 days is most likely
due to long-term processes, such as synthesis of new channel components and not simply
unmasking silent channels.

The time course of development of L-type Ca2+ channel involvement in release of AC h
from LEMS motor nerve terminals was examined further using NMJ preparations from mature
mice injected with LEMS plasma for varying lengths of time. Nerve-stimulated release of ACh
was reduced significantly following injection of mice with plasma from two patients
(designated arbitrarily as “Patients 1 and 2") after 1- 20 days in comparison to animals injected
with plasma from disease-free controls. However, L-type Ca2+ channels only became involved
in nerve-stimulated release of ACh following injection of plasma for more than 20 days.
Interestingly, plasma from a third patient with LEMS (designated “Patient 3") only significantly
affected release of ACh after 10 days of treatment and did so without inducing the involvement
of L-type Caz’ channels. The reasons for the inability of this patient’s plasma to induce L-type
Ca2+ channel involvement in ACh release are unclear, but may relate to the antibody titer in the
plasma, which was not measured. It is possible that if the antibody titer of this particular
patient’s plasma was lower than that of the other two patients a longer exposure time would
be required. However, L-type Ca2+ channels did not become involved in ACh release even after
30 days of using this treatment with this patient’s plasma.

Qualitative differences among the antibody specificity may also exist. This has been
shown previously in studies in which antibodies to other proteins involved in ACh secretion

such as synaptotagmin occur in plasma of some LEMS patients (Leveque et al., 1992;

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Takamori et al., 1995; Takamori etal. , 2000a). Although antibody titer specificity for subtypes
of VDCC was not directly measured in experiments comprising this dissertation, based on
firnctional studies, it appears that all patients’ plasma contained antibodies for P/Q-type Caz"
channels because all three patients’ plasma eventually impaired ACh release. However, it is
possible that plasma from “Patient 3" possessed antibodies directed against not only P/Q-, but
L-type Ca2+ channels as well. Therefore, L-type Caz’ channel involvement in ACh release from
motor nerves exposed to this patient’s plasma might not become apparent. The involvement
of L-type Caz’ channels in release of ACh from motor nerves exposed to the other patients’
plasma suggests that their antibody titers specific for L-type Caz’ channels would have to be
quite small or even non-existent. However, the possibility that these patients’ plasma do not
contain antibodies specific for L-type Ca2+ channels can not be fully excluded.

The time differences necessary to reduce significantly release of ACh by the various
patients’ plasma may firrther reflect differences in antibody titers. Based upon the findings of
this dissertation, I would predict that “Patient 3" possessed the lowest antibody titers specific
for P/Q-type channels. Alternatively, the VDCC-specific antibodies may have different avidities
among the LEMS patients’ plasma. In such a condition, antibody titers may be equal among
the patients’ plasma, but varying avidities for VDCC would differentially affect the time and
extent of reduction in the release of ACh. If each LEMS patient’s plasma contained similar
antibody titers with differing avidities, then it appears that plasma from “Patient 3" contained
antibodies with the lowest avidity. However, based upon the work presented in this dissertation
a distinction between antibody titers and avidities among the different patients’ plasma can not

be made directly.

179

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In conjunction with the effect of each individual patients’ plasma on release of ACh
during low frequency nerve-stimulation, preparations obtained from mice injected for 1-20 days
with plasma from “Patient 1" or “2" induced significant facilitation of ACh release during high
frequency nerve-stimulation, whereas that of “Patient 3" did not induce facilitation until after
day 10. Based upon these findings, it appears that a significant reduction in release of ACh
during low frequency nerve-stimulation is necessary in order for facilitation of ACh release to
occur. This finding lends support to the idea that Cali-dependent mechanisms must exist at
the motor nerve terminal to induce closure of VDCC during periods of intense nerve-
stimulation, and facilitation only occurs when these mechanisms are not activated. This idea
is not unique, and Cab-dependent inactivation of multiple subtypes of VDCC is known to
occur (Tareilus et al., 1994). Although the fimction of this mechanism is not fiilly understood,
it is believed to act not only as a means to control tightly transmitter release (Gutnick et al.,
1989), but also to prevent cytotoxicity and cellular death that results from the cytoplasm being
overloaded with excess Ca2+ (Choi, 1988).

In addition to becoming involved in release of ACh during low frequency nerve-
stimulation, L- type Ca2+ channels also appear to comprise a component of the facilitation
observed following passive transfer of LEMS in the 20 day treatment regimen. However, the
extent of involvement of L-type Caz‘ in release of ACh during low frequency stimulation
appears greater than that which occurs during high frequency nerve stimulation. Although the
reasons for this discrepancy are unclear, it is possible that mechanisms exist which reduce L-
type VDCC function during high frequency nerve-stimulation to a greater extent than during

low frequency nerve-stimulation.

180

In corroboration with the observations reported by other investigators, MEPP
frequencies or amplitudes measured from NMJ preparations in buffers containing physiological
concentrations of K+ were not altered by exposure of the motor nerve to any of the LEMS
patient or control plasma. This finding further supports the idea that the LEMS antibodies act
on the presynaptic motor nerve terminal without altering vesicle size or contents. Although one
could make an argument that LEMS antibodies affects vesicle size, contents and ACh receptors
located at muscle end-plates such that MEPP amplitudes appear unchanged, these structures
have been shown to be unaffected in LEMS (Molenaar et al., 1982; Lang and Vincent, 1984).
Furthermore, the findings that MEPP frequency is unaltered during passive transfer suggest that
intraterminal Ca2+ buffering mechanisms are unaltered in LEMS.

In contrast to the observations during nerve-stimulation, L-type VDCC were not
involved in spontaneous release of ACh at NMJ preparations obtained from mice injected with
any of the LEMS patient or control plasma. This finding has multiple implications: 1) it is
possible that the number of functional L-type channels is not as high as the number of functional
P/Q-type channels, 2) L-type channels may be located at sites distinct from ACh release sites,
3) and/or mechanisms exist which have differential effects upon L- and P/Q-type channels
depending on the membrane potential. Most likely, the lack of involvement of L-type VDCC
in spontaneous release of ACh is due a combination of the all of the above mentioned reasons.
However, experiments performed for this thesis did not examine the densities of L-type
channels expressed during long-term exposure of motor nerves to LEMS plasma. Although
involvement of L-type Ca2+ channels in release of ACh was examined, correlations between

channel function and density can not be made. For example, L- and N-type channels are

181

expressed at adrenal chromaffin cells of rats in equal densities, however, secretion of
catecholamines is reduced by L-, but not N-type channel antagonists (Lopez 1994). Thus,
functional involvement of L-type Caz’ channels in release of ACh may not be directly related
to the density of channels found on the nerve terminal. However, experiments comprising this
thesis were performed, which used pharmacological methods to determine the approximate
location of L-type Ca2+ channels on the motor nerve terminal. DM-BAPTA, a rapid chelator
of C a2“, was loaded into motor nerve terminals in order to test the hypothesis that P/Q-, but not
L-type channels are in close proximity to ACh release sites. This was based on the premise that
coupling between the normal complement of P/Q-type VDCC and release sites is so tight that
entry of Ca2 ’ through these channels would activate release of ACh before being bound by DM-
BAPTA (Urbano et al., 2001). However, Ca2+ which entered through VDCC located at sites
distinct from the release apparatus would be bound by DM-BAPTA prior to activating release
of ACh. Thus, DM-BAPTA did not affect nerve-stimulated release of ACh from control
preparations. However, the involvement of L-type VDCC in ACh release was abolished from
motor nerve terminals loaded with DM-BAPTA. Based upon the observations that P/Q- type
C az‘ channels represent the normal VDCC subtype controlling nerve-stimulated release of ACh
from mature mouse motor nerves before and after control-plasma exposure and DM-BAPTA
has no effect on release of ACh fiom untreated (Urbano et al. , 2001) or control-plasma exposed
motor nerves implies an extremely tight coupling between P/Q-type Ca2+ channels and ACh
release sites. L-type VDCC, on the other hand, appear to be located at a site distinct from the
release sites. However, based on the findings in this dissertation it is unclear if newly

synthesized L-type Ca2+ channels are inserted into the motor nerve terminal membrane or

182

expressed on intraterminal structures (for example, the endoplasmic reticulum). Support for
an intraterminal location of L-type Ca2+ channels is based on the following: bay K 8644
increases the frequency of spontaneous release of ACh at mouse NMJ preparations in Ca2*-free
medium (Pancrazio et al., 1989), and immunostaining of motor nerve terminal membranes
obtained from mature rats does not reveal the presence of L-type Ca2+ channels comprised of
either or”)... subunits (Westenbroek, et al., 1998). It is possible that L-type Ca2+ channels
located in the motor nerve terminal membrane of mature rats are comprised of 011 subunits of
the S, R, or some other unidentified class or that the number of L-type Ca2+ channels is too low
on the motor nerve terminal membrane to detect using immunostaining methods. Nevertheless,
in the presence of membrane potentials close to the resting value of -80 mV, influx of Ca2+
through a small number of L-type channels may be buffered by intracellular proteins and
sequestration mechanisms prior to diffusing to ACh release sites. On the other hand, during
strong membrane depolarization to levels more positive than - 50 mV , which increases the
probability of many L-type channel openings, a large amount of Ca2+ enters, which may
overwhelm intracellular buffering mechanisms, and thus, affect release of ACh.

The prolonged period of time necessary for L-type Ca2+ channels to become involved
in ACh release following injection of LEMS plasma supports the idea that involvement of L-
type channels is not due merely to unmasking of normally silent channels already present on the
motor nerve terminal. This idea is also supported by the findings that L-type Ca2+ currents are
not present at mature mammalian motor nerve terminals exposed to control plasma for 30 days
when function of P/Q-type channels is blocked transiently by oo-Aga IVA (Xu et al., 1998).

Processes such as formation of new channel subunits and assembly are most likely required for

183

firnctional L-type channels to become involved in ACh release during LEMS. This finding is
similar to those obtained at motor nerve terminals during reinnervation (Katz et al., 1996) or
recovery from botulinum toxin-induced poisoning (Santafe and Uchitel, 2000), which reflect
processes that occur during maturation of motor nerves. Thus, immature motor nerves also
possess multiple subtypes of VDCC that are involved in release of ACh, but whose involvement
is reduced until the mature motor nerve only has one primary subtype of VDCC involved in
ACh release (Sugiura and Ko, 1997; Rosato Siri and Uchitel, I999; Santafe et al., 2001;
Rosato-Siri et al., 2002; Santafe et al., 2002). However, LEMS antibodies have not been
shown to damage the nerve terminal or induce-sprouting of immature motor nerves (F ukunaga
et al., 1983; Tsujihata et al., 1987) and thus, it is unclear if mechanisms underlying L-type Caz’
channel expression are similar to those involved during motor nerve maturation.

The finding of differential localization of multiple VDCC subtypes is not unique to
LEMS. BAC cells possess Q-type Ca2+ channels located in close proximity to catecholamine
release sites and L-type Ca2+ channels located at a distinct site (Lara et al., 1998). Similar
findings have been observed at motor nerve terminals from neonatal (0-4 day old) rats (Rosato-
Siri et al., 2002); P/Q- and N-type channels both appear to be involved in ACh release,
however, P/Q-, but not N-type channels remain involved in release of ACh from BAPTA-AM
loaded nerve terminals, providing evidence for differential localizations of these channel
subtypes. Differential localizations of multiple VDCC subtypes may allow fine tuning of
transmitter release under varying stimuli or physiological conditions.

Although the normal lack of involvement of L-type Ca2+ channels in release of ACh

from mature mammalian motor nerves is not firlly understood, it is possible that P/Q-type Caz’

I84

channels may be better suited for the precise nature of ACh release. Using a fluorescent C a”
ion indicator it has been observed that, entry of Ca2+ through VDCC involved in release of ACh
occurs in microdomains instead of a general diffuse pattern (Llinas et al., 1992). This control
is related to the biophysical properties, location, and other secondary mechanisms that affect
channel function. As such, the large single channel conductance of L-type channels and slow
rate of voltage-dependent inactivation may not allow precise control of Ca2+ influx to occur in
microdomains. This in turn may prevent tightly controlled release, thus requiring that L-type
Ca” channels be located away from release sites. Differential localizations of multiple VDCC-
subtypes may also reflect the amino acid sequences of comprising each channel subtype (Atlas,
2001); amino acid sequences and conformational 3-dimensional structure of P/Q-type Ca2+
channels may allow tight interactions with other proteins, such as synaptotagmin, that are
directly associated with the release apparatus, whereas L-type Ca2+ may lack the necessary
coupling sites. L-type Ca2+ channels found on non-motor nerves of the mammalian system are
also generally believed not to be tightly coupled with fast transmitter release. Thus, it is not
surprising to find L-type channels located at a site distinct from that of P/Q-type channels.
However, in retinal bipolar cells, L—type Ca2+ channels appear to be associated with the release
apparatus (Tachibana 1999). This finding may reflect differences in the 011 subtype comprising
L-type channels in retinal bipolar and those L-type channels or other proteins involved in ACh
release fiom LEMS motor nerves. Currently, the subunit composition of native L-type Ca2+
channels involved in ACh release from motor nerves following passive transfer of LEMS is
unknown. In addition, VDCC have been shown to be modulated directly by interactions with

proteins comprising the release apparatus. Thus, normally silent L-type channels, which may

185

be located at sites distinct from the release apparatus, may not be involved in the release of
AC h under normal conditions due to lack of modulation by proteins associated with the release
apparatus.

The location of VDCC may be directly related to the function of the channel during
development. Channels destined to be the primary subtype involved in ACh release from mature
motor nerves are more closely associated with the release apparatus, while channels located at
distinct sites have additional firnctions. L-type Ca” channels on immature motor nerve
terminals may not only aid in release of ACh during periods of incomplete expression of P/Q-
type channels, but also be involved in repressing development of motor nerves destined to be
eliminated— firnctions, which could only arise due to the location of the channel. It also has
been proposed that motor nerves from mice deficient in NCAM (neuronal cell adhesion
molecule) exhibit two separate vesicle recycling pathways that resemble those found on
immature and mature motor nerves (Polo-Parada et al., 2001). Interestingly, L-type VDCC
found in pre-terminal regions appear to be involved in the immature vesicle recycling pathway,
but not involved in transmitter release from NCAM deficient motor nerves. It is possible that
during exposure of motor nerve terminals to LEMS plasma, L-type Ca2+ channels assume the
role they played during maturation based solely upon their location at the motor nerve terminal.

Based on the findings described thus far, a hypothetical model (Fig. 6.1) has been
proposed to explain the involvement of L-type Ca2+ in ACh release from motor nerves exposed
to LEMS plasma. Normally, tonic excitatory signals originating from the motor nerve terminal
or post-synaptic muscle membrane activate the motor cell body to initiate the production and

transport of L-type channel subunit proteins. Once these L-type Ca2+ channels subunits reach

186

the necessary site at the motor nerve terminal cytoplasm, they are assembled and inserted into
the membrane. However, normally the excitatory signals that stimulate the cell body to begin
channel production are inhibited tonically by multiple mechanisms, which may include signals
originating from the pre- and post-synaptic structures. Consequently, Ca2+ influx through P/Q-
type channels and ACh release may directly or indirectly act as negative regulators of the tonic
excitatory signals to the cell body and/or as negative regulators of L-type channels already
present at sites distinct from the release apparatus. During down-regulation of P/Q-type
channels by antibodies from patients with LEMS, entry of Ca2+ through P/Q-type channels and
release of ACh are reduced. This in turn leads to loss of signals that normally inhibit the tonic
. excitatory stimulation of L-type channel production in the cell body. Thus, in LEMS expression
of L-type Ca2+ channels is increased. However, it is unclear if production of new P/Q-type
channels also occurs in LEMS. It has been observed that during exposure of [MR cells to N-
type VDCC antibodies, channel turnover is increased (Passafaro et al., 1992). In conjunction
with the vital role which P/Q-type Ca2+ channels play in release of ACh, it seems plausible that
in LEMS, synthesis of new P/Q-type channels occurs in addition to production of new of L-
type channels. However, P/Q-type Ca2+ channel subunit production and assembly may not be
able to keep up with the continual loss of P/Q-type channels that occurs during exposure to
LEMS antibodies. In such a situation, it would appear, therefore, that only new L-type channel
subunits are synthesized.

Along these lines, Ca2+ influx has been observed to play a vital role in gene expression
in many cellular model systems. For example, the amplitude and frequency of Ca2+ entry into

B- and T-lymphocytes, respectively has been demonstrated to diferentially affect the expression

187

of multiple genes (Dolmetsch et al., 1997, 1998). The spatial and temporal effect of Ca” entry
on nuclear responses also is VDCC subtype-specific—expression of syntaxin-1A in HEK293
cells is dependent on the presence of P/Q-, but not L-, N-, or T-type VDCC (Sutton et al.,
1999). On the other hand, activity of L-type Ca” channels, induces phosphorylation of CREB
(C a3‘/cA1\/IP response element binding protein) in hippocampal neurons, whereas P/Q- and N-
type channel activity does not alter CREB phosphorylation (Hardingham et al., 1999;
Nakazawa and Murphy, 1999). Thus, loss of functional VDCC as that which occurs in LEMS
would likely alter expression of proteins comprising multiple VDCC subtypes.

The reason that synthesis of a VDCC subtype not normally involved in release occurs
in LEMS is unclear, but could reflect in the mechanisms controlling all VDCC synthesis. It is
possible that under conditions in which the levels of P/Q-type Caz’ channels are reduced, signals
activate a global increase in expression of multiple VDCC subtypes at the motor nerve terminal.
Furthermore, it is also unclear why L-type channels are found at the motor nerve terminals, but
not normally involved in release of ACh. It is possible that these channels are involved in some
unidentified function at the motor nerve terminal and unrelated to release of ACh. However,
under certain environmental conditions, these channels indirectly and accidentally become
involved in ACh release strictly due to their location. Alternatively, these channels may merely
represent residual channels, whose presence decreased, but was not eliminated totally during
maturation.

Although relocation of newly synthesized and pre-existing L-type channels may occur
in LEMS, this hypothesis was not tested in this dissertation. It appears unlikely, however,that

the only mechanism involved is that of relocation of pre-existing channels. This is based upon

188

the long time necessary for L-type channels to become involved in release of ACh from LEMS-
exposed motor nerve terminals. Experiments involving immunostaining with 011 subunits
subtypes believed to comprise L-type channels of NMJ preparations obtained from mice
injected with LEMS or control plasma for varying days, may clarify the role of relocation.

In addition to location, other means of differential modulation must exist in order to
ensure precise control of ACh release when multiple subtypes of VDCC are present on the
motor nerve terminal. These may include: differences in specific accessory subunits comprising
the channel, relationship with other non-VDCC receptors or channels, and/or diverse second
messenger pathways. Although the specific accessory subunit subtypes comprising VDCC at
the mature mammalian motor nerve terminal are unknown, firnction of VDCC is influenced by
the subtypes of the accessory subunits comprising the channel (Singer et al., 1991; Neely et
al., 1993; Chien et al., 1995; Brice et al., 1997; Brice and Dolphin, 1999). Furthermore,
differential modulation of multiple VDCC subtypes acts through other neurotransmitter
receptor subtypes, including both noradrenergic and opioid-type receptors. For instance,
stimulation of a, and B1 adrenergic receptors increases nerve-stimulated release of ACh from
rat motor nerves, by activating L- and N-type Ca2+ channels, respectively (Wessler et al., 1990).
In contrast, activation of mu-opioid receptors on acutely dissociated DRG neurons reduces Ca2+
currents by acting through G-proteins (Moises et al. , 1994). Ca2+ entry through L-type channels
also appears to be limited by Ca2’-dependent inactivation at mature mammalian motor nerve
terminals. Although P/Q-type Caz’ channels located in many cell types are inactivated during
increasing intracellular Ca2+ levels (Tareilus et al., 1994), this inhibitory mechanism does not

seem to affect P/Q-type C az’ channel dependent-release of ACh from mature mammalian motor

189

 

    

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re
\1

Figure 6.1. Hypothetical model depicting the development of firnctional L-type Ca2+ channels
at adult mouse motor nerve terminals. L-type Ca2+ channel subunits are produced in the motor
nerve cell body (1) in response to tonic excitatory signals originating from pre- (2) and/or post-
synaptic (3) structures. L-type Ca” channel subunits are then transported (4) along the axon
and assembled (5) into intact channels at the motor nerve terminal. Signals from pre- and post-
synaptic structures can also excite one another to enhance input signals to the cell body (6).
However, Caz’ entry (7) through the normal complement of P/Q-type Ca2+ channels inhibits
both the tonic excitatory pre-synaptic signal (8) and function of L-type Ca2+ channels already
present at the terminal (9). ACh released in response to Ca” entry through the normal
complement of P/Q-type channels also inhibits the tonic excitatory pre- and post-synaptic
signals (10). Therefore, in the presence of the normal complement of P/Q-type Ca2+ channels,
production of L-type Ca2+ channel subunits is terminated. LEMS antibodies reduce the number
of fimctional P/Q-type Ca2+ channels, and subsequently reduce both entry of Ca2+ into the
terminal and nerve-evoked release of ACh. Thus, in LEMS, inhibitory signals normally
generated by both Ca2+ entry through P/Q-type channels and P/Q-type Ca2+ channel-dependent
ACh release are lost, which in turn may enhance the production and function of L-type Ca2+
channels.

190

nerve terminals during low-frequency nerve-stimulation. Furthermore, mechanisms affecting
phosphorylation of VDCC have been observed to modulate channel function. Release of 5-
hydroxytryptamine (5-HT) from nerve terminal preparations (synaptosomes) isolated from
Spinal cords of adult rats is increased by protein kinase C—dependent activation of L-type Ca2+
channels (Talley et al., 1997). Activation of protein kinase C by stimulation of muscarinic
receptors also increases L-type Ca2+ channel activity at major pelvic ganglia of adult rats
(Sculptoreanu et al., 2001). Moreover, inhibition of protein phosphatases, which normally
dephosphorylate proteins, involves L-type Ca2+ channels in ACh release from mature
mammalian motor nerves (Urbano et al., 2001). Alterations in stimulus parameters also affect
channel function by acting through phosphorylation-dependent mechanisms; amplitudes of Ca2+
currents are enhanced during intense depolarization by increasing phosphorylation of L-type
Ca2+ channels (Sculptoreanu et al., 1995). Prolonged depolarization of mature mammalian
motor nerve terminals activates protein kinases through stimulation of A2 A adenosine receptors
and in turn, unmasks L-type channels involved in release of ACh (Correia-de-Sa et al., 2000).
Although the exact mechanism has not been examined, intense depolarization of adult mouse
motor nerves induced by 3,4 DAP, which blocks voltage-dependent K+ channels, enhances ACh
release by activating and prolonging the activation of P/Q- and L-type VDCC (Hong and Chang
1990). Based on these observations, it seems likely that following synthesis of L-type Ca2+
channel subunit proteins and formation and insertion of newly assembled firnctional channels
into the motor nerve terminal membrane during exposure to LEMS plasma, more immediate
mechanisms also exist, which enhance or permit involvement of these newly expressed channels

in ACh release. The location of L-type channels at sites distinct from the release machinery

191

from LEMS-exposed motor nerve terminals most likely necessitates a large influx of Ca2+
through L-type channels in order to affect release of ACh. While the exact secondary
mechanism is not certain, it is possible that mechanisms which aid in prolongation of membrane
depolarization and thus prolongation of channel openings, enhance L-type channel involvement
in release of ACh from LEMS motor nerve terminals. More specifically, loss of activation of
KCa channels may enhance involvement of L-type Ca” channel-dependent release of ACh
during LEMS. This idea is based on multiple observations. KCa channels are colocalized with
P/Q- and N-type channels at adult mouse and frog motor nerve terminals, respectively, and
directly influence the openings of VDCC by aiding membrane repolarization (Mallart, 1985;
Robitaille and Charlton, 1992; Robitaille et al., 1993; Xu and Atchison, 1996; Protti and
Uchitel, 1997). Furthermore, intense and prolonged membrane depolarization by electrical
stimulation or block of voltage-dependent K+ channels with 3,4 DAP involves L-type VDCC
in release of ACh from mature mammalian motor nerve terminals. It is possible that in LEMS,
the decrease in the number of P/Q-type VDCC leads to a decrease in openings of colocalized
Kc. channels and prolongs depolarization of the motor nerve terminal membrane. The overall
effect of prolonged membrane depolarization could potentially be to enhance the involvement
of L-type VDCC in release of ACh by increasing the duration of channel openings and thus,
increasing Ca2+ entry through these channels.

Incubation of naive neuromu scular preparations obtained from mature mice with the KC,
channel antagonist, iberiotoxin, increased the nerve-stimulated release of ACh without altering
MEPP amplitudes. However, iberiotoxin did not affect the spontaneous asynchronous release

of ACh until the motor nerve temrinal membrane was depolarized to approximately -42 mV

192

by exposure of NMJ preparations to buffers containing 20 mM KCl. Although all
measurements pertaining to membrane potentials were made from the muscle, membrane
potentials of the motor nerve terminal innervating the muscle were presumably similar.
However, this may not be entirely true— mechanisms controlling membrane potentials may vary
between muscle and nerve leading to differences in potentials between the two cell types.
Nevertheless, the small size and inaccessibility of the motor nerve terminals precludes direct
measurements, and thus, membrane potentials of the motor nerve tem1inal were assumed to be
close to those measured from the muscle.

Enhanced release of ACh by iberiotoxin was much greater following electrical
stimulation of the nerve than following Ki-induced depolarization of the nerve. This
discrepancy most likely reflects than manner in which the motor nerve terminal was depolarized.
Electrical stimulation of the nerve transiently depolarizes the motor nerve terminal membrane
to ~ 0 mV. At such positive potentials, loss of KC, channel activation would affect the open
probability of a greater number of VDCC than that observed when the membrane potential is
~ -42 mV. Thus in the presence of iberiotoxin, release of ACh is higher during electrical
stimulation of the nerve. In addition, electrical stimulation of the nerve induces a large
depolarization of the membrane that occurs rapidly and transiently, whereas, under the
conditions used in this dissertation, Ki-induced depolarization of the motor nerve terminal
produces modest and continuous membrane depolarization. During continual membrane
depolarization, involvement of a large proportion of VDCC in release of ACh may be limited
by voltage-dependent and/or other secondary channel inactivating mechanisms.

During fimctional antagonism of Kc. channels, a significant number of normally silent

193

L-type channels become involved in ACh during electrical or K*-induced nerve (at KCl
concentrations of 20 mM) stimulation. Thus, it is possible that in LEMS as the number of
functional P/Q-type Ca2+ channels decreases, activation of KC,I channels also decreases. This
decrease in activation of Kc. channels, in turn, enhances involvement of newly synthesized
and/or normally silent L-type VDCC already present at the motor nerve terminal in release of
ACh. In support of this hypothesis, it has been observed that exposure of BAC cells to LEMS
plasma not only reduces the amplitude of P/Q-type Ca2+ current, but that of KC, current as well
(Kim et al., 1998). Although it is possible that in LEMS functional KCa channels are
downregulated in manner similar to that of P/Q-type Ca2+ channels, many studies have shown
that KC, channel densities are not generaly affected (Garcia and Beam, 1996; Garcia et al.,
1996)

L-type C a2 ’ current involved in release of ACh during LEMS may be influenced directly
or indirectly by KC, channels. Loss of P/Q-type channels may decrease activation of colocalized
KCa channels, and in turn, enhance the extent and duration of global depolarization of the motor
nerve terminal membrane. Alternatively, expressed L-type VDCC at the motor nerve temtinal
during exposure to LEM S plasma may not be influenced by Kc. channels because these channels
may not be colocalized with one another. In support of the latter argument, differential
colocalization of VDCC subtypes and KCa channels has been observed at chick parasympathetic
and sympathetic nerves (Wisgirda and Dryer, 1994). L— and N— type channels are both found
on chick parasympathetic and sympathetic nerves, however, N—type channels are colocalized
with KCa channels on chick sympathetic nerves, whereas L—type channels are colocalized with

Km channels on parasympathetic nerves.

194

Based on pharmacological sensitivities, KC, channels found on mature mammalian motor
nerve terminals are most likely of the BK type and are sensitive to intracellular Ca2+ and to
depolarization as well. However, the effect of depolarization on these KCa channels is enhanced
greatly by the presence of Caz’; activation of KCal channels in the presence of reduced Ca2+ entry
as in LEMS still occurs via direct depolarization, but to a lesser extent than would normally
occur. Thus in LEMS, loss of P/Q- type Ca2+ channels most likely leads to reduced, but not
complete loss of KCa channel activation. It would therefore seem likely that in LEMS, lack of
colocalization of Kc. channels with newly synthesized L-type Ca2+ channels would have a
greater effect on L-type Ca2+ channel involvement in release of ACh than would reduced
activation of Kc. channels due to decreased Ca2+ entry through P/Q-type channels. This
hypothetical model is represented in Figure 6.2 and most likely acts in concert with the
hypothetical model depicted in Figure 6.1 to maximize involvement of L-type Ca2+ channels in
release of ACh in LEMS.

Although expression and involvement of L-type Ca2+ channels in release of ACh occurs
following passive transfer of LEMS to mice, other VDCC channel subtypes may also become
involved in release of ACh. Nerve-stimulated release of ACh from motor nerves obtained from
mice genetically altered to lack P/Q-type channels is controlled by Ca2+ entry through multiple
VDCC subtypes, including the N-type. However, based on antibody specificities in LEMS,
expression of other VDCC subtypes, particularly N-type channels, may not become apparent.
During LEMS, synthesis of newly formed N-type channels also may occur, but due to a
stronger preference of LEMS antibodies for not only P/Q-, but N- in comparison to L-type Ca2+

channels, the turnover of N-type channels may not occur fast enough to allow them to become

195

1 r"!

involved in release of ACh.

The involvement of L-type channels in release of ACh from motor nerves following
passive transfer of LEMS to mice likely occurs in humans as well. Although direct evidence
for a similar findings in humans has not been reported, indirect evidence supporting the role of
L-type Caz" channel development at motor nerve terminals in LEMS has been observed. For
example, a relationship between the development of LEMS and serum levels of the L-type
Ca2+ channel antagonist, diltiazem has been reported in a patient with myocardial ischemia
(Ueno and Hara, 1992). Additionally, use of the L-type Caz’ channel antagonist, verapamil has
been implicated to induce respiratory failure in a patient with LEMS (Krendel and Hopkins,
1986). It is likely, therefore, that patients with LEMS may benefit from therapies that
specifically target and increase currents through newly formed L-type Ca2+ channels.
However,more work will need to be done, specifically using tissues from patients with LEMS
in order to clarify this issue.

The distinct localization of individual subtypes of VDCC at motor nerve terminals
during LEMS and the resulting temporal overlap of multiple Ca2+ domains may act as a means
to maintain a short-term plasticity until more complex mechanisms can occur (Rosato—Siri,
2002). Thus, in LEMS, L-type channels may assume the role of lost P/Q-type channels in

order to maintain a given level of ACh.

196

 

Control LEMS

 

  
   

P/Q‘type channels —
L‘tYPe channels
KC, channels

 

Figure 6.2. Hypothetical model representing the role of KC, channels in LEMS at the mature
motor nerve terminal of mice. In Control environments, P/Q-type Ca2+ channels are the
primary channel subtype controlling ACh release from motor nerve terminals of mature mice.
Although Ca2+ entry through few L-type Ca2+ channels located at sites distinct from the release
apparatus occurs during membrane depolarization, the Ca2+ is buffered by intracellular
mechanisms prior to becoming involved in ACh release. Activation of KCa channels during
membrane depolarization and increasing intracellular [Ca2+] allows efflux of K’, which aids in
membrane repolarization. In turn, the probability of L-type VDCC openings and release of ACh
are decreased. In addition to K+ efflux through KC, channels, some L-type channels present at
the motor nerve terminal remain closed during membrane depolarization due to the influence
of other secondary mechanisms. In LEMS, the number of firnctional P/Q-type channels is
reduced, while expression of L-type Ca2+ channels at sites distinct fi'om the release apparatus
is increased. Furthermore, newly expressed L-type Ca2+ channels may not be colocalized with
KC, channels. During membrane depolarization, entry of Ca2+ through P/Q-type channels and
thus, subsequent activation of KC, channels is reduced. In turn, the membrane remains
depolarized for longer periods of time, which increases the duration of VDCC openings. In
conjunction with increased expression of L-type channels, entry of Ca2+ overwhelms buffering
mechanisms and participates in ACh release. However, due to buffering mechanisms and
channel localization, not all of the Ca2+ entering through L-type channels is involved in ACh
release. Adapted and modified from Smith et al., 1995.

197

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