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FUNCTIONAL AND PHARMACOLOGICAL
CHARACTERIZATION OF SIXTY-FOUR PARA SODIUM
CHANNEL VARIANTS FROM DROSOPHILA
MELANOGASTER IN XENOPUS OOCYTES

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2/05 p2/CIRC/DaIeDue.indd-p.1

 

FUNCTIONAL AND PHARMACOLOGICAL CHARACTERIZATION OF
SIXTY-FOUR PARA SODIUM CHANNEL VARIANTS FROM DROSOPHILA
MELANOGASTER IN XENOPUS OOCYTES
By

Rachel Lee O’Donnell Olson

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Entomology

2006

ABSTRACT

FUNCTIONAL AND PHARMACOLOGICAL CHARACTERIZATION OF SIXTY-
FOUR PARA SODIUM CHANNEL VARIANTS IN XENOPUS OOCYTES

By

Rachel Lee O’Donnell Olson

Extensive alternative splicing and RNA editing of para, the voltage-gated
sodium channel of Drosophila melanogaster, has been documented. However,
the functional consequences of these post-transcriptional modifications have not
yet been investigated. In this study we report the isolation, expression, and
functional Characterization of sixty-four full-length para CDNA clones. The sixty-
four clones were grouped into twenty-nine splice types based on alternative exon
usage. Functional analysis of these variants revealed a broad spectrum of
voltage-dependences of activation and to a lesser extent, voltage dependences
of inactivation. Sequence comparison of three functionally distinct variants
uncovered novel A to I editing sites. Furthermore, differential sensitivities to an
a-scorpion toxin, thorlT, were revealed. The observed functional and
pharmacological diversity of para variants supports the notion that alternative
splicing and RNA editing are the two major mechanisms for generating sodium

channel diversity in insects.

ACKNOWLEDGMENTS

I would like to express my sincere thanks to Dr. Ke Dong for providing me
with this opportunity, her guidance, support, and encouragement throughout the
course of my master's research.

I would also like to thank Dr. Robert Hollingworth, Dr. Suzanne Thiem, and
Dr. Edward Walker for their guidance and support.

Appreciation is also extended to the members of Dr. Ke Dong’s laboratory.
I would like to thank Dr. Weizhong Song and Dr. Yuhze Du for teaching me
electrophysiological techniques, Mrs. Yoshiko Nomura for her assistance and
instruction in molecular biology techniques, and Dr. Zhiqi Liu for cloning sixty-four
para variants providing the foundation for my thesis work. A special thanks is
also extended to Dr. Kristopher Silver for his critical review of my thesis.

I would finally like to express my appreciation for the support and
encouragement I received during the course of this research from my husband

Rory Olson, and my parents Ruth and Jim O’Donnell.

iii

TABLE OF CONTENTS

List of Tables and Figures for Chapter 1 .............................................. vi

List of Tables and Figures for Chapter 3VII

Chapter 1: Literature Review .................................................................... 1
|. Discovery of Sodium Current .................................................... 1
ll. Isolation of the Sodium Channel Protein ..................................... 1
III. Molecular Biology of the Sodium Channel ................................... 2
“LA Cloning of the First a—Subunit cDNAs ......................................... 2
"LB Functional Expression of the or-Subunit ....................................... 2
INC The Molecular Basis of Sodium Channel Function ........................ 3
|||.C1 Outer and Inner Pores, Selectivity Filter ...................................... 3
|||.CZ S4 Segments; Activation ......................................................... 4
|||.CS IFMT Motif; Inactivation ........................................................... 5
IV. Mammalian Voltage-Gated Sodium Channels .............................. 7
WA Tissue-Specific Expression ...................................................... 7
NB Developmental-Specific Expression ........................................... 8
WC Unique Electrophysiological Gating Properties of

Mammalian VGSCs ................................................................ 8
V. Insect Voltage-Gated Sodium Channel ....................................... 9
VA Isolation of the Drosophila Voltage-Gated Sodium Channel Gene....10
V.B Conservation of Alternative Splicing of the Insect VGSC Genes ...... 13
V.B1 D. melanogaster ................................................................... 13
v.32 D. vin’lis ............................................................................... 14
was Musca domestica ................................................................... 15
V.B4 Blattella germanica .................................................................. 15
V.C Alternative Splicing Generates Distinct Gating Properties of

Insect Voltage-Gated Sodium Channels ..................................... 16
VD RNA Editing Generates Diversity in IVGSCs ............................... 19
V.E Electrophysiological Analysis of RNA Edited Variants of BgNav ....... 20
VI. Pharmacology of Voltage-Gated Sodium Channels ....................... 21
Chapter 2: Specific Aims, Rationale, and Significance ........................... 25-26

Chapter 3: Functional and Pharmacological Characterization of
Sixty-Four Para Sodium Channel Variants in Xenopus

Oocytes ........................................................................ 27

Abstract ........................................................................................ 27
Introduction ................................................................................... 28
Materials and Methods ..................................................................... 29
Synthesis of First Strand cDNAs ............................................... 29

PCR and Cloning of para full-length cDNAs ................................. 29

iv

Expression of para channels in Xenopus Iaevis oocytes ................ 30

Electrophysiological Recording and Analysis ............................... 31
thaIT sensitivity assay ........................................................... 32
Results ......................................................................................... 33
Molecular Analysis of 64 full-length CDNA clones ......................... 33
Functional Expression of 64 Variants in Xenopus oocytes .............. 33
Sequence Analysis of Three Functionally Distinct Variants ............. 34
Examination of the sensitivity of Para variants to thalT ................ 39
Examination of the senstitivities of Para variantsto thalT ...................... 40
Discussion .................................................................................... 43
Functional Versus Non-Functional Para Variants .......................... 43
Identification of 29 para Splice Types ......................................... 45
Frequency of alternative splicing of insect sodium channels. . .. ........45
Identification of Three Novel RNA Editing Sites ........................... 46
Identification of High-Voltage Activated and Low-Voltage
Activated Para Variants ................................................. 48
Identification of an thaIT-resistant Para variant .......................... 50
Literature Cited .............................................................................. 51

LIST OF TABLES AND FIGURES FOR CHAPTER 1

Figure 1.

Table 1.

Table 2.

Figure 2.

Table 3.

Table 4.

Topological transmembrane organization of the
voltage-gated sodium channel ......................................... 4

Tissue-specific and developmental-specific expression

patterns of the nine mammalian voltage—gated sodium

channels .................................................................... 9
Gating properties of nine mammalian SCs .......................... 10
Topology of the insect SC containing positions of axons. . 14
Alignment of the predicted amino acid sequences for

optional exons a, b, e, f, h, i, and j and of mutually

exclusive exons old and k/l ............................................. 16-17

Neurotoxins that act on sodium channels ........................... 22

vi

LIST OF TABLES AND FIGURES FOR CHAPTER 3

Figure 1.
Table 1.
Figure 2.
Table 2.
Table 3.

Figure 3.

Figure 4.

Figure 5.

Table 4.

Table 5.

Identification of 29 Splice Types ....................................... 35
Activation and inactivation properties of 32 variants ............ 36
Amino acid changes in variants 13, 18, and 50 .................... 38

Amino acid changes observed in variants 13, 18, and 50 ....... 39

Effects of 1 nM thdIT on para variants ............................ 40
Peak sodium current traces of variant # 8 before and after

application of thalT ..................................................... 41
Dose response curves of variants 8 and 21 ........................ 41

Amino acid changes in variants 8 and 21 compared to

GenBank # M32078 ...................................................... 42
Sequence comparison of variants 8 and 2143
Exon frequencies of Para, VSSC1, BgNav ......................... 47

vii

CHAPTER 1: Literature Review
I. Discovery of Sodium Current

Action potentials are electrical signals by which excitable cells, such as
neurons, receive, conduct, and transmit information. In a series of elegant
experiments, Alan Hodgkin and Andrew Huxley demonstrated that action
potentials are the result of ions moving through the plasma membrane (Hodgkin
and Huxley, 1952 a, b). Furthermore, they established sodium as one of the ions
responsible for the production of action potentials and hypothesized that sodium
selective pores activate (open) and inactivate (close) in response to changes in
membrane potential. Because of their significant discovery the Nobel Prize was
appropriately awarded to Alan Hodgkin and Andrew Huxley.
ll. Isolation of the Sodium Channel Protein

The proteins that comprise the voltage-gated sodium channel (SC) have
been purified from a variety of tissues and species (review by Catterall, 2000).
Using the high affinity binding of the SC protein to a radioactive-labeled
tetrodotoxin, the protein was first purified from the electric organ of the eel,
Electrophorus electricus (Agnew et al. 1980). This purified SC consists of a
single polypeptide of an approximate weight of 270 kDa. Subsequent photo-
affinity labeling experiments using a sodium specific neurotoxin, a-scorpion toxin,
led to the discovery of two peptides (36 and 260 kDa) from synaptosomal
membranes of the rat brain. The isolated peptides are denoted as a (primary)
and B (auxiliary) subunits of the SC. The a polypeptide corresponds to the 270

kDa polypeptide isolated from eel (Beneski and Catterall, 1980).

Ill. Molecular Biology of the Sodium Channel

With the development of molecular biology techniques, our understanding
of the molecular biology of voltage-gated sodium channels has greatly advanced.
The major achievements and discoveries are summarized below.
III. A Cloning of the First (Jr-Subunit cDNAs

Sodium channel oz-subunit cDNAs were isolated from electric eel by
screening expression libraries of electroplax eel mRNA using oligonucleotides
encoding the electric eel SC and antibodies against the eel SC protein (Noda et
al., 1984). Sequencing the entire polypeptide from the deduced cDNAs revealed
the primary amino acid structure of the SC. The secondary structure of the
a—subunit was determined to have four homologous domains (Dl — DIV), each
consisting of six transmembrane segments (S1 - S6) (Noda et al., 1984; review
by Catterall, 2000).
III. B Functional Expression of the (Ir-Subunit

Expression of the pore-fonning a-subunit alone generates sodium currents
in Xenopus oocytes and mammalian cell lines (Noda et al., 1986). However,
channel gating and expression are modulated by the inclusion of 3 subunits and
result in emulating channels in vivo (reviewed by Catterall, 2000). A total of four
3 subunits have been identified in mammals (B1, B2, 83, & 34) (Yu et al., 2003).
These subunits are hypothesized to function as cell adhesion molecules involved
in cell-to-cell interactions. It is important to note that the expression of oz and 3

subunits varies between tissues (reviewed by Isom, 2001). For example, the

skeletal muscle sodium channel is a complex of a and B1 subunits, whereas, the
mammalian brain is comprised of a, B1, and 32 subunits.
III. C The Molecular Basis of Sodium Channel Function

Molecular mechanisms pertaining to the structure-function relationship of
mammalian SCs have been and are currently under investigation. Below is our
current understanding of the molecular basis of SC function.
Ill. C1 Outer and Inner Pores, Selectivity Filter

The outer pore (extracellular opening) and selectivity filter (amino acids
that confer the channel to be sodium selective) of the channel is formed by the
membrane re-entrant loops connecting segments 5 and 6 of domains l-lV
(Catterall, 2000). The neurotoxins tetrodotoxin ('l'l'X) and saxitoxin (STX) block
the SC by binding to the outer pore and thereby preventing sodium ion entry into
the cell (Hille, 1984). A pair of amino acids located in each reentrant loop was
associated with TTX and STX binding along with forming the selectivity filter
(Hille, 1984; Terlau et al., 1991 ). The outer most set of amino acids, EEDD, form
the outermost ring of the channel and serve as the primary binding site of these
neurotoxins, which led to the identification of the outer pore (Fig. 1). The amino
acid residues DEKA form the selectivity filter in the innermost part of the outer
pore (Fig. 1; Terlau et al., 1991). Mutating DEKA to EEEE causes the SC to
become permeable to calcium ions (Heinemann et al., 1992). The inner pore
(intracellular portion) of the SC is proposed to be formed by the 86 segments
(Fig. 1; Striessnig et al., 1990), which is based on evidence that local anesthetics
(LAs) enter and bind to residues in the S6 segments and block the pore from the

intracellular side (Hille, 1984).

      

    

Inactivation Inner
Particle Pore

Figure 1 A-B Topological transmembrane organlzation of the voltage-gated
sodium channel. A. The a—subunit of the SC consists of four homologous domains (DI-
DIV) each containing six transmembrane segments (S1-$6). Voltage-sensors are
formed by the S4 segments in each domain (grey). The outer pore and selectivity filter
are formed by the linker connecting 85-86 of all four domains (striped). The sodium-
selective-filter is formed by the amino acids DEKA. The inactivation particle containing
the IFMT motif is located on the D3-D4 loop. The inner pore is formed by the
arrangement of the segments 5 and 6. B. Schematic transmembrane arrangement of
Dl-DIV of the a-subunit forming the channel pore. The linker connecting 85-86 of each
domain form the outer pore and contain the amino acids DEKA (top view). The S6
segments form the inner pore and are depicted by the bottom portion of the $6 cylinders
being tilted inwards.

lll. C2 S4 Segments; Activation
A depolarization of the membrane electric field leads to activation of the
sodium channel (Hodgkin and Huxley 1952a). Each S4 segment contains four to

eight positively charged arginine or lysine residues separated from each other by

two neutral residues. These positively charged S4 segments serve as a voltage
sensor and respond to potential changes across the plasma membrane (Fig. 1;
Noda et al. 1984). Two comparable models have been proposed to explain the
mechanism underlying activation; the sliding helix and the helical screw
(Catterall, 1986; Guy and Seetharamulu, 1986). Both models assume that the
positive residues of the S4 segments form ion pairs with negatively charged
residues located in neighboring segments. The binding of ion pairs are released
upon membrane depolarization, causing a conformational change of the channel
resulting in opening of the pore (Catterall, 1986; Guy and Seetharamulu, 1986).
Mutating the positive residues of the voltage sensor to neutral residues altered
the voltage-dependency of gating, providing strong evidence for the two
proposed activation models (Stuhmer et al., 1989; Kontis et al., 1997). The
outward movement of the S4 segments in response to depolarization was
detected in an elegant set of cysteine scanning mutagenesis experiments, further
establishing the role of S4 as voltage sensors in sodium channels (Refs. in
Catterall, 2000).

III. C3 The IFMT Motif; Inactivation.

Inactivation of the sodium channel occurs within a few milliseconds after
activation, closing the channel pore (Ulbricht, 2005). Site-directed antibody and
site-directed mutagenesis studies revealed that the short intracellular linker
connecting DIII to DIV plays an integral role in fast inactivation (Fig. 1) (Refs. in
Catterall, 2000). A contiguous hydrophobic triad of isoleucine, phenylalanine,
and methionine (IFM) positioned in the middle of this linker are crucial for fast

inactivation (West et al., 1992). Site-directed anti-peptide antibodies to this linker

(Vassilev et al., 1988, 1989), deletions of amino acids from this loop (Patton et
al., 1992), as well as mutating the IFM triad to glutamate (QQQ) inhibited
inactivation (West at al., 1992). Additional experiments demonstrated that
peptides containing the IFM motif serve as pore blockers to inactivation deficient
channels and completely restore inactivation, providing further evidence
supporting the significant role of the IFM motif in inactivation (Eaholtz et al.,
1994; McPhee 1998; Eaholtz et al., 1999). Recent experiments indicate that
there is a fourth amino acid, threonine, in addition to the IFM triad, that is
essential for fast inactivation (IFMT) (Rohl et al., 1999).

Inactivation occurs with the occlusion of the pore resulting in the block of
further ion entry into the cell (review by Goldin, 2003). The small linker
connecting Dlll to DIV (containing the IFMT motif) is responsible for occlusion of
the pore by binding to hydrophobic residues near the intracellular portion of the
channel. Scanning mutagenesis experiments indicate that the docking site of the
IFMT motif consists of the 84-85 linkers of DI" and DIV along with the
cytoplasmic portion of DIVSB. It is hypothesized that the conformational change
ensued by the S4 segments result in the exposure of the hydrophobic residues
contained in the 84-85 segments which then interact with the IFMT motif, looking
it into place.

Binding of the IFMT motif to its docking site is thought to be facilitated by
glycine and proline residues that flank the short intracellular linker connecting Dlll
to DIV. Glycine residues confer flexibility in protein structure as proline residues
offer structural rigidity and a bent conformation to the linker (refs. in Zhao et al.,

2004). The flexibility of glycine and the structural rigidity of proline serve as‘

‘molecular hinges’ for the small linker containing the IFMT motif. Thus, the linker
connecting DIII to DIV has been described as a ‘hinged lid’ with the IFMT motif
acting as a ‘latch’, locking the lid into place by binding to the intracellular portion
of the pore and preventing further ion entry into the cell (described as a ‘hinged
lid’ model; review by Catterall, 2000).
IV. Mammalian Voltage-Gated Sodium Channels

Voltage-gated sodium channel genes have been identified in various
vertebrates and invertebrates such as humans, rats, leaches, fish, and several
species of insects (review by Goldin, 2002). Nine different SC a-subunit genes,
Nav1.1 — Nav1.9, have been identified in mammalian systems (Goldin et al.
2000; Catterall et al. 2003). These nine mammalian SC proteins share greater
than 50% amino acid homology (Goldin et al. 2000). These 803 are expressed
in different tissues and cell types and are localized in different sub-cellular
compartments. Each has unique gating properties thought to fulfill distinctive
roles in a given cell type. Mutations in the SC genes have been associated with
various mammalian diseases including myoclonic epilepsy of infancy and
hyperkalemic periodic paralysis (Claes et al., 2001; Kelly et al., 1997).
NA Tissue-Specific Expression

The nine isoforms of the mammalian SC exhibit distinct tissue distribution
patterns. Transcripts of most isoforms are present in many different tissues,
however," the abundance of each isoform is profound in only select tissues
(review by Goldin, 1999). lsofonn Nav1.1 is abundantly expressed in the central
nervous system (CNS) along with Nav1.2 and Nav 1.3. Additionally, Nav1.1 and

Navt .3 are localized to the somas of neurons, while Navt .2 was detected in the

nodes of Ranvier and in unmyelinated axons. Nav1.4 is specifically expressed in
skeletal muscle and Nav1.5 is independently expressed in cardiac myocytes.
lsoforrn Nav1.6 is most abundant in the CNS in comparison to the peripheral
nervous system (PNS) but is also detected in dorsal root ganglia (DRG) neurons.
Nav1.7 is mainly expressed in the PNS, being present in DRG neurons, Schwann
cells, and in neuron-endocrine cells. The last two isoforms, Nav1.8 and Nav1.9,
have been identified in the PNS DRG neurons and trigeminal ganglia.
IV.B Developmental-Specific Expression

Besides tissue-specific expression patterns, developmental specific-
regulation of mammalian 805 have been documented for seven of the nine
isoforms (Nav1.1 - Nav1.7; review by Goldin, 1999). Nav1.1 becomes detectible
shortly after birth and increases in number until adulthood. Nav 1.2 is
accumulated in embryonic nodes of Ranvier in some CNS tracts. The
transcription level of isoform Nav1.3 peaks at birth and becomes undetectable in
adulthood. Transcripts of Nav 1.5 are detectable in neonatal skeletal muscle but
are replaced by Nav 1.4 in adult skeletal muscle. Nav 1.6 is the most abundantly
expressed isoform found in the CNS during adulthood, but, is expressed in lower
numbers during development. Transcripts of Nav 1.7 are located in the neonatal
cortex of the CNS The remaining two isoforms, Nav 1.8 and Nav 1.9, have not
yet been described to display developmental-specific expression patterns (review
by Goldin, 1999).
MC Unique Electrophysiological Gating Properties of Mammalian $08

The nine SC variants in mammals exhibit significant differences in

functional properties. These differences likely contribute unique functional roles

in distinct tissues or cell types. Specifically, the voltage required for activation
and inactivation varies among the isoforms (Table 2).

Table 1. Tissue-specific and developmental-specific expression patterns of
the nine mammalian voltage-gated sodium channels

 

 

SC Primary Tissue Developemental
Type Distribution Profile
Nav1.1 CNS adult
Nav 1.2 CNS adult
Nav 1.3 CNS fetus
Nav 1.4 skeletal muscle adult
Nav 1.5 heart muscle adult
Nav 1.6 CNS adult
Nav 1.7 PNS neonatal
Nav 1.8 PNS

Nav 1.9 PNS

 

Table modified from Goldin, 1999
V. Insect Voltage-Gated Sodium Channel

It is important to study insect 80$ for three key reasons. First, 803 are
targeted sites of several Classes of insecticides, including DDT, pyrethroids, and
indoxacarb (Soderlund, 2005). These insecticides are widely used in the control
of agricultural and medically related pest species. However, resistance to current
insecticides is a global problem. Mutations in the SC gene have already been
shown to confer resistance to pyrethroids and DDT (Soderlund, 2005). Second,
as presented below, the insect SC gene undergoes extensive post-transcriptional
modifications including alternative splicing and RNA editing events. At the
present time, we do not understand the contributions of alternative splicing and

RNA editing in the regulation of neuronal excitability. Third, though there is great

similarity in the structure-function relationships between the insect and
mammalian channels, pharmacological differences have been reported (Cestele
and Catterall, 2000). Thus, by better understanding the molecular basis of
differential sensitivities of insect and mammalian $03 to various neurotoxins,
valuable information will be provided toward the development of safe and
selective insecticides.

Table 2. Gating properties of nine mammalian SCs
lsoforrn Activation (mV) Inactivation (mV) Notes

 

 

Nav 1.1 -33 -72
Nav 1.2 -24 -53
Nav 1.3 -23 to -26 -65 to -69
Nav 1.4 -26 -50.1 or -56
-30 rat
Nav 1.5 -47 or -56 -84 or -100 F as the major anion in
intracellular solution
-27 -61 aspartate as the major anion in
intracellular solution
Nav 1.6 -8.8 -55 cut-open oocyte clamp; mouse
-17 -51 “ “ + B1& B2
-26 “ “ + inactivation removed
-37.7 -97.6 macropatch clamp; rat
Nav 1.7 -31 macropatch; rat
-45 -65 TTX sensitive DRG neurons
-78 two-electrode clamp; rat
-60.5 whole-cell clamp
-39.6V whole-cell clamp (+81)
Nav 1.8 -16 to -21 ~-3O DRG neurons; rat
Nav 1.9 -47 to -54 -44 to -54 DRG neurons; rat

 

Modified from Catterall, Goldin, and Waxman (2005).

VA Isolation of the Drosophila Voltage-Gated Sodium Channel Gene
In 1987 the Qrosophila sodium channel 1 (DSC1) was isolated by

screening a Drosophila genomic library with a CDNA probe encoding the eel SC.

10

The deduced amino acid sequence of DSC1 revealed high homology to that of
the previously characterized vertebrate SCs. The secondary structure of DSC1
was deduced to contain four homologous domains, each containing six
transmembrane segments like that of mammalian SCs. Importantly, the S4
segments of each domain of the 0801 channel also contained positive residues
separated by two uncharged resides resembling the ‘voltage sensors’ described
in mammalian sodium channels (Salkoff et al., 1987).

In the same year, 1987, Ganetzky and colleagues isolated a second SC gene,
para, by analyzing a temperature-sensitive paralytic phenotype of Drosophila
mutants. These paralytic temperature-sensitive mutant flies undergo
instantaneous paralysis when temperatures are elevated to 29°C but fully
recover when restored to ambient temperature (22°C) (Suzuki et al. 1971; Siddiqi
and Benzer, 1976). The observed paralysis was hypothesized to be the result of
a temperature sensitive blockade of action potential conduction (Siddiqi and
Benzer, 1976; Wu and Ganetzky, 1980). This para gene was isolated using P-
element transposon tagging (Loughney et al., 1989). The complete Para protein
was deduced from six overlapping CDNA clones and revealed four homologous
domains like that described of other 80s as well as being highly homologous in
the transmembrane regions (60%) to those of the rat brain channel (Loughney et
al., 1989). In an independent study, the D. melanogaster genomic library was
searched for sequences similar to rat brain 803. This study resulted in the
isolation of partial cDNAs corresponding to para and DSC1, suggesting that there

are two genes encoding SCs in insects (Ramaswami and Tanouye, 1989).

11

In situ hybridization and immunochemistry studies revealed distinct
tissue and developmental distribution patterns of para and DSC1. Using para
and DSC1 CDNA restriction fragments as probes, the expression patterns of para
and DSC1 transcripts were analyzed in in-situ hybridization experiments. The
results of this study indicate that para is ubiquitously expressed throughout the
nervous system in all developmental stages, while DSC1 is detected in all stages
except that of the embryonic stage (Hong and Ganetzky, 1994). Additionally,
para is expressed throughout the CNS and PNS while DSC1 is restricted to a few
cells in either system (Hong and Ganetzky, 1994; Castella et al., 2001 ).

When functionally expressed the para transcripts were found to carry
the inward voltage-dependent sodium current (Genneraad et al., 1992).
Functional analysis of Para in Xenopus oocytes indicates that the biophysical
properties of the Para channel are similar to those of mammalian SCs (Warmke
et al., 1997). Also, various toxins that act on mammalian SCs also act on Para,
examples include permethrin and deltamethrin (Vais et al., 2001).

An ortholog of DSC1, BSC1, was cloned from the German cockroach, B.
gennanica. The sequence homology between BSC1 and DSC1 is high, 81%,
78%, 80%, and 88% in domains I, II, III, and IV, respectively (Liu et al., 2001).
The functional expression of BSC1 in Xenopus oocytes revealed that the BSC1
channel is more permeable to Ca2+ than to Na+ and exhibits unique gating
properties different from those of sodium channels, indicating that BSC1 encodes
a Cay-selective cation channel (Zhou et al., 2003, Liu et al., 2001). The
functional analysis of DSC1 has not yet been published, however, personal

communication with Dr. W. Song indicates that the DSC1 Channel is also

12

calcium-selective and possesses gating properties similar to those of the BSC1
channel (Dr. W. Song, a postdoctoral associate in Dr. K. Dong‘s laboratory). In
summary, DSC1 does not encode a sodium channel, and para is the only SC
gene in insects.

V.B Conservation of Alternative Splicing of the Insect Sodium Channel
Genes

Because of the involvement of SC mutations in pyrethroid resistance in
many important insect and arachnid pest species, CDNAs (partial or full-length) of
the SC gene have been isolated from many arthropod species (Soderlund,
2005). However, alternative splicing and RNA editing of the insect SC has
mainly been studied in four insect species: D. melanogaster, D. virillis, Musca
domestica, and Blattella gennanica.

V.B1 D. melanogaster:

As mentioned previously, there exists a single SC gene, para, in D.
melanogaster (Loughney et al., 1989; O’Dowd et al., 1989). Alternative splicing
of para has been proposed to be the key mechanism influencing SC diversity
(Loughney et al., 1989; O’Dowd and Aldrich, 1988; O’Dowd et al., 1995;
Thackeray and Ganetzky, 1994, 1995; Warrnke et al., 1997). Seven alternative
exons (a, b, e, f, h, i, j) and four mutually exclusive exons (old and k/l) have been
identified (Loughney et al., 1989; O’Dowd et al., 1995; Thackeray and Ganetzky,
1994, 1995; Warrnke et al., 1997; Lee et al., 2002; Tan et. al., 2002a). The
secondary structure of the insect SC along with the positioning of alternative

splicing is presented in Figure 2.

l3

In D. melanogaster alternative exons 0, e, and f have been shown to be
developmentally regulated. The percentage of transcripts in embryos containing
exons 0, e, and fwere 1.7%, 3.4% and 87%, respectively, but changed to 24%,

28% and 6.7%, respectively in adults (Thackeray and Ganetzky, 1994).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

It 3 l

j i a
Figure 2. Topology of the Insect SC containing positions of exons. Positions of
sequences encoded by optional exons a, b, e, f, and h as well as mutually exclusive
exons c and d in the sodium channel protein are highlighted in colored boxes. The
letters below the boxes are the exons to which the boxes belong.

d/c f h l/k

V. 32 D. virilis

The alternative exons of para identified in D. melanogaster are also found in
D. virilis (Thackeray and Ganetzky, 1995). These alternative exons are highly
conserved in amino acid sequence and are presented in Table 2 below. The
protein sequence identities of the entire coding regions of D. virilis and D.
melanogaster are 98.8% homologous, while the alternative exons are 99.5%
homologous in amino acid structure (Thackeray and Ganetzky, 1995). Exons c,
e, and f of D. virilis are developmentally regulated in a similar fashion to D.
melanogaster. D. vin'Iis embryos contain 0%, 9.2% and 70.2% exons 0, e, and f,
while adults contain 17.7%, 25%, and 11.3% respectively (Thackeray and

Ganetzky, 1995). It was determined that 75% of D. melanogasater embryos and

I4

57.3% of D. virilis embryos share the same exon usage; a+, b+, d, e-, f+ or a-,
b+, d, e-, f+ (Thackeray and Ganetzky, 1994, 1995).
V.B3 Musca domestica
Like that of D. melanogaster and D. virilis, the Musca domestica (house

fly) SC, Vssc1 (Voltage-sensitive sodium channel), contain exons a through h. In
addition to these exons, two novel mutually exclusive exons k and l were
identified in Vssc1. By searching the reported para sequence and performing a
BLAST search exons k and I were subsequently identified in para (Lee et al.,
2002). Exons b, f, h, and j of VSSC1 are identical in amino acid structure to
Para, while exons a, d, e, and i have less than five changes and exon c has eight
changes (Lee et al., 2002). Interestingly the regulation of alternative exons
between D. melanogaster and M. domestica species is similar. Exons a, b, c, d,
e, f, and i show comparable exon frequencies among the transcripts (Lee et al.,
2002). The frequencies of exons h, j, k, and I among para transcripts have not
yet analyzed.
V.B4 Blattella germanica

The cockroach SC, BgNav, is 78% and 76% identical in amino acid
framework to Para and Vssc1 respectively (Dong, 1997). When comparing
alternative exon sequences between D. melanogaster, D. virilis, M. domestica,
and B. gennanica, the sequences from B. germanica are quite divergent (refer to
Table 3). Exon a of BgNav contains fewer amino acids in comparison to exon a
of para,, exon d is missing, and exons e, f, and i of BgNav variants contain more

amino acid changes than D. virilis, and M. domestica when compared to para. It

15

is interesting to note that the three fly sequences are more closely related to
each other than with the cockroach sequence.

Three mutually exclusive exons have been identified in BgNav; G1, GZ,
and G3. G1 is equivalent to exon l of para, G2 corresponds to exon k of para
and GS is unique to BgNav, having not been identified in Para or other insect
SCs. Exons Gt, GZ, and G3 of the cockroach are abundantly expressed in
embryonic stages II and Ill, but were found in much lower frequencies in adults.
Exon G1 is localized to the nerve cord, coxa and the remaining leg (femur, tibia,
and tarsus). Exon G2 is the same as G1 but is expressed in the ovary and gut.
Exon G3 is localized to the coxa and the remaining leg (Tan et al., 2002a).

V.C Alternative Splicing Generates Distinct Gating Properties of Insect
Voltage-Gated Sodium Channels

Studies on mammalian 803 show that phosphorylation of protein kinase A
(PKA) sites in the cytoplasmic linker connecting DI and DH modulates channel
function (references in O’Dowd et al., 1995). The functional role of alternative
splicing in para was initially investigated by O’Dowd and colleagues (O’Dowd et

al., 1995).

Table 3. Alignment of the predlcted amino acid sequences for optlonal exons a, b,
e, f, h, i, and j and mutually exclusive exons cld and kll. Asterisks represent
sequence homology and gaps were introduced for optimal alignment (dashes). The
para and Vssc1 amino acid sequences were obtained from Lee et al., 2002, and
correspond to GenBank accession #s M32078 and X96668, respectively. The D. virilis
sequences (Thackeray and Ganetzky, 1994) corresponds to GenBank accession #s
U26343 and U26722. The cockroach sequences were obtained from Song et al., 2004
and Tan et al., 2002, and correspond to the GenBank sequence U73583. Exon d has not
been identified in BgNav (Personal communication with Mrs. Nomura in Ke Dong's
laboratory).

16

Table. 3 Amino acid sequence alingent of optional exons

 

he
0

Pan!
VCscf
Duflfifis
BgNav
Pan!
Vksc1
DuWfifis
BgNav
Pan:
VCsc1
Dufififls
BgNav
Pant
thwc1
Duflfifis
BgNav
Pan!
VCscf
Duflflfis
BgNav
Pan!
vssc1
Dufififis
BgNav
Fan!
V%301
Duflflfis
BgNav
Pant
Vksc1
Dufififis
BgNav
Pan!
Vksc1
Duflfifis
BgNav
Pan:
V%sc1
Duflfifls
BgNav
Pan:
VCscf
Duflfifls
BgNav

TSLSLPGSPPNLRRGSRSSHK

***********I*********

*********************

A*****(a) [truncated exon]
VSVYYPPT

********
********
**I*****
LRVFKLAKSWPTLNLLISIMGRTVGALGNLTFVLCIIIFIFAVMGMOLFGXNYTD
*************************************************VFY8VT
*******************************************************
*****************************************************y*
LRVFKLAKSWPTLNLLISIMGRTMGALGNLTPVLCIIIPIFAVMGMQLFGKNYHD
****it***********************************************I*

*‘k***‘k*'k***********************************************

GBRTNQISWINSB

***I*********

***I*********

**_-**3*-swx*
-GKGVCRCISA

-**********

-**********

DARE—RDTDLDLT
HIGHSINHQDNRLBHILNGRGLSIQ
******ag**********n******

******************n******

VSVI-Q-RQPAPTTAHQAT-KVR-KV8T
****-*_********p-**_***-****
****-*-********p-**-***-****
VP*F*DTKTA*KS*F*FA¥QRNLVK
PRYGRKKKQKB

***********

***********

GDF*****K**
L8LINLAKVWSGADDVPAIRSNRTLRALRPLRKVSRWBGMK

******V*****LN*IKV***********************
********I*A**A*I************************R «32)
VSLINPVISLVGAGGIQAIKTNRTLRALRPLRANBRNDGIR

*******'k*iA'k'k****************************

***************************************** (G1)

 

l7

O’Dowd and colleagues focused their study on exons a and i located
along the same linker previously described to have PKA sites in mammalian
SCs. Analysis of sodium current density in cultured embryonic neurons
suggested that the presence of exon a was necessary for detection of sodium
currents. They speculated that sodium current density is regulated by
phosphorylation events because of a potential PKA site in exon 3 (O’Dowd et al.,
1995). However, a later study investigating the role of exon a using Xenopus
oocytes showed that the presence or absence of exon a did not alter expression
of the sodium current (Warmke et al. 1997). To date, the functional consequence
of exon a remains elusive.

The functional importance of exon b has been examined in BgNav
transcripts using the two-electrode voltage clamp system (Song et al., 2004).
Deletion of exon b greatly enhanced the amplitude of sodium currents, whereas
the inclusion of this optional exon dramatically reduced the amplitude of sodium
currents in Xenopus oocytes. Interestingly, exon b contains a consensus
sequence for phosphorylation that is hypothesized to serve as an on-off switch
regulating neuronal excitability (Song et al., 2004).

Another study of cockroach SCs looked into the functional consequences
of mutually exclusive exons Gt, 62, and G3. Distinct gating and
pharmacological properties were Observed for transcripts containing G1 and G2,
however, transcripts containing G3 were non-functional due to the addition of a
stop codon. Transcripts containing exon G1 activate and inactivate at more
hyperpolarized membrane potentials in comparison to transcripts containing exon

GZ. Channel sensivity to deltamethrin is greatly increased with the inclusion of

18

G1. Transcripts containing exon G2 have a 10-fold reduced sensitivity to
deltamethrin than those containing Gt (Tan et al., 2002b).
V.D RNA Editing Generates Diversity in Insect Sodium Channels

RNA editing along with alternative splicing has been shown to generate
molecular diversity in insect SCs. RNA editing is an important post-
transcriptional modification that generates site-specific alterations of amino acids,
in addition to insertions and deletions (references in Palladino et al., 2000a). A to
l RNA editing is the result of deamination by the enzyme Adar (adenosine
deaminase acting on RNA). This editase converts A to I through a process of
hydrolytic deamination and has been reported to edit the SC in Drosophila
melanogaster. Ten A to I RNA editing events have been identified in Drosophila
para transcripts, and it appears that the frequency Of editing events dramatically
increase with age (references in Palladino et al., 2000a).

D. melanogaster mutants lacking Adar exhibit a variety of behavioral defects,
providing profound evidence in support of the significance of A to I editing.
Mutant larvae appeared normal in both locomotion and behavioral assays, while
adults exhibit severe locomotion deficits and altered behavioral patterns.
Abnormal behavioral phenotypes became increasingly acute with age. Tremors,
strange body posture, slowness, difficulty mating, balance along with inefficient
righting abilities, and circling behavior persistently worsened with age. Analysis
of brain structure revealed brain lesions that intensified with age. Analysis of
larval brains appeared normal while abundant lesions were present in adults.

The extensive locomotive and behavioral defects seen in these mutants suggest

19

that ADAR editing events in the nervous system are necessary to perform normal
function (Palladino et al., 2000b).

In the German cockroach, B. gerrnanica, A to l and U to C RNA editing of
BgNav has been reported. Similar to that of alternative splicing, tissue-specific
and developmental-specific regulation of these RNA editing events has also been
documented (Song et al., 2004). Two described U to C RNA editing events in
BgNav undergo tissue-specific regulation; a leucine to proline change in DIIIS1
and a valine to alanine change in DIVS4. The tissues of ovary, gut, nerve cord,
and leg were analyzed and both changes were found to only occur in the first two
tissues listed (Song et al., 2004). Enzymes that catalyze or mediate U to C
editing have not yet been identified. Also, an A to l editing event resulting in an
isoleucine to methionine change in DIVS4 is regulated in a tissue specific
manner. Transcripts of this A to I editing event were isolated from nerve cord
and leg (Song et al., 2004).

In addition to tissue-specific regulation, the A to l editing of DIVS4 is also
developmentally regulated. Only the unedited transcript was found in the
embryonic stage I while both edited and unedited were detected In two
embryonic (II and III) and two immature (nymph l and II) stages.

V.E Electrophysiological Analysis of RNA Edited Variants of BgNav

RNA editing of BgNav along with alternative splicing produces 803 with
distinct gating properties (Tan et al., 2002a; Liu et al., 2004, Song et al., 2004).
Changes in current, activation, and inactivation are among some of the obvious
results of RNA editing events. Examples include a U to C modification resulting

in an F to S change at the C-terrninal domain that was found to impair

20

inactivation resulting in persistent current. It is speculated that the C-terrninal
domain interacts with the IFM motif or docking receptor (discussed earlier) and
modulates the kinetics of inactivation (Liu et al., 2004). An A to l editing event
resulting in a lysine to arginine change in D182 causes a depolarizing shift in the
voltage-dependence of activation by nearly seven millivolts (Song et al., 2004).
To date, RNA editing events of the insect SC have only been documented and
functionally characterized in the cockroach system. The U to C and A to l RNA
editing events previously mentioned are the only two editing events that have
been demonstrated to confer altered gating properties. RNA editing could be a
major mechanism for increasing the diversity of sodium channel function. More
research in this area needs to be done in order to characterize the importance of
RNA editing and to understand the physiological roles it may play.
VI. Pharmacology of Voltage-Gated Sodium Channels

Sodium channels are integral transmembrane proteins responsible for the
rising phase of action potentials (Catterall, 2000). Because of their fundamental
role in neural excitability they are a target site of neurotoxins produced by
animals and plants for defense and preying strategies as well as therapeutic
drugs (Wang and Wang, 2003). Binding of toxins to 80$ change normal channel
functional and can result in modifications ranging from blocking the channel pore
to altering normal activation and inactivation processes (Wang and Wang, 2003).
These toxins are grouped into nine types according to their distinct effects and
receptor sites on the sodium channel (Table 4). The molecular determinants of

receptor sites one through five, seven, and nine have been identified (Table 4).

21

Table 4: Neurotoxins that act on sod__i_gm channels

 

Receptor Neurotoxins Physiological Location
Site Effects
1 Tetrodotoxin Binds to extracellular 85-6 loop of
saxitoxin pore; block Na” ion D1, D2, D3, D4
p-conotoxin entry
2 Batrachotoxin Persistent activation Segment 6 of
veratridine D1, D2, D3, D4
grayanotoxin
3 a-scorption toxins Block Inactivation 83-4 loop of D4
sea anemone toxins 85-6 loop of D1, D4
4 B-scorpion toxins Prolong activation, 81-82 loop of D2
hyperpolarized shift 83-84 loop of DZ
of activation
5 Brevetoxins Persistent Activation Segment 6 of D1
ciguatoxins Segment 5 of D4
6 6-conotoxins impair Inactivation Unknown
7 DDT, Block activation Segment 6 of D1,
pyrethroids depolarized shift of D2, D3
resting potential
8 Conus stratius Prolong activation Unknown
toxin
9 Local anesthetics, Block Inactivation, Segment 6 of D1 ,
prevent ion entry D2, D3

 

Modified from Wang and Wang (2003).

The most relevant toxin this study is thalT, an alpha scorpion toxin

extracted from Leiunrs quinquestn'atus hebraeus. Alpha scorpion toxins bind to

receptor site three and remove fast inactivation resulting in prolonged sodium ion

entry into the cell (Gordon et al., 1996). These neurotoxins are classified into

22

four groups based on pharmacological properties and structural similarities
(Gordon et al., 1996). The ‘classical’ alpha scorpion toxins are highly active on
mammalian 803 and weakly active on insect 803. The prototype for classical
alpha scorpion toxins is AaH ll because of its high affinity towards mammalian
brain synaptosomes (Gordon et al., 1996). Those toxins that are weakly toxic to
both insects and mammals are represented by qu IV. These toxins
competitively inhibit the binding of AaH II in mammalian systems as well as
thalT in insect 808 (Gordon et al., 1996). “Alpha-like scorpion toxins’ are
active on both mammalian and insect SCs. Interestingly though, alpha-like toxins
Born Ill and Born IV compete with thodT for binding on insect 803, but not AaH
ll of mammalian VGSCS. However, because of their “classical” inhibition of
inactivation and competitive binding with thalT, they are termed ‘alpha-like’.
Those toxins that are highly toxic to insects and weakly toxic to mammals are
considered insect-selective. thodT is significantly more toxic to insect SCs than
to mammalian 808 (Gordon et al., 1996).

The a-scorpion toxins are small polypeptides of 63 to 70 amino acids
cross-linked by four disulfide bridges (Cestele and Catterall, 2000). thaIT is
greater than ten fold more active against insect 803 than mammalian SCs (Lee
et al., 2000). Because the effect of thalT binding to insect SCs resembles the
classical scorpion toxins suggests that the receptor site on insect 803 would be
similar to mammalian 803. However, because thozIT exhibits selective
tendencies towards insect sodium channels and not mammalian sodium
channels indicates that there are unique structural features of the insect sodium

channel that confers selective binding. It has been proposed that there are

23

distinct but overlapping receptor sites in insect and mammalian 80s for the
binding of mammalian- and insect-selective toxins (Gordon et al.,1996). While
photoaffinity labeling, site-directed mutagenesis studies, and site-directed
antibodies against individual extracellular loops revealed Dl85-86, DlVS5—S6,
and DIVS3-S4 are involved in alpha-scorpion toxin binding on mammalian
sodium channels (references in Rogers et al., 1996), the critical amino acid

residues involved in the binding to insect 803 have not been identified.

24

CHAPTER 2: Specific Aims, Rationale, and Significance

Specific Aims:

The Iong-terrn goals of this project are to understand the functional
diversity of insect voltage-gated sodium channels (SCs); to elucidate the
molecular interactions between 803 and neurotoxins particularly insecticides;
and to understand the biological role of alternative splicing and RNA editing of

insect 8C genes. The specific Objectives of my masters thesis project are:

Objective 1. Expression and functional characterization of all 64 Para variants

using a two-electrode voltage clamp /Xenopus oocyte expression system.

Objective 2. Pharmacological analysis of the functional sodium channel variants

using the a-scorpion toxin, thaIT.

Rationale and Significance of Objectives:

There exists one gene encoding 803 in insects. Transcripts of this gene
undergo extensive alternative splicing and RNA editing events, generating
functionally and phannacologically distinct sodium channels. A single composite
full-length clone of para is currently available for functional, pharmacological, and
molecular analysis. This clone was constructed by ligating two overlapping
CDNA fragments (Warmke et al., 1997), and may not represent a true splice type

or the variety of sodium channels in vivo.

25

Dr. Zhiqi Liu, a postdoctoral associate in Ke Dong’s laboratory, isolated
sixty-four full-length clones of para by RT-PCR of the entire coding region. It is
likely that these Clones represent a suite of in-vivo splice types. Sequence
analysis of the full-length clones should reveal novel alternative splicing and RNA
editing sites. Comparison of alternative exon use and RNA editing events
between para and German cockroach SC genes would aid in the identification of
common and distinct features of these events. Expression and functional
analysis of the full-length CDNA clones in Xenopus oocytes is likely to reveal
Para variants with unique gating properties similar to and differing from the
properties previously identified in the German cockroach SCs (Tan et al., 2002;
Song et al., 2004; Liu et al., 2004). This information will be the foundation for
future experiments that will outline the importance of RNA editing and alternative
splicing in vivo by taking advantage of the unique genetic tools available only in
D. melanogaster.

The SC is the primary target of various neurotoxins, including insecticides.
Sodium channels exhibit distinct pharmacological profiles in both insect and
mammals (Zhao et al., 2005; Song et al., 2006). This thesis will focus on the
alpha scorpion toxin, thaIT for pharmacological analysis. thoIlT is an insect-
specific scorpion toxin and little is known pertaining to the binding site of thole
on insect 80s. The response of Para variants to thalT application will be
determined. Identification of thaIT susceptible and resistant variants will
provide valuable resources towards the analysis of molecular determinants that

are critical for toxin binding and action.

26

CHAPTER 3: Functional and Pharmacological Characterization of Sixty-
Four Para Sodium Channel Variants in Xenopus Oocytes
ABSTRACT
Extensive alternative splicing and RNA editing of the Drosophila
melnaogaster voltage-gated sodium channel (SC) gene, para, has been
documented. However, the functional consequences of these post-
transcriptional modifications have not yet been investigated. In this study we
report the isolation, expression, and functional characterization of sixty-four full-
length para CDNA clones. The sixty-four clones were grouped into twenty-nine
splice types based on alternative exon usage. Functional analysis of these
variants revealed a broad spectrum in the voltage-dependence of activation with
the voltages for half-maximal activation ranging from -6.8 mV to -46.8 mV. The
voltage dependence of inactivation, however, was less variable. Sequence
comparison of three functionally distinct variants uncovered three novel A to l
editing sites. Furthermore, we revealed differential sensitivities of these variants
to thalT, an a-scorpion toxin isolated from Leiurus quinquestn‘atus hebraeus.
The observed functional and pharmacological diversity of para variants supports
the notion that alternative splicing and RNA editing are the two major

mechanisms for generating sodium channel diversity in insects.

27

INTRODUCTION

Voltage-gated sodium channels (SCs) are integral transmembrane
proteins responsible for the generation of action potentials across the
membranes of excitable cells. The mammalian SC consists of a large,
membrane bound, pore-forming, voltage-sensing a-subunit of approximately 260
kDa, along with several smaller auxiliary )9 subunits. The a-subunit is composed
of four repeated homologous domains (I-lV), each containing six hydrophobic
transmembrane segments (S1-86). Mammals have nine genes that encode for
functionally and pharmacologically distinct SCs each with unique tissue,
developmental, and cellular-specific expression patterns (see Chapter 1 for
details; review by Catterall, 2000).

Insect SCs, unlike mammalian counterparts, are encoded by a single
gene. The Drosophila melanogaster SC, para, was isolated from temperature-
sensitive paralytic mutants using P-element transposon tagging (Loughney et al.,
1989). The complete Para amino acid sequence was deduced from six
overtapping CDNA clones and was predicted to have extensive homology
(greater than 58 percent for each four domains) to mammalian SCs (Loughney et
al., 1989). Alternative splicing and RNA editing of para are predicted to be two
major mechanisms for increasing molecular and functional diversity of insect SCs
(Loughney et al., 1989; O’Dowd and Aldrich, 1988; O’Dowd, 1995; Thackeray
and Ganetzky, 1994, 1995; Wannke et al., 1997; Palladino et al., 2000), but, the
functional consequences of alternative exons and RNA editing sites have not
been determined in para. However, the importance of alternative splicing and

RNA editing of the German cockroach SC gene BgNav in creating insect SC

28

functional diversity has been recently documented (Tan et al., 20023; Liu et al.,
2004; Song et al., 2004).

Prior to our study a single variant of para has been functionally
characterized in Xenopus oocytes (Warmke et al., 1997). However, this
composite full-length CDNA clone may not represent a splicing type in vivo
because it was constructed by ligating two overlapping partial CDNA clones. In
this study we report the isolation, expression, functional and pharmacological
characterization of sixty-four full-length para CDNA clones.

MATERIALS AND METHODS
Synthesis of first strand cDNAs

Total RNA was isolated using lnvitrogen TRlzol Reagents (lnvitrogen,
Rockville, MD). Isolation of mRNA was performed using Promega polyA+ RNA
isolation kit (Promega, Madison, WI). First-strand CDNA was synthesized from
mRNA using a Para-specific primer (D-3’-1; 5’ - TAC TCA TGC TAA TAC TCG
CG -3’ ) based on the sequence of a region immediately down stream of the stop
codon (Genbank accession M32078), and 8uper8cript ll RNase H-reverse
transcriptase (lnvitrogen, Rockville, MD). First strand CDNA synthesis reaction
conditions were: incubation at 42°C for 2 min. followed by a 60 min. incubation at
48°C. RNA was removed using RNaseH followed by a 20 min. incubation at
37°C.

PCR and cloning of para full-length cDNAs

To amplify the entire coding region of para (6 kb) by PCR, a fonIvard
primer (D-para-Kpnl - 5’-CGG GGT ACC GCC ACC ATG GCA GAA GAT TCC
GAC TCG-3’) and a reverse primer (D-para-Xba - 5’-GCT CTA GAT ACT GCT

29

AAT ACT CGC G—3’) were designed based on the 5’ and 3’ sequences of the
para open reading frame. Xba and Kpnl restriction enzyme site sequences
(underlined) were added to the forward and reverse primers, respectively, to
facilitate cloning. A Kozak sequences (double underlined) was added to the
primer D-para-Kpnl for high-efficiency translation. The reaction mixture of
amplification (50 pl) contained 0.5 pl CDNA, 50 pmol each of primers D-para-Xba
and D-para-Kpnl, 200 pM each dNTP, 1 U eLONGase (lnvitrogen), 1.5 mM
MgCIz, and 1X PCR reaction buffer. PCR amplification was carried out as
follows: 1 cycle of 94° C for 1 min.; 33 cycles of 94° C for 30 sec., 58° C for 30
sec., 68° C for 8 min.; one cycle of 68° C for 15 min. The PCR products were
purified using QIAEX ll Gel Extraction Kit (QIAGEN Sciences, Maryland) and
cloned in pGH19, an oocyte expression vector. Plasmids were transformed into
competent bacterial cells, Stbl2 (lnvitrogen), and sixty-four positive colonies were
isolated.
Expression of para sodium channels in Xenopus Iaevis oocytes

Oocytes were obtained surgically from oocyte-positive female Xenopus
Iaevis (Nasco, Ft. Atkinson, WI). Females were anesthetized with Tricaine (3-
aminobenzoic acid ethyl ester) at Zg/Iiter, and several lobes of the ovary were
surgically removed. Follicle cells surrounding the oocytes were loosened by
collagenase treatment (1 mg/ml Type IA collagenase, Sigma 00., 8t. Loise, MO)
in calcium-free ND-96 medium (96 mM NaCl, 2mM KCI, 1 mM MQCIz, and 5 mM
HEPES adjusted to a pH of 7.5) then the remaining follicle cells were removed
with forceps. Isolated oocytes were incubated in N096 medium (96 mM NaCl,

2mM KCI, 1 mM MgCl2, and 5 mM HEPES, 1.8 mM CaClz, 50 [1ng gentamicin,

3O

5mM pyruvate, and 0.5 mM theophylline, adjusted to a pH of 7.5) (Goldin, 1992).
Healthy oocytes stages V and VI were used for cRNA injection.

Plasmid DNAs containing the para and TipE (auxiliary subunit of the para
construct, refer to Chapter 1 for details) constructs were linearized by Notl.
cRNAs were prepared by in vitro transcription with T7 polymerase (mMESSAGE
mMACHINE kit, Ambion). TipE and para cRNAs were co-injected (0.25 ng — 10
ng) into Xenopus oocytes. TipE facilitates robust expression of para current in
oocytes (see Chapter 1 for details). Oocytes with peak sodium current of 0.5 uA—
2 (IA were used for functional analysis using the two-electrode voltage clamp.
Usually 0.5 pA- 2 uA of sodium current can be detected in the oocyte one to five
days following cRNA injection. Typically, by the sixth day, the oocyte condition
was too poor for recording currents.

Electrophysiological recording and analysis

Sodium currents were recorded using a standard two-electrode voltage
clamp system (Kontis and Goldin, 1993). Voltage and current borosilicate glass
electrodes were filled with 3 M KCI and manipulated for a resistance of less than
0.5 MI). Amplifier OC72SC (Warner Instrument Corp. Hamden CT), Digidata
1320A, and pCLAMP 8.2 software (both of Axon Instrument, Foster City, CA)
were used for current measurement. All experiments were performed at room
temperature (20 to 22°C). Capacitive transient and linear leak currents were
corrected using the PIN subtraction technique.

The voltage-dependence of conductance (G) was calculated by measuring
peak currents at test potentials ranging from -80 to +65 mV in increments of 5

mV and dividing by (V-Vrev), where V is the test potential and Vrev is the reversal

31

potential for sodium. Reversal potentials were determined from the I-V curves
and peak conductance values were normalized to the maximal peak
conductance (Gmax). Peak conductance values were fitted with a Boltzmann
equation of the form G=1-[1+exp(V-V1/2)/k]'1. Where V is the potential of the
voltage pulse, Vuz is the half-maximal voltage for activation, and k is the slope
factor.

The voltage-dependence of inactivation was determined using 200ms
inactivating prepulses ranging from -120 mV to -15 mV in 5 mV increments from
a holding potential of -120 mV. Peak current amplitude during the test
depolarization was normalized to the maximum current amplitude, and plotted as
a function of the pre-pulse potential. The data were fit with a Boltzman equation
of the form l=lmax*[1+(exp(V-V1/2)/k)]'1, where I."W is the maximal current, V is the
voltage pulse potential, V1,; is the voltage at which half of the current is
inactivated, and k is the slope factor.
thaIT sensitivity assay

thalT was generously provided by M. Gurevitz (Tel Aviv University,
Israel). The method used for application of thalT in the recording system is
identical to that described by Tan et al. (2002). Briefly, disposable recording
chambers were made in Petri dishes with hot glue dams (1-1.5 ml volume).
thalT was delivered to the oocyte recording chamber by hydrostatic force using
glass capillary tubes. The toxin-induced effects were quantified by the ratio of

the current amplitude 20 ms into depolarization (I20) over the peak current (lpeak).

32

RESULTS
Molecular analysis of 64 full-length cDNA clones

Prior to our study, eleven alternative exons were identified in the para
transcript (Thackeray and Ganetzky, 1994; O’Dowd et al., 1995; Warmke et al.,
1997; Lee et al., 2002). Seven of them are optional (a, b, e, f, h, i, j), and four
are mutually exclusive (Old and UK; Fig.1A). In this study, Dr. Zhiqi Liu isolated
sixty-four para full-length CDNA clones using RT-PCR. Determination of the
alternative exon usage in these variants was done by using PCR or sequencing.
Our analysis revealed twenty-nine splice types (Fig. 1B). The most abundant
splice type is
a, b, d, i, j, I. It is interesting that out of seven optional exons and four mutually
exclusive exons that only twenty-nine splicing types were observed because
there is a possibility of forming 20,160 splicing types. Most of the variants
contain exons a, d, i, j, | suggesting that there is selection pressure for these
exons.
Functional expression of 64 variants In Xenopus oocytes

All sixty-four of the para variants were co-injected with TipE into Xenopus
oocytes for functional analysis using the two-electrode voltage clamp expression
system. Twenty-six of the variants did not generate any sodium current even
eight days after cRNA injection. Another six variants produced detectable
sodium currents, but the currents were too small (less than 0.5 M) for functional
analysis. Sodium currents from the remaining thirty-two variants were sufficient
for functional analysis (0.5 IA -2 IA). We determined the voltage-dependence of

activation and inactivation of these thirty-two variants.

33

As presented in Table 1, the voltages for half maximal activation (V1,2)
varied greatly among the functional constructs. We divided the variants into
three groups based on their voltage dependence of activation. Five variants, 50,
55, 48, 32, and 53, required more positive membrane potentials for activation
with a Vm of -6.8 mV to 15.69 mV and were designated as high-voltage activated
(HVA) variants. Nine variants, 49, 10, 45, 27, 62, 22, 2, 21, and 18, activated at
more negative membrane potentials with V1/2 of -26 mV to -46 mV, and were
termed low-voltage activated (LVA) variants. The majority of the variants had a
VW value of -20 mV to -25 mV, and were labeled mid-voltage activated (MVA)
variants. Voltages for half maximal inactivation (V10) were less variable for most
of the variants with a range of -32 mV to -50 mV. Variant 18 was unique in that it
inactivated at strongly hyperpolarized potentials (-65 mV) and also activated at
extremely hyperpolarized potentials (-47 mV).

Sequence analysis of three functionally distinct variants

Three para variants were sequenced, each representing one of the three
activation groups; LVA (variant 18), MVA (variant 13), and HVA (variant 50).
Each variant contained unique alternative splicing and RNA editing sites. Variant
13 belonged to splice type fourteen (c, i, j, I), variant 18 belonged to splice type

seven (a, c, j, l), and variant 50 belonged to splice type nineteen (d, e, f, i, j, k).

34

 

 

 

 

 

 

[1A

 

 

 

 

3
15543332222221111111111111111

Vanant
Number

k W WWW W EE k
IEEEEEEEEEEEEEEEEEEMEEEEEE EQI
ELI“ 6 “mm m H mm I. e
m m a e as WWW SSW .emm a
IWII E lella I... W E Ewmiid

It bEEEE E E E E EE W E E E b

an an awn an an :::::m

l/k
/
/

WA

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

d
d

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

.m. 0.123456789012345
T1234567891111111111222222

U LI]
T-U

ta . . mm a
v/.- -mmmmmm mmmmm mmmmmmmm mmm

Splicing
26

A. A topological view of the sodium

channel with positions of alternative exons indicated by colored boxes (A). 3. Schematic

presentation of alternative exon usage in each splice type.
35

Identification of 29 splice types.

Figure 1.

Table 1. Activation and inactivation properties of 32 functional variants.

 

 

 

 

 

 

 

 

Variant Activation Inactivation
V1,2(mV) k (mV) n Vuz (mV) k (mV) n

50 -6.80 11.74 5.20 1 1.10 12 60.83 1 1.69 6.11 1 0.68 18
55 -9.94 1 3.72 9.39 1 4.56 6 —50.19 1 2.95 5.42 1 0.85 10
48 41.42 1 2.34 7.33 1 0.79 14 34.90 1 3.86 5.47 1 1.06 14
32 -14.78 1 3.24 7.27 1 0.96 21 42.32 1 5.22 7.31 1 6.20 39
53 ~15.69 1 2.05 8.16 1 1.06 10 -45.46 1 1.30 7.46 1 0.63 12

T 49.37 1 1.81 6.88 1 1.20 20 -39.81 1 2.88 6.06 1 1.50 37
38 -20.49 1 4.63 7.94 1 1.37 8 -46.72 1 0.46 4.84 1 0.55 7
17 -2057 1 2.72 6.78 1 1.31 13 46.38 1 2.69 4.98 1 0.43 18
58 -21.60 1 2.68 6.33 1 1.22 23 40.99 1 2.02 5.28 1 0.65 24
24 -21.60 1 2.07 6.90 1 1.19 21 43.95 1 2.14 5.36 1 0.72 25
7 -22.40 1 2.48 7.44 1 1.19 11 47.41 1 2.78 5.12 1 0.70 15
19 -2229 1 3.07 8.68 1 1.57 19 45.39 1 2.27 5.44 1 0.79 25
11 -22.71 1 3.75 8.11 11.83 19 47.32 1 2.12 5.55 1 0.42 23
13 -2274 1 3.33 3.46 1 0.64 11 -32.07 1 2.17 3.85 1 0.49 17
39 -23.26 1 4.58 5.49 1 1.54 19 -39.87 1 2.90 5.29 1 1.10 13
41 -23.70 1 3.33 4.50 1 0.83 24 40.09 1 2.65 4.54 1 1.01 24
24 -23.77 1 3.72 6.69 1 0.85 14 -48.31 1 2.30 5.32 1 0.48 15
31 -2403 1 2.98 5.15 1 0.80 12 45.06 1 1.97 4.83 1 0.74 22
47 -25.34 1 4.34 4.70 1 1.25 20 42.87 1 2.59 4.82 1 0.33 20
57 -25.66 1 2.23 5.80 1 1.23 3 46.07 1 3.08 4.87 1 0.36 15
56 -25.72 1 4.06 5.94 1 1.39 20 -35.05 1 3.09 4.94 1 0.42 21
8 -25.75 1 1.95 6.38 1 1.58 8 47.11 1 2.20 4.92 1 0.33 11
44 -2599 1 4.49 3.00 1 1.30 24 -39.21 1 1.46 4.18 1 0.44 24

_49’— -26.25 1 2.66 5.08 1 1.01 17 43.51 1 1.59 4.85 1 0.30 19
10 -26.33 1 5.77 3.95 1 1.19 18 -38.95 1 1.64 4.84 1 0.51 19
45 -27.13 1 3.80 5.81 1 1.04 24 45.15 1 2.64 5.35 1 0.21 24
27 -27.16 1 3.92 4.91 1 1.13 21 41.86 1 3.18 5.02 1 0.30 22
62 -29.08 1 3.58 3.68 1 0.87 17 42.88 1 1.82 4.49 1 0.37 18
22 -29.53 1 2.48 10.36 1 2.07 13 49.19 1 1.95 6.48 11.19 13
2 -29.67 1 7.27 4.35 1 1.19 33 42.01 1 2.45 5.05 1 0.52 37
21 -31.66 1 3.69 4.32 1 0.90 20 43.66 1 2.07 4.55 1 0.29 25
18 -46.56 1 4.55 6.17 1 1.06 17 -65.42 1 2.42 4.43 1 0.46 24

 

 

Sequence comparison of variants 13, 18, and 50 with the GenBank Para
sequence (accession number M32078) revealed fifteen, twelve, and eight amino

acid changes, respectively (Figure 2 and Table 2). In an earlier study, ten A to I

36

editing sites were identified in para (Palladino et al., 2000a). Five of the
observed Changes from this study match five of the previously reported A to l
editing sites (Palladino et al., 2000). Because PCR mistakes are unlikely to
occur in multiple variants, we have identified and additional three A to I editing
sites (observed in multiple variants; Table 2A). Additionally, two nucleic acid
Changes were observed in multiple variants that resulted in a guanine to cytosine
change. However, whether these are the result of alternative splicing or a novel
type of RNA editing remains to be determined.

Nucleic acid changes that occur in a single variant may be the result of
RNA editing, alternative splicing, or a mistake made during PCR. Fifteen distinct
nucleic acid changes were observed in variants 13, 18, and 50 (Table 23). One
of these changes, N1822S, corresponds to a previously recognized A to l editing
site (Palladino et al., 2000a). The remaining fourteen changes need to be further

investigated to determine the foundation of change.

37

E2990 D1300N M1376V T1532A I1661V |1691V N18228

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  
  

   

  
  
 

 

 

 

 

 

 

 

 

 

 

 

Variant
13
R520 UU
Q471R R12960 L1363F N1565K |167O I1679V
E2990 K303R W319R I1661V |1691V A1731V
An
U
L1363F |1670M H1676
E299Q 89 2P D1300N I1661V |1691V N18228
, AA
X
Variant
50
R520 UV U 'L'

      

R12960 L1363F S1587N |1670 I1679V

 

Figure 2. Amino acid changes in variants 13, 18, and 50. The secondary structure of
variants 13, 18, and 50 along with the position of amino acid change compared to the
GenBank sequence (M32078). For example, R520 indicates R in the Genebank
sequence and Q in variants at the amino acid position 52. Amino acid changes occur in
more than one variant are indicated by the darkened circles. Triangles represent
variant-specific amino acid changes.

38

Table 2. Amino acid changes observed in variants 13, 18, and 50.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Variant Variant Variant Nucleo- Location RNA Report-
13 18 50 tide Editing ed
Change Type A to l
A
R 52 Q R 52 Q R 52 Q g-a N.T. A to l *
E 299 Q E 299 Q E 299 Q J—C l85-l86 ?
Q 471 R Q 471 R a-l I-ll Atol
R 1296 Q R1296 Q g-a ll-lll A to l
D 1300 N D1300 N J-a IIIS1 A to l
L 1363 F L 1363 F g—C lll82-lll83 ?
l 1661 V I 1661 V a-g IVS1-IVS2 Atol
l 1670 M l 1670 M a-g IVS2 A to l
I 1691 V l 1691 V a-g IVS3 Atol
B
I 260 T a-g 184-185
K303R a-g l84-l85
W319 t-c |85-IS6
S 922 P t-c IIS4
L1363F g-c I|82-IIS3
M 1376 V ag Ill83
Q 1450 R Q llIS5
T 1532 A a-g IllS5-III86
N 1562 K t:g Ill-IV
S 1587 N g-a Ill-IV
H1676R a-g IV82-IVS3
I 1679 V a;g IVS3
A 1731 V c-t IV84
N 1822 S a-g lVS5-IVS6 *
l 1850 M EL IVS6

 

 

 

 

 

 

 

 

 

Note: N.T. represents the N-terrninal domain. Question marks (7) indicate that the
mechanism underlying the amino-acid changes are unknown. Asterisks (*) represent
the previously reported A to I editing sites in para (Palladino et al., 2000).

Examination of the sensitivity of Para variants to thalT
thalT, an alpha scorpion toxin isolated from Leiurus quinquestn'atus
hebraeus, binds to receptor site three (refer to Chapter 1) and removes fast

inactivation resulting in prolonged action potential propagation (Chen et al.,

39

 

2000). thadT is polypeptide of 66 amino acids that is ten fold more selective to
insect 803 than to mammalian SCs (Lee et al., 2000).

To examine the sensitivities of the functional variants of para to 1 nM
thalT, we determined the ratio of Izo/lpeak before and after toxin application
(Figure 3; Table 3). Although inactivation was affected by 1 nM thalT for all
variants, some variants are more sensitive than others. For example, variant 21
is most resistant to thalT compared to all other variants with only 29% of
inactivating current inhibited by 1 nM thalT. Variants 8 and 58 are among the
most sensitive ones with inactivation being completely abolished by toxin
application. To quantitatively compare the effect of thalT between variants 8
and 21. dose-response curves were created (Fig. 4). While the E050 of variant 8
could not be generated due to an inadequate number of data points, Figure 4
indicates that variant 8 is apparently more sensitive than variant 21 (E050 of 2.58
nM) to thalT.

Table 3. Effects of1 nM thalT on para variants

 

 

 

 

Variant '20/lpeak (%) 1 SD n Variant I20/Ipeak (%):|; SD n
21 29113 12 19 62:18 3
62 34 :I: 13 4 55 66 :I: 22 3
2 58133 27 13 78136 13
10 34 :I: 13 4 11 79 :l: 16 3
22 34 :I: 13 4 8 80 :l: 25 18
47 29 :t 13 12 17 81 :l: 9 4
56 34 :l: 13 4 50 85 i: 37 2
39 58 :I: 33 27 7 86 :I: 12 2
48 60 j: 15 6 31 88 1
18 61 :I: 17 6 44 88 1
25 56 :I: 14 5 27 93 1
45 58 1 24 94 :t 1 4
49 58 1 52 105 :I: 7 8
41 59 :I: 0 2 32 105 1
57 62 1: 27 4 58 105 :t 9 2

 

 

 

 

40

 

1 nM thalT

I
I -1-0011A
1.84 “A 1

I

Figure 3. Peak sodium current traces of variant #8 before and after application of
1 nM thalT. Peak current was measured by depolarizing the oocytes membrane to -
10 mV for 20 ms from a holding potential of -120 mV. Peak current was determined
before the application of 1 nM thalT and ten minutes after the application of 1 nM
thaIT.

 

 

 

 

140 -

“2% 20 1 "

a. 1
‘g 004 I f II 1'
=- I
C 80 I -- 31/
.2 f/
= 1 /_/
.0 e
E 60 ‘ '//’/
s _. T
5 40 -b ///-l . var'ant # 21
2 ‘ --”’
0 20 1 I-‘/” I varlant # 8
0- ,.~ .
0 V V ' V v

 

'1 I I “"10
thalT Concentration (nM)

Figure 4. Dose response curves of variants 8 and 21. Dose-response curves were
fit with a Hill equation of the form y = Vmx [x"/(k" + x")]. Variant 21 has an E050 of 2.58
nM. More data points were needed to determine the EC50 of variant 8.

41

Variants 8 and 21 were selected for sequence analysis because of their
differential sensitivities to thodT. The results of this analysis are presented in
Figure 5 and Table 4. Eight of the observed changes correspond to the
previously recognized RNA editing sites. Three of the changes are identical to
the earlier mentioned possible RNA editing sites and five are novel. Variant 8
has one novel amino acid change (K1455R) and variant 21 has two novel amino
acid changes (|1519V and H1772Y) that are located on the extracellular loops of

the pore and that may interact with the binding of thodT.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

52990 K1455R I1661V |1691V N1822S
Variant
8
R5
L1363F |1670 I1679V
£2990 D1300N I1519V I1661Vl1691V N18228
A An
Variant s
21
R520 UU U

  

|260VH487N DB 66 L1363F |1670M I1679V H1772

R12960

Figure 5. Amino acid changes in variants 8 and 21 compared to GenBank #
M32078. This figure depicts the topology of variants 8 and 21. Circles indicate common
changes. Triangles represent unique changes in the transmembrane regions. and stars
indicate unique changes in the extracellular portion.

42

Table 4. Sequence comparison of variants 8 and 21

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Variant 8 Variant 21 Nucleotide Location
Change
R 52 Q1 R 52 Q“ g-a N.T.
I 260 v2 a-g lS4-SS
E 299 Q‘ E 299 Q1 g-c lSS-ISG
H 487 N3 c-a |SG-llS1
D 656 G3 a-g ISG-lIS1
R 1296 Q" j-a IISG-IIIS1
D 1300 N1 g-a IIIS1
L 1363 F“ L 1363 F“ g-c llISZ-lllS3
K 1455 R4 a-g lllS5-SG
I1519V" a-g lllS5-36
I 1661 v1 I 1661 VT a-g Ivs1-Ivs2
l 1670 MI I 1670 M‘ a-g IVS2
I 1679 v2 I 1679 v2 a3 IVS3
I 1691 v1 I 1691 VI a-g IVS3
H 1772 Y4 c-t Ivss-se
N 1822 s2 N 1822 $2 a-g IVS5-IVS6

 

Note: 1. Amino acid changes that correspond to previously recognized RNA editing
sites of variants 13, 18, and 55. 2. Amino acid changes that correspond to possible
RNA editing sites of variants 13, 18, and 55. 3. Unique editing sites. 4. Unique editing
sites that are situated on the extracellular portion of the pore and that may interact with
the binding of thalT.

DISCUSSION:
Functional versus non-functional Para variants in oocytes

In this study, we co-expressed the sixty-four cDNA clones of para with
TipE to achieve robust expression of sodium currents as previously
demonstrated by Warmke et al. (1997). Half of the total variants, thirty-two, had
sodium currents large enough for functional analysis (1-2 M). An additional six
variants were functional, however, did not produce sufficient sodium currents for
functional analysis (<1 IA). The remaining twenty-six variants did not generate
any detectable sodium current (0 [.LA).

Recently, four TipE-homologous genes (T EH1-4) were identified from D.

melanogaster (Derst et al., 2006). The co-expression of three of these genes

43

 

(THE 1-3) with para (the para construct was obtained from Dr. J.C. Cohen; refer
to Warmke et al., 1997) in Xenopus oocytes also elevated sodium current
expression. It would be of interest to determine whether TEH1-4 can boost the
low level expression of the six variants in our study. RT-PCR analysis indicated
that the expression patterns of TEH1 and TEH4 are under tissue-specific
regulation (Derst et al., 2006). Distinct tissue distribution patterns have also
been observed for alternative splicing and RNA editing of BgNav variants (Tan et
al., 2002; Song et al., 2002). The possible tissue-specific co-expression of para
variants with TipE or THE1-4 in vivo remains to be investigated.

The presence of exon b in para and the ortholog B in BgNav hampers the
expression of BgNav and Para channels expressed in oocytes (Song et al., 2004;
Song, Personal Communication). Interestingly, four of the six variants with low
levels of sodium current expression contain exon B/b, suggesting that this exon
could be the cause of poor expression. However, the majority of our functional
variants also contain this exon (eighteen out of thirty-two), suggesting that the
expression of sodium current is not solely regulated by exon B/b.

At present, we do not know why the twenty-six variants are not functional
in Xenopus oocytes. Sequencing of the twenty-six nonfunctional variants will
reveal whether these variants contain stop codons in the open reading frame
thereby generating non-functional channels. It is also possible that these
variants require one of the other auxiliary subunits, such as TEH1-4, to facilitate
expression, or that unidentified sequence features in these variants down-

regulate sodium current expression.

 

Identification of twenty-nine Para splice types

Theoretically 20,160 splicing types could be formed out of the seven
optional exons and four mutually exclusive exons. Interestingly though, we only
isolated twenty-nine splice types. Although we may have failed to isolate some
low frequency splicing types, we believe these twenty-nine represent the majority
of transcripts in vivo. A previous study investigating exon use of BgNav variants
identified 20 splice types from a total of 69 variants (Song et al., 2004). These
results suggest that alternative exon usage is regulated, and not formed by
random combinations.

Since molecular analysis of a large pool of insect SC full-length cDNA
clones has been conducted in both para (this study) and BgNav (Song et al.,
2004), a direct comparison of splice types can be made between the two. The
most abundant splice type of para (twenty percent) is a, b, d, iI i, kI and I, while
the most abundant splice type (fifty-five percent) of BgNav is a. f. i. k and l.
lntriguingly, the most abundant para splice type was not isolated at all from
BgNav variants (Song et al., 2004). This discrepancy is due in part to the
differences in the frequency of exon usage of several alternative exons
(described above).

Frequency of alternative splicing of insect sodium channels

Previous studies show that alternative splicing sites of SC genes are
extremely conserved among D. melanogaster, D. virilis, M. domestica, and B.
gerrnanica (See Chapter 1). However, the frequencies of alternative exon use
are less conserved (Table 5). In general, similar exon frequencies can be

observed for closely related species, D. melanogaster and D. virilis, and even for

45

M. domestica SCs. The frequency of exon use between B. gennanica, D.
melanogaster, D, virilis and M. domestica SCs, however, are more divergent.
For example, exon a is frequently used in D. melangoaster, D. virilis, M.
domestica and B. gennanica SC variants while exon e is not. Exons b, d, and i
are expressed in greater than sixty percent of the para (both D. melanogaster
and D. vin'Iis) and M. domestica variants. However, exons b and i are not
commonly used in BgNav transcripts and use of exon d has not yet been
identified in BgNav. Exons c and fare not frequently used in D. melangoaster, D.
vin'lis, or M. domestica SC transcripts, but are present in over ninety-eight
percent of BgNav transcripts. Exon h is used in low frequencies in para, both of
D. melanogaster and D. virilis, and in BgNav, however, this exon is present in all
transcripts of Vssc1. The frequency of expression of mutually exclusive exon j is
high for para and BgNav transcripts (eighty-nine and seventy nine percent
respectively) but low for Vssc1 (D. virilis has not been analyzed for the frequency
of exon j)
identification of Three Novel RNA Editing Sites

Prior to this study, ten A-to-l editing sites were identified in para (Palladino
et al., 2000), and two were reported in BgNav variants (Liu et al., 2004; Song et
al., 2004). Five of the ten A-to-l editing sites identified in para were also
identified in the three sequenced variants (13, 18, and 50). Additionally, we
identified three novel A-to-I editing events, resulting in three amino acid changes,
two l-to-V and one l-to-M change. Potentially there are more RNA editing sites in
these three sequenced variants, however, alternative splicing or a mistake made

during PCR can not be ruled out. Investigations into the additional changes

46

observed revealed three possible U-to-C editing. In a previous study three U-to-
C editing sites were reported in BgNav variants (Liu et al., 2004; Song et al.,
2004). Additionally, there are nine possible A-to-l editing sites and two novel
changes that do not conform to either A-to-l or U-to-C editing.

Table 5. Frequencies of Exon Usage in Para, VSSC1, BgNav

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Exon Para1 Para2 D. virilisr VSSC1‘ BgNav!s
A 71.9 57.3 54.4 91-98 98.5
B 60.9 ~80 ~70 100 17.4
C 29.7 <30 ~20 ~0 100
D 70.3 >70 ~80 ~100 0
E 18.8 ~25 ~25 <10 17.2
F 9.4 6.7 ~10 ~10 98.5
H 3.1 21 100 0
I 85.9 ~85 81 100 5.8
J 89.1 <25 79.7
K 3.1 78-100 5.8
L 96.9 <25 92.7

1 . This study

2. O'Dowd et al., 1995; Thackeray and Ganetzky 1994,

3. Thackeray and Ganetzky, 1995

4. Lee et al., 2002;.

5. Song et al., 2004

Estimates are indicated by ‘~' marks, ‘>’ means greater-than, and ‘<’ means less
than.

It is worthwhile mentioning here that a previously identified U-to-C editing
event in both para and BgNav was not detected in any of these sixty-four
variants. This U-to-C editing event results in persistent current (Liu et al., 2004)
and none of our thirty-two functional variants exhibited persistent currents.
Obviously we have missed this variant in our cloning due to the low frequency of
this editing event. For the same reason, we may have missed the isolation of
other variants which have low editing frequency and play critical roles in

regulating inactivation kinetics in vivo.

47

 

Identification of High-Voltage Activated and Low-Voltage Activated Para
Variants

The functional analysis of our sixty-four para cDNA clones revealed a
broad spectrum in the voltage-dependences of activation among the variants. In
an earlier study performed on BgNav variants a broad range in the voltage-
dependency of activation was also identified (Song et al., 2004). The range of
the voltage-dependences of activation for the BgNav variants was from -24.9 mV
to -43.9 mV (Song et al., 2004). In this study we, for the first time, identified
several LVA and HVA sodium channels. Variant 18 is an LVA channel with a
voltage—dependence of activation of -46 mV. Variants 50 and 55 are extreme
HVA channels with voltage dependences less than -10 mV. An additional
sixteen of our variants activate at potentials less than that previously reported for
BgNav variants (less than -24.9 mV). The molecular basis of the LVA and HVA
gating characteristics remained to be identified.

Mammalian SCs exhibit distinct voltage dependences of activation
(Chapter 1 — Table 1). In general, those isoforms, that are predominantly located
in the central nervous system (CNS; Nav 1.1, 2, 3, and 6) activate at more
depolarized (i.e., positive) potentials than those predominantly located in the
peripheral nervous system (PNS; Nav 1.7, 8, and 9). The cardiac sodium
channel, Nav 1.5, and isoform Nav 1.9 activate at more hyperpolarized (i.e.,
negative) potentials than the remaining mammalian sodium channels (Chapter 1
- Table 2). The half-maximal voltages for activation of Nav1.5 (heart SC) are -45
or -46 mV and Nav1.9 (DRG neurons) are -47 or -54 mV, similar to that of the

Para variant 18.

48

The terms, HVA and LVA, are well-established to describe distinct calcium
channel families (review by Yunker, 2003). LVA calcium channels, such as
Cav3.1, 2, and 3, activate at hyperpolarized potentials near resting membrane
potential, while HVA calcium channels, such as Cav1.1, 2, 3, and 4, require
strong depolarization above resting membrane potential in order to activate the
channel (review by Catterall et al., 2005). LVA calcium channels are responsible
for the repetitive firing of action potentials in pacemaker cells while HVA channels
are involved in excitation-contraction coupling, hormone release, and
neurotransmitter release (review by Catterall et al., 2005).

Whether these Para LVA channels play a similar role and have similar
tissue distributions as mammalian Nav 1.5 and LVA calcium channels remains to
be determined. Both Nav1.5 and LVA calcium channels are localized in the heart
muscle and both facilitate spontaneous firing. While we do not know the tissue
distribution pattern of variant 18, it may be expressed in tissues and cells with
pace-making activities. Studies of cockroach terminal abdominal ganglia DUM
neurons characterized pacemaking activities as the result of LVA sodium
channels (Lapied et al., 1998). These LVA sodium channels activated at
potentials of -50 mV, which corresponds to the resting membrane potential of
DUM neurons in cockroach terminal abdominal ganglia. It was hypothesized in
this study that background channels are essential in the instigation and
continuation of spontaneous firing because activation of these channels
depolarize membrane potentials which in turn activate additional channels and

result in action potential propagation. It would be of interest to determine the

49

tissue distribution of variant 18, and determine if this variant is responsible for the
pace-making activities observed in the DUM neurons.
Identification of an thalT-resistant Para variant

The specific amino acids of the SV that are critical for thorlT binding
remain elusive. Identification of an thorlT-resistant variant, #21, from our survey
of thalT sensitivity of sixty-four Para variants provided valuable information for
further elucidating the molecular determinants of the thalT binding site on the
sodium channel.

Photoaffinity labeling, site-directed mutagenesis studies, and site-directed
antibodies against individual extracellular loops revealed DlSS—SG, DlVS5-86,
and DlVSS-S4 are involved in a-scorpion toxin binding on mammalian sodium
channels (references in Rogers et al., 1996). We found a single novel amino
acid change in variant 8 that is located in the extracellular loop connecting DlllSS
to Dll|86 that may confer the observed sensitivity of this variant to thalT binding
(K1455R). The insensitive variant 21 has two novel amino acid changes (|1519V
and Y1772H) that are located in the extracellular loops connecting $5 to $6 of
Dlll and DIV that may interact with thalT and inhibit binding of the toxin to the
channel resulting in a resistant variant. Future mutagenesis studies are needed

in order to verify the involvement of these amino acids in thalT binding.

50

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Beneski DA, Catterall WA. 1980. Covalent labeling of protein components of the
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Castella C, Amichot M, Berge JB, Pauron D. 2001. DSC1 channels are
expressed in both the central and the peripheral nervous system of adult
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Catterall WA. 1986. Molecular properties of voltage-sensitive sodium channels.
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Catterall WA. 2000. From ionic currents to molecular mechanisms: the structure
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Catterall WA, Goldin AL, Waxman SC. 2003. lntemational Union of
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