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This is to certify that the

dissertation entitled

Caz+ -CH9N_NEL PROTEIN FUNCTION AND REGULATION: POSSIBLE

ALTERED Ca -CHANNEL PROTEIN FUNCTION IN FORMATION OF PSE
TURKEY

presented by

LI-JU WANG”

has been accepted towardsfulfillment
of the requirements for

Ph.D. degreein FOOD SCIENCE

AIM Mum

Major profesinr

Date 4-19-96

 

MS U i: an Affirmative Action/Equal Opportunity Institution 0-12771

 

 

LIBRARY
Michigan State
University

 

 

 

PLACE ll RETURN BOXto remove We checkout from your record.
TO AVOID FINES return on or betore date due.

  

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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jF—T—T

”Affirm“. ActionlEqueJ Oppommttylnetltwon

 

Cay-CHANNEL PROTEIN FUNCTION AND REGULATION: POSSIBLE
ALTERED Ca2*-CHANNEL PROTEIN FUNCTION IN FORMATION OF PSE
TURKEY
By

Li-Ju Wang

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Food Science and Human Nutrition

East Lansing, Michigan
1 996

 

ABSTRACT

Cay-CHANNEL PROTEIN FUNCTION AND REGULATION:
POSSIBLE ALTERED Cay-CHANNEL PROTEIN FUNCTION IN
FORMATION OF PSE TURKEY

By

Li-Ju Wang

The turkey industry is experiencing pale, soft, exudative (PSE) meat
quality problems which resemble that of PSE pork, a significant fraction of
which is associated with a genetic defect of the Cay-channel protein in the
sarcoplasmic reticulum (SR). The aim of this research was to test the
hypothesis that there is a genetic defect in the turkey SR, Cay-channel
protein(s) which is responsible, in part, for the turkey PSE problem.
[3H]ryanodine binding studies indicated that the average Ca2+-channel protein
content or Bm from a group of commercial turkey heavy SR preparations
(110 pmol/mg; n=7) was lower than that from a group of genetically
unimproved turkeys (4.01 pmol/mg; n=7). Average channel protein contents
determined from crude total membrane fraction were also significantly
different between the two types of turkeys. Ca2*-channel protein in heavy
SR from commercial turkey heavy SR exhibited a higher binding affinity for
[3H]ryanodine when compared to that from unimproved turkey heavy SR
(Kd=12.2 versus 20.5 nM), suggesting that differences exist in the channel

Protein activity or its regulation in these two populations. The [3H]ryanodine

binding to the Ca2+ channel protein from both types of turkeys was activated
at a threshold concentration of approximately 0.2 uM Ca2+, and reached a
plateau of binding over the range of 3 to 30 pM free Ca2+, whereas it was
only slightly inhibited at 1 mM Ca2+. Calmodulin (CaM) affinity labeling
experiments indicated that the SR Cay-channel protein from both commercial
and unimproved turkeys bind CaM in Cay-independent fashion under
physiological conditions. However, unlike the SR from unimproved turkeys,
the Ca2+-channel protein was not the main CaM receptor in SR from
commercial turkeys. Instead, a 75 kDa protein present in most of the
commercial turkey SR preparations was the most abundant CaM receptor.
These results support the hypothesis that there is a genetic basis for the PSE
problem in the turkey industry.

To My Beloved Father

iv

ACKNOWLEDGMENTS

I wish to give special thanks to my major advisor, Dr. Gale M.
Strasburg, for his supporting and encouraging during the Ph.D. program. His
positive attitude and enthusiasm for research are an inspiration. He is an
excellent teacher, and also a good friend. I appreciate all he did for me in
past four and half years.

Also, I appreciate all the help from the members of the guidance
committee: Dr. Alden M. Booren, Dr. John E. Linz, Dr. James J. Pestka, and
Dr. William Wells.

This project would not have been possibly finished without Dr.
Richard J. Balander and Dr. Cal I. Flegal who helped to obtain the raw
materials for this research.

I am also grateful for the fiiendship of numerous graduate students
whom I have had the pleasure of working with at Michigan State.

The love, and supporting of my family are the foundation for all my
achievements. I also want to express gratitude to my husband, Yu-Shiun

Lin, for his love and support.

TABLE OF CONTENTS

List ofl‘ ables ............................................................................................... ix
List of Figures .............................................................................................. x
Introduction .................................................................................................. 1
Chapter 1. Literature Review ...................................................................... 4
1. Structure of Muscle Tissue ............................................................ 4
A. Muscle Structure and Membrane Systems ........................ 4
B. "Foot" Structure .................................................................. 7
1. Dihydropyridine Receptor ........................................ 7
2. The SR Cazi-channel Portein ................................... 11
II. Excitation-Contraction Coupling .................................................. 12
III. The Structure of the Ca2+ -channel Protein ................................ 15
IV. The isoforms of the Ca” -channel Protein ................................ 19
V. The Modulators and Phosphorylation of the Ca2+ -channel Protein
................................................................................................... 21
A. Modulators of the Ca2*-channel Protein ........................... 21
I. Calmodulin ................................................................ 23
2. Ryanodine ................................................................. 29
3. Calcium, Magnesium, and Adenine Nucleotide ...... 32
4. Caffeine ..................................................................... 36
B. Phosphorylation of the Ca2*-channel Protein .................... 37
VI. Diseases Relatived to the Skeletal Cay-channel Protein Gene:
Porcine Stress Syndrome and Malignant Hyperthermia ........ 41
Chapter 2. Biochemical properties of the SR CaZI-channel protein from
genetically unimproved turkey skeletal muscle ..................... 46
I. Introduction .................................................................................... 46
11. Materials and Methods ................................................................. 49
A. Materials ............................................................................. 49
B. Methods .............................................................................. 49
III. Results and Discussion ............................................................... 56

vi

vii
A. Purification of SR from Turkey Skeletal Muscle ............. 56
B. Cay-dependent of [3H]ryanodine Binding Assays ........... 56

C. Ryanodine-dependent of [3H]ryanodine Binding Assays . 60
D. Camodulin Binding Activity of Ca2+-channel Protein Isoforms

.......................................................................................... 62

E. Quantitation of Camodulin-binding Activity of Ca2+-channel
Protein in Turkey Skeletal Muscle ............................... 68

IV. Conclusions ................................................................................. 80

Chapter 3. The biochemical basis for PSE quality problems associated with

turkey ....................................................................................... 82

I. Introduction .................................................................................... 82
II. Materials and Methods ................................................................. 85
A. Materials ............................................................................. 85

B. Methods .............................................................................. 85

III. Results and Discussion ............................................................... 91
A. Electrophoretic Analysis of Skeletal Muscle Heavy SR from
Unimproved and Commercial Turkeys ........................ 91

B. Ca2*-dependent of [3H]ryanodine Binding to the Cay-channel
Protein ............................................................................ 94

C. Ryanodine-dependent of [3H]ryanodine Binding to SR
Vesicles from Unimproved and Commerical Turkeys

.......................................................................................... 98

D. Calmodulin Affinity Labeling of SR Vesicle fiom
Unimproved and Commercial Turkeys ....................... 108

E. Partial Characterization of 75 kDa Protein of Commercial
Turkey Skeletal Muscle Heavy SR ............................. 113

IV. Conclusions ................................................................................ 118
Chapter 4. Calmodulin interaction with the purified cardiac Cay-channel
protein(B-likeisoform) ......................................................... 120

I. Introduction ................................................................................... 120
II. Materials and Methods ................................................................ 122
A. Materials ............................................................................ 122

B. Methods ............................................................................. 122

III. Results and Discussions ............................................................. 127
A. Calmodulin Binding Activity of the Purified Cardiac Ca2+-
channel Protein ............................................................ 127

B. Stoichiometry and Affinity of Rh-CaM with Purified Cardiac

viii

Ca2+-channel Protein .................................................... 129

IV. Conclusions ................................................................................ 136
Conclusions ................................................................................................. 138
Future Research ......................................................................................... 141

List of References ....................................................................................... 143

LIST OF TABLES

TABLE PAGE
1.1 Modulators of the sarc0plasmic reticulum Ca2+4channel protein 24
2.1 Equilibrium Constants for Rh-CaM Interaction with the Ca2+-channel

Protein in Turkey Skeletal Muscle Heavy SR vesicles ................... 78
3.1 Densitometric Analysis of SDS-PAGE of Unimproved and Commercial

3.2

4.1

Turkey Heavy SR .............................................................................. 93

Equilibrium Constants of [3H]Ryanodine Binding of Crude and Heavy
SR fi'om Unimproved and Commercial Turkeys at 10 M or 300 [1.1M
[Cay] ................................................................................................. 103

Equilibrium Constants for Rh-CaM Interaction with the Purified

Cardiac Cab-channel Protein in the presence of .1 mM CaCl2 + 2 mM
MgCl2 ................................................................................................ 134

ix

LIST OF FIGURES

FIGURE PAGE
1.1 Diagrammatic representation of macroscopic and microscopic muscle
structure .............................................................................................. 5
1.2 Diagrammatic representation of sarcoplasmic reticulum and T tubules,
and their relation to the myofibrils of mammalian skeletal muscle ........
............................................................................................................... 6
1.3 Model of the triad junction .................................................................. 8
1.4 Model of the subunit structure of the dihydropyridine receptor ..... 10
1.5 A two component model for the calcium release process in
nonmammalian vertebrate skeletal muscle ....................................... 22
1.6 Comparison of the amino acid sequences of calmodulins in their four
domain structures ............................................................................... 25
1.7 The crystal structure of calmodulin .................................................. 27
1.8 Ryanodine structure showing positions of tritium labels (9,21) ..... 30
2.1 SDS-polyacrylamide gel of SR preparations from unimproved turkey
and porcine skeletal muscles ............................................................. 57
2.2 Ca2+ dependence of [3H]ryanodine binding to genetically unimproved
turkey skeletal muscle heavy SR vesicles ........................................ 59
2.3 Ryanodine dependence of [3H]ryanodine binding to unimproved turkey
skeletal muscle heavy SR vesicles at 10 uM Ca2+ .......................... 61
2-4 [Mg2+] and [Ca2+] dependence of affinity labeling of turkey skeletal

2.5

2.6

2.7

2.8

2.9

3.1

3.2

3.3

3.4

3.5

3.6

xi
muscle heavy SR with wheat germ [‘2’I]-Bz-CaM under different
buffer conditions ................................................................................ 63

[Mg21] and [Ca2+] dependence of affinity labeling of turkey skeletal
muscle heavy SR with chicken (Q143C) [‘”I]-Bz-CaM under different
bufi‘er conditions ................................................................................ 66

Titration of Rh-CaM with turkey skeletal heavy SR vesicles under
different conditions ............................................................................. 70

[KCl] dependence of binding of Rh-CaM to turkey skeletal heavy SR
vesicles under different [Ca2+] conditions ........................................ 71

Titration of skeletal heavy SR vesicles with Rh-CaM in the presence
of EGTA ............................................................................................ 74

Titration of skeletal heavy SR vesicles with Rh-CaM in the presence
of CaCl2 .............................................................................................. 76

SDS-polyacrylamide gel of heavy SR preparations from unimproved
and commercial skeletal muscles ...................................................... 92

Ca” dependence of [3H]ryanodine binding to commercial turkey
skeletal muscle heavy SR vesicles ................................................... 95

Ca2+ dependence of [3H]ryanodine binding to unimproved and
commercial turkey skeletal muscle heavy SR vesicles ................... 96

Ryanodine dependence of [3H]ryanodine binding to unimproved and
commercial turkey skeletal muscle heavy SR vesicles at 10 uM Ca2+....

Ryanodine dependence of [3H]ryanodine binding to unimproved and
commercial turkey skeletal muscle crude SR vesicles at 10 uM Ca2+ .....
............................................................................................................. 101

Ryanodine dependence of [3H]ryanodine binding to unimproved and
commercial turkey skeletal muscle crude SR vesicles at 300 uM Ca2+...
............................................................................................................. 106

3.7

3.8

3.9

4.1

4.2

4.3

xii
[Mg2+] and [Ca2+] dependence of affinity labeling of commercial turkey

skeletal muscle heavy SR with chicken (Q143C) [‘”I]-Bz-CaM ...........
............................................................................................................. 109

[Mg2+] and [Ca2+] dependence of affinity labeling autoradiogram of
commercial turkey skeletal muscle heavy SR with wheat germ [”51]-
Bz-CaM under different buffer conditions ...................................... 1 12

Comassie blue and Stains-all stained SDS-polyacrylamide gel of heavy
SR preparations from unimproved and commercial skeletal muscles .....
............................................................................................................. 114

[Mg”] and [Ca2+] dependence of affinity labeling of the purified
cardiac Cay-channel protein with wheat germ [‘”I]-Bz-CaM under
difi‘erentbufi‘erconditions ............................................................... 128

Titration of Rh-CaM with the purified cardiac Ca2+-channel protein ......
............................................................................................................. 130

Titration of the purified cardiac Cay-channel protein with Rh-CaM in
the presence of CaCl 2 and M gCI2 .................................................... 132

INTRODUCTION

The US. turkey industry has grown rapidly in response to increased
consumer demand for lean processed meat products. To meet this demand,
producers have intensely bred for growth efficiency and heavily muscled
* animals. In recent years, the turkey processing industry has been
experiencing severe meat quality problems which closely resemble pale, soft,
exudative (PSE) pork. Meat from these birds tends to be very exudative, and
shows more rapid decline pH, sofier texture, poorer bind and higher cooking
losses compared to non-stressed birds (Sosnicki, 1993). Factors which
increase the incidence of the PSE condition in turkey include stress such as
exposure to heat or cold, and loading turkeys into trucks and transporting
them to the slaughter plant (Froning et al., 1978). These stresses are
reminiscent of the observations of factors which increase the incidence of
PSE pork.

A substantial fraction of PSE pork arises from pigs with porcine stress
syndrome (PSS), an inherited skeletal muscle disorder which affects 10-20%
of the swine population in the US. (Vansickle, 1989). This syndrome results
in substantial economic losses to farmers and processors, owing to death of
animals from stresses of transportation , heat, crowding, etc., before reaching
market, as well as losses to processors because of formation of PSE meat
(Louis, 1993). While PSS has been observed in many swine breeds, the
prevalence of this disorder correlates strongly with genetic selection of

leaner, more heavily muscled and faster growing animals (Zhang et al.,

I

sue:
lid
10- It

has

shits

least :

DIODE

1992).

Biochemical studies indicated that skeletal muscle fibers fiom the PSS-
susceptible pig have abnormal Ca2+ regulation (Mickelson et al., 1986;
Mickelson et al., 1988; Endo et al., 1983). The specific defect was traced
to an abnormality which was subsequently identified as a point mutation in
the skeletal muscle Cay-channel protein (Arg615 to Cys) (Fujii et al., 1991).

The similarities in factors which produce PSE meat from turkey with
that of PSE pork strongly suggests that genetic selection for desirable growth
traits in both species may have resulted in inadvertent selection for a mutated
form of the SR Caz”-channel resulting in undesirable meat quality
characteristics. Furthermore, the fact that turkeys subjected to the same
environmental stresses show different subpopulation of responses lends
additional support for a genetic basis for at least some of the incidence of
PSE turkey.

The hypothesis guiding this study is that a defect in Ca2+ regulation
exists in a subpopulation of commercial turkeys which is responsible for at
least some of the incidence of PSE meat. This defect may take the form of
a mutation in the Cazi-channel protein resulting in altered Ca2+ release
properties. . The first objective of this study was to purify and characterize
the SR from turkey skeletal muscle. The second objective was to define the
biochemical basis for PSE problems associated with commercial turkey. The
third objective was to define the affinity and stoichiometry of the cardiac
Ca2‘-channel protein for CaM, as a model for behavior of one of the turkey
skeletal muscle isofonns.

The dissertation was organized in a series of chapters. The first
chapter is a review of the literature which forms the basis for the studies

conducted for this thesis. Each subsequent chapter addresses a specific

3

objective as indicated above, and is organized as a manuscript with its
specific introduction, materials and methods, results and discussions, and
conclusion sections. The last common sections are Conclusion, Future
Research, and List of References in Journal of Food Science format for the

entire dissertation.

CHAPTER 1
LITERATURE REVIEW

1. The Structure of Muscle Tissue
A. Muscle Structure and Membrane Systems

Muscle tissue consists of muscle fibers, connective tissues, blood
vessels, nerve fibers and blood fluid. The muscle fiber is a cell which forms
the structural and functional unit of muscle tissue. The membrane
surrounding the muscle fiber is called the sarcolemma. The muscle fiber
comprises a highly organized network of contractile elements referred to as
myofibrils (Figure 1.1).

Periodically, along the length of the fiber and around its entire
circumference, invaginations of the sarcolemma form a network of tubules
called transverse tubules (T tubules) which run more or less perpendicularly
to the long axis of the muscle fiber (Figure 1.2). The T-tubules and
cistemae form junctions with a closely meshed membrane network
surrounding each myofibril called the sarcoplasmic reticulum (SR) (Figure
1.2).

The SR consists of several distinct elements: longitudinal tubules,
fenestrated collar, and terminal cistemae. The longitudinal tubules are
oriented in the general direction of the myofibrillar axis. The fenestrated
collar forms a perforated sheet in the H zone regions of the sarcomere. The
terminal cistemae are the rather large, sac-like SR structures which form

junctions on both sides of the T-tubule. The structural element comprising

4

epirnyeium

 

 

Figure 1.1.Diagrammatic representation of macroscopic and microscopic
muscle structure (adapted from Chrystal], 1970).

  
  
 

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Myofilnments } One Sarcomere ' Triad
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cistemae tubule tubule

Figure 1.2 Diagrammatic representation of sarcoplasmic reticulum and
T tubules, and their relation to the myofibrils of mammalian skeletal
muscle (adapted from Judge et al., 1989).

dezsi
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7

the T-tubules and its adjoining cistemae is referred to as the triad.

SR can be separated into two fiactions on the basis of differences in
density using sucrose gradients. These fractions are referred to as light and
heavy SR. The heavy SR fraction is composed of the junctional membrane
(which contains calsequestrin and the Ca2*-channel protein), triads, terminal
cistemae, and some longitudinal tubules. The light SR fraction is almost
entirely composed of longitudinal tubule membrane vesicles. The dominant
protein in light SR is the Ca2+ pump protein (Meissner, 1975; Franzini-
Armstrong, 1980).

B. The SR "Foot" Structure

The foot structure is a protein bridge which spans a gap of about 1.2-
1.4 nm at the junction between the T-tubules and the terminal cistemae of
SR (Franzini-Armstrong, 1970). The major proteins at the triad junction
include the dihydropyridine (DHP) receptors in the T-tubule, which act as
voltage-sensors, and Caz‘lchannel proteins of the SR, which act to release
Ca2+ in the sarcoplasm (Figure 1.3) (Agnew, 1988; Agnew, 1989;
McPherson and Campbell, 1993), as well as triadin, which is a 95 kDa
protein whose function is unknown (Kim et al., 1990; Caswell et al., 1991).
Analysis of the triadin amino acid sequence derived from cDNA indicates
that only 47 amino acids of the protein are cytoplasmic, with the bulk of
protein located in the lumen of the SR (Figure 1.3) (Knudson et al., 1993a).
The lumenal region of triadin is highly positively charged. It may interact
with negative charges on calsequestrin, the high capacity, moderate affinity
Ca2*-binding protein that is localized in the lumenal region of junctional SR
(Knudson et al., 1993b).

1. Dihydropyridine (DHP) receptor

The DHP receptor is so named because this complex binds drugs

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Dihydropyridine Receptor
8"“ I 0 u m N
11 (1 A
T—tubule ml. e11 1 01d e e m t a e e e
VU v V vU

NH.

 

COOH
CW \:yenodlne Receptor /— Foot new

 

 

 

 

 

 

 

 

 

 

 

e—Moouletoryflegton
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coon—J m” can” --coou

 

 

Figure 1.3 Model of the triad junction (adapted from McPherson and

Campbell, 1993).

9

which share a common dihydropyridine basic structure. Examples of these
drugs include nitrendipine, azidopine and PN200-110 (isopropyl 4-(2,1,3-
benzoxadiazol-4-yl)-1,4-dihypro-2,6-dimethyl-methyoxy-carbonylpyridine—3-
carboxylate). The molecular picture of the DHP receptor is that of a large
complex consisting of four or five protein subunits. Leung et a1. (1988)
showed that the purified DHP receptor from rabbit skeletal muscle contains
four protein components of 175 kDa, 170 kDa, 52 kDa, and 32 kDa when
analyzed by SDS-PAGE under nonreducing conditions. Under reducing
conditions, however, it was shown that the 170 kDa subunit comprised a
140 kDa peptide linked by disulfide bonds to smaller polypeptides of 32-29
kDa (Cooper et al., 1987). Later, studies (Catterall, 1988; Campbell et al.,
1988) indicated that there are five peptide subunits which constitute the DHP
receptor, orl (175 kDa), a, (143 kDa), [3 (52 kDa), y (32 kDa), and 5 (27
kDa). The size of the or, subunit predicted from the cDNA clone is 212
kDa, which differs considerably from the size of 175 kDa observed after
purification of DHP receptor (DeJongh et al., 1989). The a, subunit is the
central functional component of the complex; it has four homologous
domains with six proposed transmembrane segments in each domain. The
or, subunit is present in a complex with an intracellularly disposed [5 subunit
and a glycosylated transmembrane y subunit. The (1,, [3 and 7 subunits also
interact with a disulfide-linked glycoprotein complex consisting of a2 and 8
subunits (Figure 1.4). The a, and [3 subunits are substrates for
phosphorylation by CAMP-dependent protein kinase, which can regulate the
Ca2+ ion conductance activity of the DHP receptor. (Catterall, 1988; Catterall,
1991).

The DHP receptor in skeletal muscle can function as a calcium

channel or as a voltage sensor for excitation-contraction coupling (Agnew,

10

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Figure 1.4 Model of the subunit structure of the dihydropyridine
receptor (adapted from Catterall, 1988).

11
1987; Adams et al., 1990; Tanabe et al., 1990; Beam et al., 1992). As

previously described, the a, subunit of DHP receptor has two forms in
skeletal muscle: a full-length translational product (212 kDa) which is
present as a minor species, and a much more abundant form that has a
truncated carboxy-terminus (DeJongh et al., 1989). Recently, Beam et al.
(1992) characterized the fimctional roles of these two proteins using murine
dysgenic myotubes (muscle precursor cells). Muscular dysgenesis is a lethal
autosomal recessive mutation in which skeletal muscle is paralyzed due to
the failure of excitation-contraction coupling resulting from the absence of
the on, subunit of the DHP receptor (Adams and Beam, 1990; Powell, 1990).
Beam et al. (1992) cloned the full length cDNA into the dysgenic myotubes.
After 2-3 days excitation-contraction coupling was restored, suggesting that
only the full-length polypeptide can serve dual functions and that the
truncated form can only function as a voltage sensor.
2. The SR Cazi-Channel Protein

Ca”'-channel proteins from skeletal and cardiac muscle SR have been
purified and biochemically characterized over the past few years. These
proteins are located primarily at the terminal cistemae of the SR which forms
the triad junction with T-tubules (Inui et al., 1987). The molecular weight
of the Cazi-channel protein is over 2,200 kDa; and it consists of a tetramer
of identical 565 kDa subunits (Lai et al., 1988; Irnagawa et al., 1987). The
structure of the Ca2’-channel protein will be described in detail later (See
The Structure of the Cap-Channel Protein).

A protein-protein interaction between Ca2*-channel proteins and DHP
receptors is the mechanism believed to account for excitation-contraction
coupling of skeletal muscle. Block et al. (1988) indicated that a direct

interaction between the foot protein (the Cay-channel protein) and a protein

12

component of junctional T-tubule membrane is observed in fiog skeletal
muscle using fi'eeze-fracture images and rotary-shadowed imaging. Freeze-
fracture images of the junctional T-tubule membranes demonstrate the
presence of diamond-shaped clusters of particles that correspond exactly in
position to the subunits of the foot proteins (or the Cazi-channel proteins).
It was suggested that a voltage-dependent conformational change in the DHP
receptor triggers a secondary conformational change in the SR Ca2*-channel
protein, leading to the opening of the channel and to an increase in
myoplasmic Ca2+ (McPherson and Campbell, 1993; Catterall, 1991).
However, at the biochemical level, a direct coupling of the Ca’*-channel

protein to the DHP receptor has not yet been demonstrated.

11. Excitation-Contraction Coupling
In the muscle fiber, under normal resting conditions, an electrical

potential is maintained at -90 millivolts, and the concentration of free
calcium in the cytosol surrounding the thick and thin filaments is very low,
about 0.1 uM (Martonosi, 1984). A muscle contraction is initiated by a
neuronal stimulus at the motor end plate of skeletal muscle or by an action
potential transmitted along the cardiac muscle fiber. This stimulus is
transferred along the sarcolemma, resulting in depolarization of the
membrane. When the depolarization of the membrane is conducted by the
T-tubule to the SR via the junctional foot protein, the change in polarization
will trigger the Ca2‘-channel protein to open and to release Ca2+ from SR
lumen to the cytoplasm, thus elevating the concentration of free Ca2+ into the
1-100 uM range (a 10-103 fold increase) (Martonosi, 1984). The Ca2+ will
bind to troponin C, which triggers a cascade of conformational changes in

the thin filament permitting actin-myosin cross-bridge formation and

13

subsequent muscle contraction. Upon cessation of the action potential, the
membrane is repolarized, and the SR Ca2+ pump (Ca2+-ATPase) transfers Ca2+
ions back to the SR lumen using energy derived fi'om hydrolysis of ATP,
causing muscle relaxation.

The process of coupling chemical and electrical signals at the cell
surface to the intracellular release of calcium and ultimate contraction of
muscle fibers is termed excitation-contraction coupling. Several hypotheses
exist to explain the mechanism of excitation-contraction coupling in smooth,
cardiac and skeletal muscle cells. However, in each of them, both voltage-
gated calcium channels (DHP receptor) in the cell surface membranes and
intracellular calcium-release channel proteins in the SR play key roles.

In skeletal muscle, the most widely accepted hypothesis of excitation-
contraction coupling mechanism is voltage-dependent calcium release. The
molecular basis of this phenomenon involves the DHP receptor as the T-
tubule voltage-sensor and the junctional foot protein as the SR Ca” release
channel (Schneider and Chandler, 1973; Fleischer and Inui, 1989; Catterall,
1991; Rios et al., 1993). When the action potential arrives at the T-tubule -

SR junction, it is accompanied by an outward movement of positive gating

charge across the T-tubule membrane. This gating-charge movement is the
electrical signature of voltage-driven conformational changes in the DHP
receptor. This transmembrane conformational change might be directly
linked to activation of calcium release through protein-protein interaction
between the DHP receptor and the SR calcium channel (Schneider and
Chandler, 1973; Pietrobon et al., 1988).

Schneider and Chandler (1973) proposed that charged molecules within
the T-tubule membrane move upon depolarization and that the movement of

these molecules provides a direct physical link between T-tubule

14

depolarization and SR Ca2+ release. Their hypothesis was based on the
observation that a voltage-dependent charge movement, which was closely
connected to muscle contraction, could be measured across the T-tubule
membrane under conditions where all ionic currents were blocked. Later, it
was observed that DHPs blocked change movement and SR Ca2+ release with
similar voltage and dose dependences. The observation suggested that the
charge movement was due to gating of the DHP receptor and confirmed its
importance in the activation of SR Ca2+ release (Rios et al., 1992).

In vertebrate skeletal muscle, however, there is another hypothesis
concerning the mechanism of Ca2+ release which involves a soluble, internal
transmitter, inositol 1,4,5-trisphosphate (InsP3). Vergara et al. (1985)
reported the results of experiments using skinned skeletal muscle from
semitendinosus muscle of the frog that suggest the InsP3 may be a chemical
intermediary between the T-system depolarization and the Ca2+ release from
SR in skeletal muscle fibers. T-tubule membrane depolarization stimulates
the hydrolysis of phosphatidylinositol 4,5-bisphosphate by a
phosphodiesterase to form diacylglycerol and InsP3. It was proposed that the
latter binds to a specific receptor on the SR membrane, presumably the Ca2+-
channel protein or an lnsP3 receptor to release calcium. InsP3 is rapidly
deactivated by the InsP3 S-phosphatase to form inositol 1,4-biphosphate.
Vergara et al. (1985) presented four pieces of evidence in support of this
hypothesis. First, InsP3 is generated by electrical stimulation of skeletal
muscles. Second, InsP3 is able to release Ca2+ from SR in skinned muscle
fibers. Third, inhibitors of InsP3 5-phosphatase, such as 2,3-bisphospho-
glycerate, Cd”, Zn”, or Ag‘, increase sensitivity to InsP3. Fourth, drugs that
inhibit InsP3 release are effective blockers of excitation-contraction coupling.

The polyamine antibiotic neomycin, which inhibits InsP3 release, is an

15

effective blocker of excitation-contraction coupling. However, the Ca2+
release initiated by InsP3 is hormonally activated and takes place on a
relatively slow time scale (Caswell and Brandt, 1989). In contrast, Ca2+
release from skeletal muscle is activated by depolarization. It begins and is
terminated within a few milliseconds of depolarization (Caswell and Brandt,
1989). Thus, current evidence does not favor the hypothesis that InsP3 is
involved with the skeletal muscle contraction.

On the other hand, there is strong evidence that Cay-induced Ca2+
release (CICR) is the mechanism for Ca2+ release in heart. According to
this hypothesis, a small transsarcolemmal Ca2+ influx via DHP receptors acts
through the induction of a Ca” release fiom the SR. The trigger for CICR
of Ca2+ is not a mere change of myoplasmic [Ca2+]fm outside of the SR
(A[Ca2*]f,,,) but a function of the rate of change of [Ca2+]free ( A[Ca2+]f,,,/ At).
An increase of [Ca2+]1m to a given level induces calcium release from the SR
when it is applied rapidly, whereas it induces calcium loading of the SR
when it is applied slowly. From the Ca2+ current graph, it was observed that
there were two Ca2+ release stages: a fast component (beat stage) and a slow
component (flat stage). This suggests that the initial relatively fast
component of transsacrolemmal Ca” current would trigger Ca2+ release; the
subsequent slow component would load the SR with an amount of Ca2+

available for release during subsequent beats (Fabiato, 1983).

III. The Structure of the Ca2’-channel Protein

As previously stated, the Ca2*-channel protein is one of dozens of SR
proteins and constitutes about 2-3% of the total SR protein. The localization
of the Ca2*-channel protein to the terminal cistemae of SR was supported by

demonstrations that the plant alkaloid, ryanodine (Jenden and Fairhurst,

16

1969), binds to terminal cistemae but not to longitudinal SR, and that the
Cay-channel blocker ruthenium red enhances Ca2+ loading in terminal
cistemae but not longitudinal SR (Fleischer et al., 1985). These results
suggested that ryanodine could be locking a channel protein in an open state
which would prevent Ca2+ accumulation in terminal cistemae. Identification
and isolation of the Ca2+ -channel protein were also facilitated through the
use of ryanodine which was shown to bind to the protein with high affinity
and to modulate its function (Pessah et al., 1986; Seifert and Casida, 1986;
Lai et al., 1989). Since the Ca2"-channe1 protein was first isolated based on
its ability to bind ryanodine, it is also called the ryanodine receptor (RYR).
Recently, Carl et al. (1995) used immunofluorescence to localize the Ca2+-
channel protein/ryanodine receptor in developing rat skeletal muscle. The
243-9 RYR polyclonal antibody raised in rabbits specifically binds to the
Cay-channel protein in the triads. These results are consistent with the
biochemical studies which indicated that the Cay-channel protein is located
in terminal cistemae of SR.

Studies of the morphology of the Ca2+ -channel protein have shown
that it has a tetragonal or clover-leaf structure (Lai et al., 1988; Lai et al.,
1989). Recently, the three-dimensional architecture of the Ca2*-channel
protein was determined using cryo-electron microscopy (cryo-EM), which
allows structural preservation to high levels of resolution without stains or
fixatives, image averaging, and conical tilt reconstruction. A three-
dimensional reconstruction is then obtained from a random field of molecules
(Raderrnacher et al., 1994; Serysheva et al., 1995). The reconstructed Ca2+-
channel protein shows a clear demarcation between the channel and
cytoplasmic assemblies, both of which display four-fold symmetry
(Radermacher et al., 1994). The cytoplasmic domain or foot protein of the

l7

Ca2+-channel protein is large (29x29x12 nm), whereas the transmembrane
assembly is small, protruding 7 nm from one of its faces. A cylindrical low-
density region, 2-3 nm in diameter, extends down the center of the
transmembrane assembly, and possibly corresponds to the transmembrane
Ca2+-conducting pathway (Radermacher et al., 1994).

Stoichiometry studies by Ferguson plot analysis following SDS-
polyacrylamide gel electrophoresis (PAGE) of partial and fully crosslinked
and incompletely denatured complexes suggest that each Ca2*-channel protein
contains four identical polypeptide chains and the molecular weight of each
subunit is ~ 400 kDa in rabbit skeletal muscle (Lai et al., 1989). Similar
results obtained by Smith et al. ( 1988) indicated that the CaZI-channel protein
purified by immunoaffinity chromatography from rabbit skeletal muscle is
a tetramer which consists of four identical 450 kDa polypeptides. The
purified Ca2*-channel protein has an apparent sedimentation coefficient of
308, indicating that it is a large, tetrameric complex with an estimated
molecular mass > 10" (Lai et al., 1989). Likewise, in gel-exclusion
chromatography, the purified Cay-channel protein elutes as a peak
corresponding to a molecular mass > 10‘ (Inui et al., 1987).

The .Cazi-channel proteins from both cardiac (Otsu et al., 1990) and
skeletal (Takeshima et al., 1989; Zorzato et al., 1990) SR have been
sequenced from their cDNAs. Analysis of the sequence of the cardiac
muscle protein indicates a molecular weight of 564,000 derived from 4969
amino acids in rabbit. The cardiac derivative is 66% identical with that of
the skeletal muscle Ca” release channel, although it contains 63 fewer
residues.

Analysis of the sequence of skeletal muscle Ca2*-channel protein

indicates that there are 10 potential transmembrane sequences in COOH-

18
terminal protein of the molecule. Two additional potential transmembrane

sequences located nearer the center of the molecule could contribute to the
formation of the Ca2+ conducting pore (Zorzato et al., 1990). In contrast,
Takeshima et al. (1989) proposed a 4 transmembrane segment model. The
cardiac Ca’+-channel protein also has up to 12 potential transmembrane
segments (Otsu et al., 1990).

The fact that the Ca’+-channel protein activity is modulated by
numerous factors (see next section) led to various efforts to predict
modulator binding sites based on sequence homology. The regions of Ca2+-
channel protein that may interact with calmodulin, an inhibitor of Ca2+-
channel protein activity, are predicted at residues 277 5-2807 , 2877-2898, and
2998-3016 in cardiac muscle (Otsu et al., 1990). In contrast, Zorzato et al.
(1990) predicted calmodulin-binding sites in skeletal muscle at residues
2807-2840, 2909-2930, and 3031-3049, whereas Takeshima et al. (1989)
predicted 2 calmodulin-binding sites at 3614-3637 and 4295-4325 in skeletal
muscle. In the cardiac Ca2‘-channel protein, a potential ATP binding domain
is identified at residue 2610-2652, and a potential phosphorylation site by the
cAMP-dependent protein kinase at residue 2809 was predicted based on the
consensus sequence (RRXS) (Otsu et al., 1990). On the other hand, the
skeletal Ca2°-channel protein has several nucleotide-binding consensus
sequences found at residues 4449-4454 or 4452-4457 (Takeshima et al.,
1989). In addition, experimental evidence for the involvement of two Ca2+-
channel protein regions in regulating Cay-induced Ca2+ release in skeletal
muscle has been obtained. In malignant hyperthennia-susceptible pigs, the
skeletal muscle Ca”-channel protein contains a cysteine instead an arginine
at position 615 (Fujii et al., 1991), which alters the sensitivity of Ca”-

induced Ca2+ release process. A second Ca2+-sensitive region was obtained

19

in 45Ca2+ and ruthenium red overlay studies with trpE fusion proteins. These
findings led to the identification of three putative Cay-binding sites at amino
acid residues 4246-4467, 4382-4417, and 4478-4512 (Chen et al., 1992;
Meissner, 1994).

IV. The Isoforms of the Cay-channel Protein

Biochemical purification and cloning have revealed at least three
different tissue-specific isoforms of the Cay-channel protein in mammalian
tissues. The Cay-channel protein in skeletal muscle is referred to as the type
1 RYR (RYRl), which is expressed primarily in skeletal muscle, although
it also has been found in the Purkinje cell of cerebellum (Ouyang et al.,
1993) and in brain (Takeshima et al., 1993). The cardiac Ca2*-channel
protein (RYR2) was initially purified (Inui et al.,l987) and cloned (Otsu et
al., 1990) from heart, but it is also expressed throughout the nervous system
(Kuwajirna et al., 1992). A third isoform (RYR3) has been cloned from
brain. It is expressed primarily in brain, but is also present in smooth and
skeletal muscles and some endothelial cells (Hakamata et al., 1992).

Whereas only one isoform has been detected in mammalian skeletal
muscle, Sutko and colleagues first identified the existence of two isoforms
(0: and B) in chicken pectoral muscles (Airey et al., 1990). Subsequently,
two isoforms of Ca2*-channel protein in skeletal muscle have been identified
in different species such as toadfish, frog, bullfrog, turtle, crocodile, and
shark (Coronado et al., 1994; Ogawa, 1994). However, even in the same
species, different muscles possibly contain different proportions of isoforms.
For example, two isoforms (or and B) of Ca2*-channel protein are expressed
in approximately equal abundance in epaxial (swimming) muscles of toadfish
(O'Brien et al., 1993) and blue marlin (O'Brien et al., 1995), whereas only

20

a. isoform exists in extraocular' muscle of both fish. Airey et al. (1990)
indicated that the B isoform is not a proteolytic fragment of the o. isoform,
but is a true muscle isoform based on immunological reactivity, distinct
peptide maps and immunofluorescent microscopy. The polypeptides were
shown to be distinct by limited proteolysis and to be subunits of different
[3H]ryanodine-binding proteins by using isoform-specific antibodies; both
proteins have similar distribution in the same fiber and were found to be
associated with the terminal cistemae of SR by immunolabeling techniques.
Additionally, the two isoforms do not necessarily exist in equal amount; both
are present in equal relative amounts in microsomes from chicken breast
muscle consisting of all white fiber types. However, the a isoform is more
abundant than B in microsomes from chicken thigh muscle (Olivares et al.,
1991)

O'Brien et al. (1995) examined the physiological properties of the a
and B isoforms from different fish skeletal muscles. They found that there
are differences in the calcium activation and inactivation properties of two
isoforms. The a isoform, the sole isoform expressed in extraocular muscle,
displays calcium concentration-dependent [3H]ryanodine binding. The Ca2+-
channel protein is activated at 0.1 uM Ca2+, shows peak activation in the 1-
10 uM range, and is subsequently inhibited by ~1 mM Ca2+. This feature of
the or RYR isoform of fish muscle is similar to that of to the mammalian
skeletal muscle (rabbit) RYR. On the other hand, the B isoform of fish
skeletal muscle more closely resembles the fish and mammalian cardiac Ca2+-
channel protein in calcium concentration-dependent [3H]ryanodine binding.
The most prominent difference of the B and cardiac isoforms from or isoform
is the lack of inactivation of [3H]ryanodine binding and the decreased

maximum open probability (P0) in the presence of millimolar free Ca2+. In

21
addition, [3H]ryanodine binding by the or isoform is selectively inhibited by
100 uM tetracaine (70% inhibition), whereas cardiac Ca2*-channel protein
and the B skeletal muscle isoform are much less affected (less than 5%
inhibition).

O'Brien et al. (1995) proposed a two component model for the calcium
release process in nonmammalian vertebrate skeletal muscle (Figure 1.5). In
this model, the skeletal-like a isoform occupies a position in SR directly
coupled to the DHP receptor or voltage sensor. The B isoform is a logical
candidate to be the calcium-coupled Ca2"-channel protein because it has
functional similarity to the cardiac Ca’+-channel protein, which operates by
Ca2*-induced Ca2+ release. An influx of Ca2+ via the depolarization of the or
isoform would trigger opening of the B channels.

Lai et al. (1992) reported that the amphibian Cay-channel protein or
and B isoforms are related to those of mammalian skeletal muscle and
cardiac muscle, respectively. Immunoblot analysis shows that polyclonal
antiserum to the rat skeletal Ca2’-channel protein preferentially cross-reacts
with the or isoform of frog skeletal Ca2*-channel protein. Conversely, two
monoclonal antibodies to canine cardiac Cay-channel protein preferentially
cross-reactwith the B isoform of frog skeletal Cay-channel protein.

Airey et al. ( 1993) showed that chicken Cay-channel protein isoforms
differ with respect to CaM affinity labeling. While chicken skeletal muscle
microsomes were incubated with azido[”’l]calmodulin, the result
demonstrated that the a isoform Ca2*-channel protein from chicken bound

CaM to a much greater extent than the B-isoform (Airey et al., 1993).

V. The Modulators and Phosphogglation of the Cay-channel Protein
A. Modulators of the Ca2*-channel Protein

22

Two-component model of calcium release

T-tubule

 

 

 

 

 

    

 

 

 

 

 

 

B RYR a RYR
2+ [
Ca SR Lumen

L

 

Figure 1.5 A two component model for the calcium release process in
nonmammalian vertebrate skeletal muscle (adapted from O'Brien et al.,
1995).

23

Regulation of Ca2*-channel protein activity has been investigated by
following Ca2+ efflux from isolated SR vesicles (Nagasaki and Kasai, 1983;
Ikemoto et al., 1985; Meissner et al., 1986; Meissner, 1986a) and by single-
channel recordings of the activity of the Ca2*-channel protein incorporated
from SR vesicle into planar lipid bilayers (Smith et al., 1985, 1986a, b).
Results of these experiments indicate the Ca’*-channel protein activity is
modulated by positive effectors such as micromolar concentrations of Ca2+
(CICR), caffeine, adenine nucleotides and nanomolar concentrations of the
plant alkaloid ryanodine, and by inhibitors such as Mg”, calmodulin,
ruthenium red and micromolar concentrations of ryanodine. In addition,
there are numerous other modulators for Ca2*-channel protein which have
been reported (Table 1.1).

l. Calmodulin (CaM)

CaM is a small, acidic protein whose molecular weight is about 17,000
Da. The complete amino acid sequence of CaM from bovine brain
(Watterson et al., 1980), rabbit skeletal muscle (Grand et al., 1981), human
brain (Sasagawa et al., 1982), scallop (Toda et al., 1981), Tetrahymena
(Yazawa et al., 1981), sea anemone (Takagi et al., 1980), spinach (Lukas et
al., 1984) and wheat (Toda et al., 1985) have been determined (Figure 1.6).
The sequences of CaM from vertebrates are virtually identical except for
some discrepancies in amide assignments, indicating that CaM has been
highly conserved throughout vertebrate evolution. The amino acid sequences
of plant CaMs contain Cys, whereas those of animal CaMs do not.
Comparison of the amino acid sequences of CaMs of vertebrates,
invertebrates, a protozoan, and plants, showed that the primary structures are
not completely conserved among all eukaryotes, but rigid conservations are

observed among vertebrates (Toda et al., 1985; Klee and Vanaman, 1982).

Table 1.1 Modulators of the sarcoplasmic reticulum Ca

24

2+.

channel protein (adapted from Coronado et al., 1994).

 

r

ill-eh

 

- "x °' War a... “a...
.mumones
Deunorubidne 1—100 “M e e ND Rabbitskeletal, rat
ardiaeaad brain
Digoxin 1-20 nM 0 ND * Sheepurdiee
Doserubicin 1-300 all 0 a + Rabbit skeletal. rat cardiac.
“cardiac
MitosaMrone 5-10 .111 4 ND ND Rabbit skeletal
lubidaaone 16-150 “M 9 ND ND Rabbit skeletal
”amines
Gentamicin 1-20 is” - ND :3 Rabbit skeletal
neon ‘ . -20 N - - '1 “It
’“" °°' “ “3.4mm“
Nylfiiue 1-10 uglml 9 ND ND Rabbitekeletal
Pretamine t uglml 9 ND ND Rabbit skeletal
Mreacine 1-100 mM ND 9 ND Rabbitslseletal
Whine 1-100 mM ND 4 ND Rabbitebeletal
Seer-tine 0.4-20 mM 0 + ND Rabbitslreletal
norm .
lensocaine 1-10 mM ND - ND Rabbttslreletal
'ne 0.1-1.5 mM ND - ND Rabbit skeletal
lb'bucaine 008-1.! mM 4 - ND Rabbit skeletal. sheep brain
W 0.1-15 mM ND 0 ND Rabbitskeletalmheqbrain
Precaine 1-20 mM - - - Rabbit skeleta: .

. _ _ - _ t . .
Tm 0.01 2 In" MMWMIII. rat cardiac
Mus-ans 21 vol ND + ND Figurines
Haletbeue 1.5%. 21- vel + 4 ND Degand pigcudiee
beam-am 2i. 25$ vol + NB ND Degaad flebdstal

'flyedd W
basic-chain eql CaA 00 “M 9 ND ND Rabbit skeletal
Wadi! 1-50 m + ND ND RabbitskeletaLchardies
Aqlamitiu. 50 it“ e ND ND Rabbitsbeletd
Pal-interwar! 1-100 m o e e Rabbitaad piceheletal
' ' 80-50 “M 9 - ND Rabbitsbeletal
0.1-10 all - - ND Rabbitelneletal
Med! 16-32 all 9 ND ND Rabbitalreletal
mm
“hes-em 0.1-son “In! ND e + Rabbitsheletangviae
cardiac ratbnra
WA 14.000 n14 ND 4 e Rabbitehdetal
We! 1-1.000 all ND - - Rabbitslteletalbenns
Weaken.
Dim 1-1,000 n14 ND - ND Rabbltslteletaldqusdiec
Incl: 1-1.000 an o - ND RabbitelreletaquaIlses
1-12 “N - ND ND skeletal
In. P 1-1.000 all e - ND Dam-tine. rabbit side“!
1-12 .14 - ND ND
ND ND Ratebelstd
“lb 10-25 I’d!" e
Disc-10 0.01-10 .14 - - ND Rabbltebeletal
1-100 .14 o e e “twig“ rat
M 0.1-1N all 0 ND ND Rabbitshletal
QdisADP n‘bees 1-1‘1 all 0 NE NE Rabbitslelstal
1-2 a." N3 - NE Shdetal
- 1-2 .01 e e e Deco-die:
Danni-lane 10 all ND - ND Rabbltebdetal
new 85-800 all - - ND RebbltfilstahsbeQ-diac
W 0.0-1 .11 ND 0 ND WW
urea #100 all e - ND Mafia:
ruse: 0.01-20 all - - ND mama-um
NIED 08-10 p.14 0 NE ND Rabfltsbaletal
Puebla-ta 0—100 all 0 + + RabbitehaletaLsebbttnsdlac
Myrna 1-‘0 a)! o e e WM
Rees banal 1-300 a“ o - e Rabbitsbaletal
Rib-Ib- red tml-IO all - - - Rabbltehddakdqndnt
Sulnuele 0.1-10 all ND 4 ND Sheepudiae
' 1 IN ND ND ND Rabbttufles
V-w-nl t-uoo nu ND - - ans-aw

 

r, Stimulation of Cab-release, [’leyanodine binding, or Cazi-release
channel open probability. -, inhibition of Ca”-rclease, [’H]ryanodine
binding, or Ca"-release channel open probability; ND, not determined;
NE, no effect. DCCD, N,N’-dicyclohexylcarbodiimide; O-HCH, 8-
hexachloro-cyclohexane'FLA36512,6-dichloro-4-dimethylaminophenyl]
isopropyl-amine; MBED, 9-methyl-7-bromoeudistornin D.

25

‘<
N
I
‘<
o
N

 

, 0
loans TTKFLGTVWWSL

Bovine Ac-A D Q L T E E
Rabbit - - - - - - -
Human , - - - - - - -
Sea Aneeone x (- B 2)- - Z Z
Scallop - - - - - - -
Tetrahyeena - - - - - - -
Spinach X (V 8 S - N - -
wheat - - - - - D -

- +1

toerer
erosion}:
I
I
O
r

I
I
O
I
OnlllII-C

Warrior—n]

23
[IrisocsOEx
1.»

Ditto.
O
s
s
s
kittens
0
I
s

2
[LIIIIII—l
O
U
I
I
[T

 

 

 

 

 

 

 

 

w _ m _ _ m _
GQNPTEAE T 0%M1NEVDADGNGTIDFPEFLTHHARKH
-------- - zm--s--s-u-a---a------------ II
- - - - - - - - - - - ..... - - - - o - - - - - - - - - - - - . - - -

- - - - - - - - - - - ----- - - -(-)o - - - - - - - - - s L - - -)- -
-------- C -- ..... -----------.-.NL.----

. d ----- .l

w so 10 pm

xoroscrr F RTAFRVFDKDGNGVISAA LRHVHTNL
-a-a-zz- - -- ----- ------r---- ------e- In
- - - - - - - - - - - ..... - - - - D - p - - - - - - - - - - - -
-------- L I---K---R--D-L-T-- ----..--
-------- L K- ----- ---Q--F---- -b%-----
mg a ..... aw.--- -- L

 

 

O
'5
-.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

[oorrtsoflh[|sssrssm]

2 3
GEK'LTDEE V DEHIREANIDGDGEWME
-------- - -- ..... ------Q----
- - - - - - - - - - - ..... o - - - - - Q - - - -
-----nz- - -- ----- a-s-n-z-a-z rv
- - - - - - - - - - . ..... o . - - - - Q - - - -
--3----- - -- ----- o-----ur---
-4---—-- -- ----- ov----or---
-------- J -H ----- EV""0M"'

 

 

 

Figure 1.6 Comparison of the amino acid sequences of calmodulins in
their four domain structures. Identical or similar residues among eight
sequences through the four domains are boxed with solid lines (adapted

from Toda et al., 1985).

26

The CaM sequence, which consists of 148-149 amino acids, can be
divided into four domains. Each domain contains one helix-loop-helix
structure which provides a Cay-binding site. The crystal structure of CaM
suggests there are two globular calcium-binding domains, each containing
two Cay-binding regions with the characteristic helix-loop-helix structure,
connected by a long central helix of nearly seven turns (Babu et al., 1988;
Taylor et al., 1991; Rao et al., 1993). The four Ca2*-binding sites can be
divided into two classes based on affinity: two high affinity sites in the C-
domain which bind Ca2+or Mg” and two low affinity sites in the N-domain
which are specific for Ca” binding (Figure 1.7).

CaM regulates a variety of calcium-dependent intracellular processes
including activation of regulatory enzymes such as certain kinases,
phosphatases, cyclases, phospodiesterases, and ATPases (see Klee and
Vanaman, 1982 for review). An important question regarding the mechanism
of action of CaM concerns which portions of this protein participate in the
interaction with individual target enzymes. O'Neil and DeGrado (1990)
suggest that as a general mechanism CaM will bind in a Ca’+-dependent
manner with high affinity and a 1:1 stoichiometry to a variety of peptides
known to form amphiphilic helices. The affinity of these peptides for CaM
correlates with their propensities for forming positively charged helices
suggesting this structural feature might be important for binding. On the
other hand, the hydrophobic helices of these peptides may interact with their
target substrates or proteins. Flexibility in the helix connecting the globular
domains of CaM will clearly facilitate CaM binding to a variety of such
sequences because the details of specific interaction in the complex can vary
depending on the precise manner in which the two globular domains

approach each other. The flexibility thus provides a mechanism by which

27

CALMODULIN

«I.
r 1,.
i“

 

 

 

 

Figure 1.7 The crystal structure of calmodulin (adapted from Babu
et al., 1985). The four Ca2+ ion are represented by dotted circles.

28

the highly conserved CaM molecule can bind to a wide variety of target
enzymes whose CaM binding sequences display considerable variability.

Previous studies have shown that Ca2+ release from the heavy SR
vesicles which contain the Ca2*-channel protein, is directly inhibited by CaM
both in skeletal muscle (Meissner, 1986a; Plank et al., 1988) and cardiac
muscle (Meissner and Henderson, 1987). The SR vesicles were passively
loaded with "Ca” in the presence of CaM and its inhibitors, followed by
measurement of the Ca2+ release rate. The results showed that CaM at a
concentration of 2-10 11M reduces Ca2+ efflux rates from SR vesicles by 2-3
fold in skeletal muscle (Meissner, 1986a). In cardiac SR vesicles, up to 6-
fold reduction of Ca2+ release was observed (Meissner and Henderson, 1987).
Subsequently, Smith et al. (1989), using the planar bilayer-vesicle fusion
technique, reported that the inhibitory effect of CaM on both the skeletal and
cardiac SR Ca’*-channel proteins resulted from reduction of the open state
probability via direct binding of CaM to the Ca2*-channel protein rather than
via phosphorylation of substrates.

More recently, fluorescence anisotropy was used to characterize CaM
interaction with the skeletal muscle Ca2+-channel protein in SR vesicles
(Yang et al., 1994). The results showed that at low Ca2+ concentrations (in
the presence of 1 mM EGTA), the tetrameric Ca2*-channel protein binds 20-
24 molecules of CaM with high affinity (Kd=8.6 nM), corresponding to 5-6
molecules of CaM per channel protein subunit. In the presence of 0.1 mM
Ca2+, approximately 4 high affinity (Kd=4.3 nM) and 16 low affinity (K5239
nM) sites per intact Cazflchannel protein were observed. In the presence of
0.1 mM Ca2+ and 1 mM Mg", a single binding site with a very high afiinity
(Kd=0.1 nM) and around 7 sites with a lower affinity (Kd=70 nM) were
obtained.

29

Similar results were observed by Tripathy et al. (1995), using
[‘”I]CaM and [3H]ryanodine binding to SR vesicles and to the purified Ca2+-
channel protein. Their data indicated that the tetrameric Ca2"-channel protein
binds CaM with high affinity (Kd= 5-25 nM). The channel tetramer binds
l6 CaM at low Ca2+ concentration (S 0.1 nM) and 4 CaM at 0.1 mM Ca2+.

However, their experimental conditions were not able to detect the CaM
binding at the low affinity sites (Kd=239 nM) of the Ca2*-channel protein.
In addition, SR vesicle-”Ca2+ flux and single channel measurements showed
that at lower Ca2+ concentrations (5 0.2 nM) corresponding to the resting
muscle state, CaM activates the Ca2+ release channel 1.5-5 fold. On the
other hand, at micromolar to millimolar Ca2+ concentrations, CaM inhibits
the Ca2+ release channel activity 3-6 fold. These results suggest that the
CaM may play dual roles in modulating SR Ca2+ release of skeletal muscle
at both resting and elevated Ca” concentrations (Tripathy et al., 1995).

In addition, using fluorescence anisotropy measurements, binding of
CaM to porcine cardiac muscle SR vesicles were determined (Strasburg et
al., 1993). In the presence of CaCl2 plus MgClz, binding of CaM to the
Cazfichannel protein in cardiac SR vesicles yielded a K,l of 13 nM and a Bmx
of 55 pmol/mg (Strasburg et al., 1993). The results suggest that there are
approximately 3 CaM binding sites per subunit in Cay-channel protein
(Strasburg et al., 1993).

2. Ryanodine

Ryanodine is a neutral alkaloid isolated from the plant Ryam'a
speciosa. The structure of ryanodine is shown in Figure 1.8. Ryanodine
first gained attention because of its pharmacological properties. Even at low
nanomolar to micromolar concentrations, ryanodine can alter the mechanical

performance of vertebrate skeletal muscle and cardiac muscle (J enden and

3O

 

Figure 1.8 Ryanodine structure showing positions of tritium labels (9,21).

31

Fairhurst, 1969). However, the effects of ryanodine on muscle are complex
and depend on muscle type, calcium activity, pattern of muscle stimulation,
as well as ryanodine concentration.

In 1986, Meissner reported that ryanodine either stimulates or inhibits
Ca2+ efllux fiom skeletal muscle heavy SR vesicles depending on the
experimental conditions. At 10 nM ryanodine, the vesicle is rendered
permeable to 4sCa2+ even in the presence of two Cay-channel protein
inhibitors, Mg’+ and ruthenium red. At ryanodine concentrations greater than
10 uM, ”Ca” efflux is inhibited in channel-activating medium (Meissner,
1986b).

The effects of high and low concentrations of ryanodine on the activity
of the Cay-channel protein were investigated using the channel protein
reconstituted from SR into planar lipid bilayers (Bull et al., 1989; Lai et
al.,l989; Chu et al., 1990). Upon addition of 10 nM ryanodine at the cis
side (SR cytoplasmic side) in the absence of ATP, the open state probability
of the Cay-channel protein increases from 0.75 to 0.94 without modifying
the current amplitude (Bull et al., 1989). However, upon addition of uM
ryanodine at the cis side, the Ca2*-channel protein shows long openings (Chu
et al., 1990) and enters into a characteristic subconductance state which has
~40% of the normal conductance (Lai et al., 1989). Upon further addition of
cis-ryanodine to millimolar concentrations, the subconductance state of the
channel protein abruptly disappears; and the channel protein enters into a
fully closed state (Smith et al., 1988; Lai et al., 1989). The observation of
two distinct effects of low and high ryanodine concentrations on conductance
behavior of the channel protein suggested the existence of high and low
affinity sites for ryanodine (Lai et al., 1989).

Ryanodine is a good probe for investigating the function of the SR

32

Ca2I—channel protein because of its specific binding properties for the Ca2+-
channel protein. Ryanodine binds specifically to the Ca’I-channel protein in
SR vesicles with a stoichiometry of 1 mole of ryanodine per mole of channel
protein tetramer (Lai et al., 1988). In addition, ryanodine binds to the Ca”-
channel protein only when the Ca’*-channel protein is in the open state
(Michalak et al., 1988; Ogawa and Harafirji, 1990; Inui et al.,l988; Pessah
et al.,l986). Therefore, the binding of 3H-ryanodine was used as a probe to
characterize the Cay-channel protein content in SR, channel structure, and
channel activity.
3. Calcium, Magnesium, and Adenine Nucleotide

Both planar lipid bilayer-single channel recording (Smith et al., 1988;
Smith et al., 1986b) and ion flux measurements (Meissner and Henderson,
1987; Meissner et al., 1986; Smith et al., 1985) have indicated that the Ca”-
channel protein is activated by micromolar concentrations of cytoplasmic
Ca2+ and by mM adenine nucleotides, and is inhibited by mM Mg”. In
addition, the results of [3H]ryanodine binding were consistent with the data
of the previous studies in SR vesicles (Pessah et al., 1987; Pessah et al.,
1986; Pessah et al., 1985; Seifert and Casida, 1986).

Smith et al. ( l986b) reported that the open state probability (P) of the
Cazfichannel protein is close to zero with nanomolar free Ca2+ on the cis side
of the bilayer. The addition of micromolar Ca2+ to the cis side (cytoplasmic)
activates the channel protein and induces rapid channel openings and
closings occurring as single events or as bursts that could last fi'om less than
one to many milliseconds. The presence of millimolar Ca2+ to the cis side
results in channel inactivation (Meissner, 1994). Thus, in the absence of
other regulatory ligands such as Mg2+ and ATP, a bell-shaped Ca2+ activation

curve of Ca” efflux from skeletal muscle heavy SR vesicles has been

33

obtained, with Ca2+ effiux being maximal in the 1-10 1.1M Ca2+ concentration
range (Fill et al., 1990; Meissner et al., 1986). In addition, the Ca2+-
dependence of [3H]ryanodine binding by skeletal SR vesicles also shows a
similar bell-shaped binding curve with a Ca2+ threshold of 0.1-1 uM, an
Optimal Ca2+ concentration at 10-100 M, and inhibition at Ca2+
concentrations > 1 mM (Irnagawa et al., 1987; Mickelson et al., 1988; Chu
et al., 1990; Zimanyi and Pessah, 1991). Binding of ryanodine to cardiac SR
has a Ca2+ threshold of ~1 uM, is optimal at 10 uM to 1 mM, but, in contrast
to skeletal Ca-channel proteins, shows little inhibition at high Ca2+
concentration (Zimanyi and Pessah, 1991; O'Brien et al., 1995).

Millimolar ATP is found to be as good as or better than Ca2+ in
activation of Ca2*-channel protein (Smith et al., 1988; Smith et al., 1986b;
Meissner et al., 1986; Meissner and Henderson, 1987; Smith et al., 1985;
Chu et al., 1990). However, in single channel measurements, both Ca2+ and
ATP are required to fully activate the channel, i.e., to increase Po close to 1.0
(Smith et al., l986b).

Smith et al. (1986b), using the planar lipid bilayer method, indicated
that millimolar ATP in the absence of Ca2+ increases the fi'equency of open
events anddecreases the duration of closed events (Po=0.6). ATP and Ca2+
together produce a synergism of activation that increases the duration of
open events and allows the channel protein to remain open nearly 100% of
the time (P°> 0.99).

The addition of 5 mM ATP or of the ATP analogue adenosine 5'-
(B,y—methylenetriphosphate)(AMP-PCP) results in intermediate Ca2+ release
rates as determined by radioisotope flux-rapid-quench-Millipore filtration
method (Meissner et al., 1986). Comparison of the activation of Ca2+ release
between skeletal and cardiac SR vesicles showed that addition of 5 mM ATP

34
to 10'9 M Ca” medium increases "Ca” efllux loo-fold fiom skeletal SR

vesicles, but only 2-fold increase fiom cardiac SR vesicles (Meissner and
Henderson, 1987).

In [3H]ryanodine binding measurements, millimolar ATP has been
shown to stimulate ryanodine binding to skeletal Ca”-chanrrel proteins
(Imagawa et al., 1987; Bull et al., 1989; Chu et al., 1990; Ogawa and
Harafuji, 1990; Zimanyi and Pessah, 1991), but to have little effect on
cardiac Ca”-channel proteins (Michalak et al., 1988; Zimanyi and Pessah,
1991). In the presence of optimal Ca” concentration (50 nM), 1 mM AMP-
PCP enhances the [3H]ryanodine binding 4-fold in skeletal SR vesicles but
only 1.3-fold in cardiac SR vesicles (Zimanyi and Pessah, 1991).

Various other adenine nucleotides (AMP-PCP, ADP, AMP, cAMP,
adenosine, adenine) potentiate Ca” release, which suggests that activation
results from binding to an effector site of the Ca”-channel protein rather than
through covalent modification of the Ca”-channel protein via a
phosphorylation reaction (Meissner, 1994).

Mg” affects the Ca”-channel protein in the opposite manner.
Meissner et al. (1986) indicated that Mg” partially inhibits Ca”- and
nucleotide-induced ”Ca” efflux of the Ca”-channel protein in skeletal SR
vesicles. Varying concentrations of Mg” were added to media containing
4 uM free Ca” and 5 mM AMP-PCP. In the presence of 0.14 mM free
Mg”, the rate constant of “Ca” efflux was reduced fiom 56 to 30 s". A
more dramatic decrease of the rate constant to 1.3 s'1 was observed when the
free Mg” concentration was increased to 4 mM. In the absence of AMP-
PCP, Ca” release was fully inhibited at 5 mM Mg” (Meissner et al., 1986).
Likewise, in single channel recordings, cis 4 mM Mg” partially inhibited
the CazI-channel activity by decreasing opening probability (Lai et al., 1988).

35

In addition, when cis 0.12 mM Mg” (4 uM tree Mg”) was added to the
Ca”-channel protein which had been activated by 1.84 mM ATP and <10
nM cis free Ca”, the single channel recording produced an unresolvable
flickering. However, 30 seconds after Mg” addition, inhibition was
essentially complete, with channel opening occurring infiequently (Smith et
al., l986b).

The cardiac Ca”-channel protein, like the skeletal channel protein, is
inhibited by millimolar Mg”. Ca”-induced Ca” release rates in the presence
or absence of 5 mM AMP-PCP were greatly reduced by 3 mM Mg”
(Meissner and Henderson, 1987). Similarly, at the single channel level, 3-5
mM Mg” quickly blocked the channel, but the block was usually transient,
with some activity returning spontaneously (Hymel et al., 1988).

Millimolar Mg” has been shown to effectively inhibit ryanodine
binding to the skeletal Ca”-channel proteins but has little effect on the
binding to cardiac Ca”-channel proteins (Pessah et al., 1985). The effect of
Mg” has been attributed to a decrease in both the apparent binding capacity,
Bum, and the binding affinity, Kd, resulting from a slowing of the ryanodine
association rate without a change in the dissociation rate (Chu et al., 1990).
Mg” inhibition of ryanodine binding may result fiom a direct competition
between Ca” and Mg” for the Ca” activation site of the Ca”-channel
protein (Pessah et al., 1987; Nagasaki and Kasai, 1983; Meissner and
Henderson, 1987).

There are two proposed mechanisms for the inhibition of Ca” release
by Mg”: (1) Mg” could competitively bind to low-affinity Ca” inhibitory
sites of the Ca”-channel protein (Kirino et al., 1983); (2) Mg” could
sterically block the Ca”-channel protein as it binds to a site near the

conduction pathway (Meissner et al., 1986; Smith et al., 1986b). At present

36

there is insufficient evidence to support or exclude either mechanism.
4. Caffeine

Caffeine enhances the Ca”-channel protein activity in "Ca” efflux and
in single channel measurements (Kirino et al., 1983; Meissner and
Henderson, 1987; Rousseau and Meissner, 1989). Caffeine mimics the
effects of cytoplasmic Ca” in that it is more effective in stimulating Ca”
release from cardiac (500-fold) than skeletal vesicles (20-fold) in low Ca”
medium. At micromolar Ca”, 10 mM caffeine, like 5 mM nucleotide,
optimally stimulates 45Ca” efflux from cardiac vesicles (2500-fold), whereas
in skeletal vesicles caffeine has a smaller efi‘ect (16-fold) (Meissner and
Henderson, 1987). Kirino et al. (1983) also indicated that caffeine enhances
"Ca” efflux about 50% in lower Ca” concentration (pCa=6-7) but it does
not in the higher Ca” concentration range. In the single-channel
measurements (Rousseau and Meissner, 1989), the cardiac Ca”-channel
protein is activated in a steady-state manner by mM caffeine on the cis side.
In the presence of cis nM free Ca” and caffeine, the Ca” channel protein is
activated by increasing the total number of open events, increasing the mean
lifetime of the short open state, and causing the appearance of a second
longer lasting open state. The caffeine-activated channel is moderately
sensitive to the voltage applied across the bilayer, is sensitive to further
activation by ATP, and is inhibited by Mg” and ruthenium red (Rousseau
and Meissner, 1989).

In addition, caffeine in the millimolar range has been found to
stimulate [3H]ryanodine binding to skeletal Ca”-channel proteins and, to a
lesser extent, to cardiac Ca”-channel protein. In the absence of Mg”, the
concentration of caffeine resulting in 50% of the maximal activation (EC,0)
is 0.26:0.03 mM for cardiac SR vesicles, whereas the ECso is 1.8:l:0.l mM

37

for skeletal SR vesicles. The result showed that caffeine is seven times more
effective at stimulating [3H]ryanodine binding to Ca”-channel protein in
cardiac than skeletal SR vesicles. In the presence of 1 mM Mg”, caffeine
is 2.5-fold more potent in the [3H]ryanodine binding to cardiac Ca”-channel
protein SR vesicles (ECw=3.52:l:0.14 vs. 8.55:1:0.61 mM); but the maximal
effect on occupancy is similar in both preparations (Zimanyi and Pessah,
1991). Chu et al. (1990) indicated that caffeine (with or without ATP)
enhances the ryanodine association rate of the Ca”-channel protein without
a change in the dissociation rate. However, in the presence of Ca” and
Mg”, caffeine appears to increase the affinity of the activation site of the
Ca”-channel protein for Ca” (Pessah et al., 1987; Kirino et al., 1983).

B. Phosphorylation of the Ca”-channel protein

In cardiac muscle, Ca” uptake activity by the SR Ca”-stimulated
ATPase is stimulated by CaM- and CAMP-dependent protein kinases,
resulting in increased rate of relaxation and increased contractility of the
heart. Cardiac contractility may also be enhanced by CaM-dependent
phosphorylation of the cardiac channel protein which could result in an
increase in the Ca”-release rate.

Takasago et al. (1991) indicated that the exogenous addition of the
CAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase
(PKG) or the endogenous CaM-dependent protein kinase (CaM-kinase)
induces rapid phosphorylation of the Ca”-channel protein in canine cardiac
microsomes. Added protein kinase C (PKC) also phosphorylates the cardiac
Ca”-channel protein but at a relatively slow rate. The phosphorylation by
PKA, PKG, and PKC increases [3H]ryanodine binding around 20%
comparing with control treatment, respectively. In contrast, the endogenous
CaM-kinase decreases [3H]ryanodine binding by 38% (added CaM alone)

38

(0.8 pmol/mg versus 1.2 pmol/mg) or 53% (added CaM and ATP) (0.6
pmol/mg versus 1.2 pmol/mg). The observations of phosphopeptide mapping
and phosphoamino acid analysis suggested that PKA, PKG, and PKC
predominately phosphorylate serine residue(s) in the same phosphopeptide,
whereas the endogenous CaM-kinase phosphorylates serine residue(s) in
different phosphopeptide(s). Thus, these results led to the suggestion that the
phosphorylation of distinct serine residues in the Ca”-channel protein up-or-
down-regulates its channel activity.

Additionally, the level of 32P incorporation in the presence of PKA,
PKC, or PKG was comparable with the maximal level of [3H]ryanodine
binding, indicating a stoichiometry of ~1 mole of phosphate per mole of the
Ca”-channel protein tetramer. In contrast, the phosphorylation by
endogenous CaM kinase was ~4 times greater than that with PKA, PKG, or
PKC (Takasago et al., 1991). However, Witcher et al. (1991) demonstrated
that phosphorylation of the channel protein by CaM-dependent kinase occurs
at a single serine residue (2809) on each subunit. Phosphorylation of the
cardiac channel protein by endogenous CaM kinase was about one-fourth the
value achieved with exogenous CaM kinase. These results indicated that the
endogenous CaM kinase phosphorylated only one of the available sites,
whereas exogenous CaM kinase phosphorylated all four of the channel
protein subunits. Phosphorylation of the channel protein, in turn, results in
increased Ca” conductance which could cause increasing muscle
contractility.

In preliminary studies, Strasburg et al. (1993) compared binding of
CaM to the phosphorylated and unphosphorylated forms of channel in SR
vesicles. There are two classes of CaM-binding sites in the Ca”-channel

protein: high affinity and low affinity binding sites. When the cardiac

39

channel protein was phosphorylated by CaM-dependent kinase, it resulted
in elimination of CaM binding to the low affinity CaM-binding sites; but
there was no effect on CaM binding to the high affinity sites. The change
of CaM binding also could result fi'om small amounts of other proteins in the
SR membrane which still bind CaM, or fi'om uncompleted phosphorylation
of cardiac channel protein.

Whereas the cardiac Ca2+-channel protein is an excellent substrate for
the multifimctional Ca2*/CaM kinase, phosphorylation of skeletal muscle
Ca2+-channel protein by endogenous and exogenous CaM kinases was found
to be more variable (Seiler et al., 1984; Strand et al., 1993; Wang and Best,
1992; Hain et al., 1994; Mayrleitner et al., 1995). Seiler et al. (1984) found
that high molecular mass proteins of junctional SR of both cardiac and
skeletal muscle, later identified as the Ca2*-channel protein, were
phosphorylated by endogenous CaM kinase or by exogenous PKA. Later,
Witcher et al. (1991) indicated that the cardiac Ca2*-channel protein is
phosphorylated in junctional SR vesicles by endogenous CaM kinase and that
this phosphorylation is stimulated several—fold when exogenous CaM kinase
is added. In contrast, the Ca”-channel protein in canine fast and slow
skeletal muscle SR vesicles is not significantly phosphorylated by either
endogenous or exogenous CaM kinase (Seiler et al., 1984). Strand et al.
(1993) also reported that there is minimal phosphorylation of intact porcine
skeletal muscle SR Ca2’-channel protein by either PKA or endogenous CaM
kinase. They found that the level of 32P incorporation of the cardiac Ca2+-
channel protein in the presence of either PKA (6.4 pmol Pi/mg SR) or CaM
(endogenous CaM kinase)(10.6 pmol Pi/mg SR) is approximately equal to or
twice the [3H]ryanodine binding activity of this preparation (5.2 pmol/mg).

In skeletal muscle, however, the level of PKA or endogenous CaM kinase

4O

catalyzed phosphorylation (0.2 pmol Pi/mg SR for PKA and 2.9 pmol Pi/mg
SR for CaM kinase) is much less than the [3H]ryanodine binding activity of
this fraction (11.6 pmol/mg). These results indicated that there are one or
two phosphorylation sites in the cardiac Ca’+-channel protein tetramer,
whereas there is less than one phosphorylation site in the skeletal channel
tetramer.

However, more recently, more extensive phosphorylation of the
skeletal Caz‘fichannel protein by endogenous or exogenous CaM kinase,
PKA, PKG, or PKC has been demonstrated (Suko et al., 1993; Mayrleitner
et al., 1995). In the skeletal Ca2+-channel protein, Ser 2843 was identified
as the major target for PKA, PKG, and CaM kinase (Suko et al., 1993). The
site is homologous to Ser 2809 of the cardiac Cazfichannel protein.
Recently, Mayrleimer et al. (1995) found that phosphorylation of skeletal
terminal cistemae of SR vesicles with PKA, PKC, or Ca2*/ CaM kinase 11
reduces the Ca2+ loading rate of terminal cistemae of SR vesicles 3-fold, 1.7-
fold and 2.1-fold, respectively. Phosphorylation stoichiometries of 1.94
(”P/tetramer) for PKA, 0.89 for Ca2*/CaM kinase II and 0.95 for PKC were
obtained.

The . effects of protein phosphorylation on skeletal muscle SR Ca”
release showed variable effects. Herrmann-Frank and Varsanyi (1993)
reported that endogenous phosphorylation of skeletal muscle SR by CaM
kinase resulted in an increase in the open probability and an increase in the
Ca2+ and ATP sensitivities of skeletal channels incorporated into planar
bilayers. Hain et al. (1993) found in single channel studies that the Ca2+-
channel protein activity can be made insensitive to block by Mg2+ when
terminal cistemae of SR, incorporated into planar bilayers, were treated with

PKA or Can CaM kinase II, and again made sensitive by treatment with

41

protein phosphatases. In contrast, an inactivation of the Ca’*-channel protein
in skeletal muscle SR resulting from CaM kinase phosphorylation was
observed (Wang and Best, 1992).

Airey et al. (1993) indicated that the or and B Ca2+-channel protein
isoform from chicken skeletal muscle are differentially phosphorylated by
CaM kinase II. The a isoform was only slightly phosphorylated relative to
that of the B isoform. This is consistent with the limited phosphorylation of
mammalian skeletal muscle Cazflchannel protein which is phosphorylated to
an insignificant extent by CaM kinase in comparison to the mammalian
cardiac muscle Cay-channel protein (Strand et al., 1993; Witcher et al.,
1991; Takasago et al., 1991; Seiler et al., 1984). The more extensive
phosphorylation of the B isoform than the or isoform in the chicken skeletal
muscle SR vesicles is consistent with the similar fimction of this isoform

with the mammalian cardiac muscle Ca’*-channel protein (Airey et al., 1993).

VI. Diseases Related to the Skeletgl Cazfichannel Protein Gene:
Porcine Stress Syndrome and Malignant Hyperthermia

Porcine stress syndrome (PSS) has been recognized as a problem in
the pork industry since the late 19503. PSS develops with exposure of
animals to cold or high temperatures, crowding, transport, and often results
in deleterious pale, soft, and exudative pork (PSE) (Harrison, 1979). In
addition, this syndrome is triggered following exposure of animals to
volatile, halogenated anesthetics such as halothane (Hall et al., 1980). PSE
meat is usually associated with stress-susceptible animals (Cheah et al.,
1984). These animals have an abnormal calcium ion homeostasis that causes
a flood of calcium into the system upon stress which activates muscle

contraction and glycolysis (Mickelson et al., 1988, 1986). PSE meat displays

42

a rapid initial posnnortem pH decline because of increased glycolysis, a
reduction in water-holding capacity, altered protein solubility, and a pale,
unstable color.

PSS is an inherited skeletal muscle disorder which is controlled by a
recessive gene with incomplete penetrance referred to as the halothane
(HAL) gene (Reik et al., 1983; Zhang et al., 1992). The presence of this
gene is associated with smaller litter size, slower growth rate, shorter carcass
length, larger longissimus muscle area, greater lean percentage, and a higher
incidence of PSE pork (Simpson and Webb, 1989; Webb and Simpson,
1986). The HAL gene, when homozygous (nn), seems to increase the water
content in lean muscle and suppress fat deposition in lean tissues while
reducing meat quality, whereas the heterozygous individuals have higher
meat quality and grow more quickly (Zhang et al., 1992).

Malignant hyperthermia (MH) is a potentially fatal genetic disease
characterized by an accelerated muscle metabolism, contracture development,
and rapidly rising temperature in response to certain anesthetics such as
halothane and depolarizing muscle relaxants such as succinylcholine
(Johnson, 1993). In humans, the trait is usually inherited in an autosomal
dominant fashion, but in halothane-sensitive pigs with a similar genotype,
inheritance of the disease is autosomal recessive or co-dominant. From
studies of an animal model of MH condition, PSS, abnormalities of the Ca2+-
channel protein were postulated as the underlying cause of this condition in
pigs (Ball and Johnson, 1993).

Biochemical studies involving genetically defined swine first suggested
the molecular basis for abnormality in PSS-susceptible animals. SR vesicles
isolated from skeletal muscle (Fill et al., 1990; Mickelson et al., 1986, 1988)
and skinned muscle fiber preparations (Endo et al., 1983) from PSS-

43

susceptible animals displayed higher rates of Ca2*-release as triggered by a
variety of agents including uM Ca2+, caffeine, and halothane. Other studies
indicated that the resting muscle Ca2+ concentration in PSS-susceptible
animals is elevated (Lopez et al., 1986). These data suggested that there is
a defect in the Cay-release channel protein of the skeletal muscle SR

The studies using isolated porcine muscle SR vesicles loaded with
“Ca” demonstrated that the rate constant for calcium release follows a bell-
shaped curve with respect to the ionized Ca2+ concentration (Fill et al., 1990;
Mickelson et al., 1986). This calcium-induced calcium release from isolated
SR vesicles of PSS pigs is approximately twice as fast as calcium release
from normal SR vesicles (Fill et al., 1990; Mickelson et al., 1988, 1986).

Another approach that has been used to examine the SR Cay-channel
protein function in PSS-susceptible pigs is [3H] ryanodine binding. When the
ryanodine-binding activities of SR vesicles derived from genetically
characterized normal Yorkshire and PSS-susceptible Pietrain pigs were
compared, it was observed that SR from the PSS-susceptible animals bound
ryanodine with significantly higher affinity than normal SR (Kd=92 nM and
265 nM, respectively), whereas the amount of ryanodine bound was not
significantly different between breeds (Mickelson et al., 1988). Furthermore,
in studies with Yorkshire-Pietrain crosses, it was observed that animals
heterozygous for the halothane-sensitivity gene displayed intermediate rates
of Ca2’-release and ryanodine affinity; i.e., CaZI-release and ryanodine
affinity correlated precisely with the presence of 0, 1, or 2 copies of the
halothane-sensitivity gene (Mickelson et al., 1989). These data suggested
that while both normal and PSS-susceptible animals possess equal channel
protein content, there could be an abnormality in the channel protein

structure. This altered channel protein, in turn, could result in defective

44

Ca’*-release and Cay-regulation in PSS-susceptible animals.

The ultimate cause of PSS in pigs has been identified as a mutation in
the skeletal muscle Ca2"-channel protein at residue 615 where an arginine
residue has been converted to a cysteine in the stress-susceptible animals
(Fujii et al., 1991). The gene for the SR Ca2"-channel protein is now known
to be on swine chromosome 6 (segment 6p11-q21) as is the well known
HAL linkage group (Harbitz et al., 1990).

Modern turkeys, like swine, have been subjected to intense genetic
selection for rapid lean muscle growth which may partially be responsible for
increased incidence of such conditions as leg weakness and edema, deep
pectoral myopathy (DPM) and focal myopathy (FM) (Sosnicki and Wilson,
1991; Wilson et al., 1990). PM of turkey skeletal muscle was detected as
a disorder characterized by segmental necrosis and hyaline degeneration
(hypercontraction or " giant" fiber) of the fast-contracting and glycolytic (PG)
and slow-contracting oxidative (SO) muscles of the pectoral and cervical
regions (Sosnicki, 1993). A PM may be associated with the incidence of
PSE-like breast muscle and alteration in the texture, cohesiveness and
juiciness of processed turkey breast muscle.

It isstriking that the turkey industry, which has intensely bred birds
for the same desirable growth traits, has recently been observing severe meat
quality problems which closely resemble PSE pork. Necrosis and
hypercontraction of muscle fibers are commonly observed in PSE prone pigs
and FM is seen in turkey muscle (Sosnicki et al., 1988; 1991a; 1991b).
Factors which increase the incidence of the PSE-like condition in turkey
include exposure to heat or cold stress, and loading turkeys into trucks and
transporting them to the slaughter plant (Froning et al., 1978). Meat fiom

these stress-susceptible birds tends to be very exudative, and shows lower pH

45
after 25 minutes post-mortem (pH 5.7) (Sosnicki and Wilson, 1992), softer

texture, poorer bind and higher cooking losses compared to non-stressed
birds (Sosnicki, 1993). Therefore, the similarities between the PSE-like meat
from turkey with that of PSE pork (which results from PSS in the pig)
strongly suggest that genetic selection for desirable growth traits in both
species may have inadvertently selected for a mutated form of the SR

channel resulting in undesirable meat quality characteristics.

CHAPTER 2
BIOCHEMICAL PROPERTIES OF THE SR Cay-CHANNEL
PROTEIN FROM GENETICALLY UNIMPROVED TURKEY
SKELETAL MUSCLE

I. Introduction

A key step in excitation-contraction coupling is the transduction of the
depolarization signal from the T-tubule to the SR, resulting in calcium
release and muscle contraction (Endo, 1977). Two membrane proteins
located in the T-tubule and junctional SR membrane, respectively, the DHP
receptor and the SR Ca’*-channel protein, regulate the process of signal
transduction. However, the precise mechanisms involved in regulation of
Ca2+ release are poorly understood. Recent studies suggest that mutations in
the SR Ca’*-channel protein result in muscle disease such as malignant
hyperthermia (MH) (Fujii et al., 1991), which in pork, contributes a
significant fraction of pale, soft, exudative (PSE) meat.

The SR Cay-channel protein has been isolated and characterized from
mammalian skeletal muscle, such as rabbit (Lai et al., 1988; Smith et al.,
1988), swine (Yang et al, 1994), and dog (Seiler et al., 1984);
nonmammalian vertebrate skeletal muscle, such as toadfish (O'Brien et al.,
1993), blue marlin (O'Brien et al., 1995), and frog ( Lai et al., 1992); avian
skeletal muscle, such as chicken (Olivares et al., 1991; Airey et al., 1993;
Airey et al., 1990).

The Cay-channel protein activity is stimulated by M Ca2+, mM

46

47

caffeine, nM ryanodine, and mM adenine nucleotides (Meissner, 1986b;
Meissner and Henderson, 1987). On the other hand, Ca’*-channel protein
activity is inhibited by mM Mg”, ruthenium red, uM ryanodine and the
intracellular Ca2*-binding protein calmodulin (CaM) (Meissner, 1986a, b;
Meissner and Henderson, 1987). The inhibitory effect of CaM on both the
skeletal and cardiac SR channel proteins results from direct binding of CaM
to the channel protein (Meissner and Henderson, 1987 ). Crosslinking studies
suggested that the binding of CaM to the porcine skeletal channel protein is
Ca2*-independent (Yang et al., 1994). These crosslinking results were
subsequently confirmed by fluorescence studies (Yang et al., 1994).

Unlike mammalian muscle which has one skeletal muscle channel
protein isoform, avian skeletal muscle contains two distinct isoforms which
are designated or and B. However, the physiological roles and the
importance of having two Caz‘i-channel protein isoforms are unclear. The
different Ca2*-channel protein isoforms reportedly differ with respect to CaM
affinity labeling (Airey et al., 1993), phosphorylation (Seiler et al., 1984;
Strand et al., 1993), sensitivity to Cay-activation (Murayama and Ogawa,
1992), and sensitivity to Ca2+ inhibition (O'Brien et al., 1995).

The increased incidence of PSE turkey in recent years has triggered
this study to characterize the biochemical defect leading to PSE meat. The
cenn'al hypothesis upon which this study is based on that an altered Ca2+
release channel is present in a fraction of the commercial turkey population
which results in production of PSE meat. To address this hypothesis the
specific objectives of this study were to develop a purification procedure for
turkey skeletal muscle SR highly enriched in Ca2+-channel protein and to
characterize a genetically unimproved population of turkeys with respect to

skeletal SR Ca2*-channel protein function. The plant alkaloid ryanodine has

48

proven very useful in the study of the Ca’*-channel protein for two reasons.
First, ryanodine binds specifically to the Ca2*-channel protein in SR vesicles
with a stoichiometry of 1 mole of ryanodine per mole of channel protein
tetramer (Lai et al., 1988). Second, altered afinity of the channel protein for
ryanodine has been used to characterize the mutation of the Cay-channel
protein in porcine MH (Mickelson et al., 1988). Thus, in this study, the
binding of [3H]ryanodine was used in this study as a probe to characterize
the Ca’*-channel protein content and channel activity in turkey SR. In
addition, these turkey skeletal SR preparations were characterized with
respect to stoichiometry and affinity of the Ca’I-channel protein for CaM, a
regulator of Ca2+ release in muscle. The results of this study provide a
model by which subsequent studies using preparations from commercial

turkeys will be compared.

49

11. Materials and Methods
A. Materials
1.Turkey
Turkeys belonging to a randomly back-crossed, genetically unimproved
line of birds ( McCartney, 1964) were obtained from the Ohio Agricultural
Experimental Station (Wooster, OH). Experimental turkeys were killed by
intravenous injection of sodium pentobarbital (12.5 mg/ml). The breast
muscles were then removed, cut into approximately one—inch cubes, and
immediately frozen in liquid nitrogen. Samples were stored at -80°C.
2. Chemicals
Benzophenone-4-maleimide and tetramethylrhodamine-x-maleimide
were purchased from Molecular Probes (Junction City, OR). Naml and
[3H]ryanodine were obtained from DuPont-NEN (Boston, MA). Ryanodine
was purchased from Calbiochem (La Jolla, CA) and Wako Chemical USA,
Inc. (Richmond, VA).
B. Methods
1. Preparation of Sarcoplasmic Reticulum Vesicles
Skeletal muscle SR was isolated from the breast muscles by the
procedure of Mickelson et al. (1986). Briefly, the frozen muscle cubes were
homogenized in a Waring blender for 60 seconds using 5 vols. (w/v) of 0.1
M NaCl, 5 mM Tris-maleate (pH 6.8). The procedure was modified to
include three proteinase inhibitors (0.1 mM PMSF, 1 pig/ml aprotinin, and
1 rig/ml leupeptin) in the homogenization buffer and in each subsequent step
of the preparation. After centrifugation of the homogenate for 30 min. at
3500 x gm“, the pellet was discarded and the resultant supernatant was
centrifuged for 30 min. at 10,000 x gm. This pellet was resuspended with
0.6 M KCl, 5 mM Tris-maleate (pH 6.8) and centrifuged for 40 min. at

50
180,000 x gm in a Beckman Ti-70 rotor. For preparation of crude total

muscle membrane fraction (crude SR), the pellet was resuspended in 10%
sucrose and then centrifuged at 180,000 x gm in Ti-70 rotor for 40 min.
The pellet was resuspended in 10% sucrose, quick-frozen and stored at
-80°C. The yield of crude SR was ~150 mg from 100 g of unimproved
turkey breast muscle.

Skeletal muscle SR was also isolated from the turkey breast muscle by
the method of Airey et al. (1993). Briefly, muscles were homogenized 3
times for 1 min. in a Waring blender in 5 mllg wet tissue weight in a
solution containing 0.3 M sucrose, 10 mM imidazole, pH 7.4, 1.1 uM
leupeptin, and 230 uM PMSF. The homogenate was centrifuged at 8,000
x gm for 14 min. and the pellet was rehomogenized and centrifuged as
above. Supematants from both centrifugations were combined. Crude
membranes collected by centrifugation at 130,000 x gm,Ix for 90 min. were
resuspended in the above solution, rapidly frozen in liquid nitrogen and
stored at -80°C.

For purification of the SR fraction enriched in the Cay-channel protein
(heavy SR), the pellet from the 0.6 M KC1 exnaction by the Mickelson et al.
(1986) procedure or from 130,000 x gm for 90 min by the Airey et al.
(1990) procedure was resuspended in 10% sucrose (w/v), 0.4 M KCl, 20 BM
CaClz, 5 mM Tris-maleate (pH 6.8) and placed on discontinuous sucrose
gradients (22%, 36%, and 45%) containing 0.4 M KCl, 20 11M CaCl,, 5 mM
Tris-maleate (pH 6.8) in all layers. The tubes were centrifuged for 5 hr at
112,000 x 8m in a Dupont Sorvall AH-629 rotor, and the material located
at the interface between each sucrose layer was removed. Fractions were
diluted at least 1:2 with 10% sucrose, and centrifuged at 180,000 x g,mm in

Ti-70 rotor for 40 min. The pellets were resuspended in 10% sucrose, quick-

51

frozen and stored at -80°C. The yield was typically 17 mg skeletal heavy
SR fi'om 100 g of unimproved turkey muscle.
2. Ryanodine Binding Assays

The ryanodine binding assays are based on that of Mickelson et al.
(1988). [3H]Ryanodine binding was performed by incubating 0.2 mg of
heavy SR protein/ml for 90 min at 37°C in media containing 0.25 M KCl,
25 mM PIPES (pH 7.0), 4 or 10 nM [3H] ryanodine, and a CaClz-EGTA-
nitrilotriacetic acid buffer to give a specific [Ca2+]fm ranging from 0.01-1000
uM. Free Ca2+ concentrations were calculated using the computer program
of Perrin and Sayce (1967), which is based on equilibrium concentrations of
metal-ligand mixtrues. This information was used to derive optimal [Ca2“]free
for subsequent [3H]ryanodine binding studies designed to obtain the Ka and
B...
unlabeled ryanodine. Samples were filtered with Whatman GF/B filters and
washed three times with 5 ml of ice-cold buffer (0.25 M KCl, 25 mM
PIPES, pH 7.0). Specific ryanodine binding was determined by subtracting

values. The ryanodine concenn‘ation was varied by addition of

the nonspecific binding obtained in presence of 100 pM unlabeled ryanodine.
The K6 and BM values for ryanodine binding by the SR preparations
were calculated from the fit of bound vs. free ryanodine using Enzfitter
computer program (Biosoft, Cambridge, UK) when ryanodine was varied
from 1-500 nM.
3. Calmodulin Purification and Derivatization
Wheat germ calmodulin was prepared as described by Strasburg et al.
(1988). Chicken CaM, site-specifically mutated at glutamine 143 to a
cysteine residue (Q143C), was obtained fi'om Dr. Albert Wang, Boston
Biomedical Research Institute. For crosslinking experiments, both wheat
germ and Q143C chicken CaM were radiolabeled with 12’l and

52

monofunctionally substituted at Cys 27 of wheat germ CaM or at Cys 143
of chicken CaM with the photo-activatable crosslinker, benzophenone-4-
maleimide (Strasburg et al., 1988). For fluorescence experiments, wheat
germ CaM was labeled at Cys 27 with tetramethylrhodamine-X-maleimide
(Strasburg et al., 1988).

4. Calmodulin Crosslinking

Affinity labeling of CaM-binding proteins in turkey skeletal SR
vesicles was performed by incubating in darkness 0.1 uM wheat germ or
Q143C [‘”I]-Bz—CaM with 100 ug of heavy SR vesicles using the buffer
conditions of Yang et al. (1994) [ 20 mM Hepes, pH 7.5, and 0.15 M NaCl],
or of Airey et al. (1993) [70 mM MOPS, pH 7 .4]. CaClz, MgClz, and EGTA
were included as indicated in the figure legends.

The mixtures were placed in plastic microcentrifuge tubes, the tubes
were covered with a plastic wrap film, and the samples were illuminated for
5 min in a Stratalinker 1800 photoreactor (Stratagene Crop., La Jolla, CA)
equipped with lamps of Am: 254 nm. After photolinking, the samples were
centrifuged in Beckman TL-100 centrifirge at 88,000 x gm“ for 20 min. The
membrane pellets were resuspended in 50 ul water. For electrophoresis, 25
pl sample .buffer containing 3.7% SDS, 12 mM EGTA, 30% glycerol,
~0.001% bromphenol blue, and 46 mM Hepes, pH 7.5 was added to each
sample.

5. Electrophoresis

The electrophoresis procedure employed was that described by
Laemmli (1970) with the modification that 4-10% linear acrylamide gradient
gels were used. Slab gels were stained with 0.05% Coomassie blue in 50%
methanol/10% acetic acid and then destained with 50% methanol/10% acetic
acid. The gels were dried and placed with Kodak Omat XAR—SX-ray film

 

 

CM

at-

of

in

Qt

EX

[IE

C0

TIC

53
in autoradiography cassettes equipped with Dupont Lightning Plus

intensifying screens.
6. Fluorescence Anisotropy Measurements

Fluorescence anisotmpy was used to characterize CaM/calcium-channel
protein interaction in SR vesicles. The principle underlying fluorescence
anisotropy is that the rapid Brownian tumbling of a small molecule is slowed
upon binding to a larger molecule to form a complex. If the smaller
molecule is fluorescent, the altered mobility of this species resulting from
complex formation can be detected by measuring the change in
depolarization of fluorescent light emitted upon excitation by polarized light
(Lakowicz, 1983).

Fluorescence measurements were performed using an SLM 4800
spectrofluorometer equipped with two detectors in T-format detector.
Samples were held in a therrnostated cell block maintained at 22°C. The
excitation wavelength of Rh-CaM was 532 nm, monochromator slits were set
at 4 nm, and emitted light was isolated using Schott OG-570 filters. During
titrations, samples were allowed to equilibrate for 4 min after each addition
of SR or Rh-CaM. All samples included 1 rig/ml aprotinin, 1 ug/ml
leupeptin, and 0.1 mM PMSF to inhibit proteolysis during the experiment.

All fluorescence measurements were performed using semi-micro,
quartz fluorescence cuvettes (4 mm x 10 mm). Prior to fluorescence
experiments, the cells were rinsed with 1 mg/ml bovine serum albumin to
minimize Rh-CaM adsorption to the walls of the cuvette. Following this
treatment, the measured anisotropy value for Rh-CaM was independent of
concentration over the range of 1 nM to 1000 nM, indicating that there was
negligible Rh-CaM adsorption to the cuvettes ( Yang, et al., 1994).

The anisotropy of ligand (Rh-CaM) is directly proportional to the

54

fraction of ligand bound to receptor (Ca2+ -channel protein). Thus, if Af is
the anisotropy of free Rh-CaM and A, is the anisotropy of fully bound
ligand, then the fraction bound, f.,, is determined fiom:

(A."Af)

f a:
b Am(l’Q) +Q(Ab) _Af

(1)

 

Where Am is the measured anisotropy for a given ligand concentration, and
q, the change in quantum yield, is the ratio of fluorescent intensity of bound
species over that of three species. If the change in quantum yield is
negligible upon binding of ligand, then the equation (1) reduces to:

_ (Am-At”)

f —_

(2)
The fraction of ligand bound, and the concentrations of bound and free Rh-
CaM are readily calculated.

The anisotrOpy of unbound Rh-CaM, Ar, was measured in the absence
of SR. The anisotropy of fully bound species, Ab, was obtained by titration
of Rh-CaM with SR vesicles, following by curve-fitting using the Enzfitter
computer program (Biosoft, Cambridge, UK) for a single class of ligand
binding site. Corrections for light scattering and background fluorescence

were made by application of the equation:

A=f;A,+f,A, (3)

where A, A| and A2 are the anisotropies of sample, the blank, and the
corrected sample, respectively. The fractional contributions, f1 and f2, of
these species were calculated from the intensities measured with the
excitation monochromator in the vertical position and the emission

monochromator at 55°. The corrected sample anisotropy, therefore, is that

55

value in the absence of background interference.

7. Protein Assay

SR protein concentrations were determined by the Lowry method

(Lowry et al., 1951). Using bovine serum albumin as a standard.
8. Statistical analysis

Comparisons of the mean Kd and Bum values for CaM binding in
different conditions were made with Student's t test.

56

III. Results and Discussions
A. Purification of SR from Turkg Skeletal Muscle

In order to study the biochemical properties of the turkey skeletal
Ca’*-channel protein, it was first necessary to optimize the preparation of the
SR membrane fraction heavy SR which is highly enriched in the Ca2+-
channel protein. Thus, heavy SR fi'om turkey skeletal muscles was purified
by two methods to obtain effective yield of the Cay-channel protein: (1) the
method of Airey et al. (1993) which was developed for chicken SR; (2) the
method of Mickelson et al. (1986) which has been used for pig. Since both
chicken and turkey are avian, it was expected that the SR purification
procedure developed for chicken skeletal muscle should also work for turkey.
However, the SDS-polyacrylamide gels of purified light and heavy SR
vesicles from turkey skeletal muscle using both purification procedures
indicated significant differences in heavy SR quality (Figure 2.1). As with
the chicken skeletal muscle SR Ca2+-channel protein (Airey et al., 1993),
turkey skeletal muscle SR Cay-channel protein has two isoforms, a and B,
which were distinguished by differences in mobility on SDS-PAGE (Figure
2.1). Comparison of the Ca2‘-channel protein yields from both methods
indicates the preparation of SR from turkey skeletal muscle by the procedure
of Mickelson et al. (1986) resulted in SR vesicles with greater channel
protein content than that obtained by the procedure of Airey et al. (1993)
(Figure 2.1, lanes 3, 5, and 7). In addition, the heavy SR preparation using
Mickelson et al. procedure without mincing of the muscle before blending
showed greater channel protein yield than that with a mincing step (Figure
2.], lanes 5 and 7). Thus, the method of Mickelson et al. (1986) was

adopted for all subsequent SR preparations.

B. Ca2*-dependence of |3H|Ryanodine Binding Assays

57

 

Figure 2.1 SDS—polyacrylamide gel of SR preparations from unimproved
turkey and porcine skeletal muscles. Lane 1: porcine skeletal muscle
crude SR; lane 2: turkey light SR prepared using Airey et al. (1993) method;
lane 3: turkey heavy SR prepared as in lane 2; lane 4: turkey light SR
prepared using Mickelson et al. (1986) method with mincing step; lane 5:
turkey heavy SR prepared as in lane 4; lane 6: turkey light SR prepared
using Mickelson et al. method without mincing step; lane 7: turkey heavy SR
as in lane 6; lane 8: molecular weight markers.

Arrows indicate the Ca"—channel protein subunits which occur as two
isoforms in turkey. The lower band of doublet in porcine SR has been
idenitified as a proteolytic degradation product of the upper band.

58

Prior to characterizing the PSE-like meat quality problems of
commercial turkeys which are highly selected for particular traits, it is first
necessary to establish the basic biochemical properties of SR from turkeys
which are derived from a randomly back-crossed, genetically unimproved
line. The [3H]ryanodine binding assay is a good approach used to
characterize the biochemical properties of the Ca2*-channel protein. Thus,
the Ca2*-channel protein activity, inactivation, and sensitivity of the
unimproved turkey were studied by using [3H]ryanodine binding assays.

The Ca” dependence of [3H]ryanodine binding to turkey skeletal
muscle heavy SR vesicles from seven individual preparations is indicated in
Figure 2.2. [3H]ryanodine binding was activated at a threshold concentration
of approximately 0.2 uM Ca2+, and reached a plateau of binding over the
range of 3 to 30 uM free Ca” (Figure 2.2). The mean [3H]ryanodine
binding at the plateau between 3 and 30 uM Ca2+ concentrations was 0.59
pmol/mg of heavy SR protein. When the free Ca2+ concentration reached
1 mM, the average [3H]ryanodine binding decreased only slightly to a mean
value of 0.47 pmol/mg of heavy SR protein, corresponding to a 20%
inhibition of ryanodine binding. Since ryanodine binding is an indicator of
Cay-channel protein activity, these results suggest that mM cytoplasmic
levels of Ca” have only a slight effect on reducing the open state of the
Ca2I-channel protein. Thus, high cytoplasmic Ca2+ levels would not be likely
to inhibit Ca" release in turkey muscle.

These results are similar to those obtained for blue-marlin skeletal
swimming muscle (which contains both a and B isoforms) and for marlin
cardiac muscle. Ryanodine binding in the fish skeletal SR system was
inhibited only about 40% at 1 mM Ca2+; the cardiac SR ryanodine binding
activity was inhibited only about 20% (O'Brien et al., 1995). In contrast, the

59

 

1.00

 

0.40

Bound. pmololmg an

 

 

 

 

0.1 1 10 100 1000

[Calcium]. ul

Figure 2.2 Ca2+ dependence of [’H]ryanodine binding to genetically
unimproved turkey skeletal muscle heavy SR vesicles. Curves represent
results from seven individual unimproved turkey SR vesicle preparations.

60

marlin superior rectus extraocular muscle SR which contains only the or
isoform, was virtually complete inactivation of ryanodine binding at 1 mM
Ca2+ (O'Brien et al., 1995). This response by the or isoform is very similar
to that of mammalian skeletal muscle (O'Brien et al., 1995; Mickelson et al.,
1988). Thus, the lack of inactivation at high Ca2+ concentrations is probably
because of the inherent properties of the B isoform which is functionally
similar to the cardiac Ca2*-channel protein isoform (Airey et al., 1993;
O'Brien et al., 1995).

Five of the seven turkey SR preparations also displayed an unusual
[3H]ryanodine binding peak in the range of 300-600 M Ca2+ in addition to
the broad peak centered around 10 M Ca2+. Other muscle preparations
which include both a and B isoforms (fish), show a Ca2+ dependence of the
Ca2*-channel protein activity reaching a single maximum at about 100 M
C3”. The unusual [3H]ryanodine binding peak shown at 300 uM Ca2+ by
some, but not all turkey skeletal Ca2*-channel protein might result fi'om
different genetic variants of the B isoform in turkeys.

C. Mine-depencgrce of PHIRvanodine Binding Assays

[3H]ryanodine-dependent ryanodine binding assays were conducted to
characterize the Ca2‘-channel protein content and channel activity of heavy
SR vesicles from turkey. [3H]ryanodine binding to turkey skeletal muscle
heavy SR vesicles was measured at various ryanodine concentrations in the
presence of 10 BM Caz’, the optimal [Ca2+] determined fi'om the previous
section (Figure 2.3). The binding capacity (Bum) and affinity (Kd) of SR
preparations were calculated using the Enzfitter program applied for a single-
site ligand binding model (Figure 2.3). The heavy SR preparations (n=7)
exhibited an average Kd of 205176 nM and a BM of 4.01:1:0.17 pmol/mg
of protein (Figure 2.3). The BM is similar with that reported for chicken

61

 

Bmax- 4.01+0.11 pmol/mg
Kd- 20.97.: M ‘

 

 

 

 

 

0.15

2 r g 010 -
0.05 r '
0.00

0.0 1.5 3.0 4.5

o 50 100 150 200 250 300

 

Bond Ryanodno. pmollmg H81!
0)
l
_._'

 

 

 

Free Ryanodine. I“

Figure 2.3 Ryanodine dependence of [’leyanodine binding to
unimproved turkey skeletal muscle heavy SR vesicles at 10 uM Car“.
The inset is a Scatchard plot of ryanodine binding to SR vesicles. Points
represent the means 1: SE of duplicates for each of seven different heavy SR
preparations. B"m and Kd values were calculated using the Enzfitter program
as indicated in Methods.

62
skeletal heavy SR, 3.45 pmol/mg protein (Airey et al., 1990).
D. Calmodulin Binding Activig of Ca2*-ghannel Protein Isoforms

Airey et al. (1993) suggested that there is differential binding of CaM
by the two channel protein isoforms. Using crosslinking methods they
showed that the a Ca2I-channel protein isoform from chicken bound CaM to
a much greater extent than the B-isoform. In addition, CaM affinity labeling
was strongly enhanced by the presence of 0.4 mM Ca2+ over that of 1.5 mM
EGTA, suggesting that CaM binding is Cay-sensitive. The observed
differences in affinity labeling patterns between the porcine channel (Yang
et al., 1994) and the avian channel proteins (Airey et al., 1993) could result
from three possibilities: I) differing buffer conditions between the two
experiments; 2) different calmodulin derivatives employed for crosslinking;
and 3) species differences in CaM-binding activity. Thus, this study tried to
clarify the reason(s) resulting in these different observations.

The two isoforms of the Ca2*-channel protein in turkey skeletal muscle
are evident in the gel electrophoretogram (Figure 2.4A). The autoradiogram
of the gel electrophoretogram (Figure 2.4B) shows that the Ca2*-channel
protein is the major protein which formed a complex with CaM in turkey
SR. The autoradiogram (Figure 2.4B) also indicates that binding activity of
both turkey isoforms for the wheat germ [‘”I]-Bz-CaM is similar in the
presence of Ca”, in contrast to that reported by Airey et al. (1993). Using
the crosslinking conditions of Airey et al. (1993) (Figure 2.4, lanes 1-6) in
which there was no added NaCl, binding of CaM was Cay-dependent,
although Mg2+ clearly enhanced binding of CaM. In contrast, using the
conditions of Yang et al. (1994) in which 0.15 M NaCl was included, CaM
binding was Ca2*-independent. This suggests that ionic strength effects

rather than species differences account for the previously reported Ca2+-

63

Figure 2.4 [Mg’*| and [Ca2+] dependence of affinity labeling of
turkey skeletal muscle heavy SR with wheat germ ["SI]-Bz-CaM
under different buffer conditions. These experiments were
conducted using wheat germ CaM with the benzophenone-maleimide
cross-linker at Cys-27, and using buffer conditions of Yang et al.
described previously (1994) [20 mM Hepes, pH 7.5, and 0.15 M
NaCl] (A and B, lanes 7-12), or Airey et al. (1993) [ 70 mM MOPS,
pH 7.4 ] (A and B, lanes 1-6). (A) Coomassie blue stained gel. (B)
Autoradiogram of dried gel. 1 mM EGTA (lanes 1-3, and 7-9) or 0.1
mM CaCl2 (lanes 4-6, and 10-12) were included in buffer with 0, 1,
or 10 mM MgC12. Molecular weight markers were indicated as kDa.

64

123456789101112

---—~-.fl~—_—~

“

 

 

 

—116
— 97.4

—66

—45

— 11 6
— 97.4

"66

Un

red

Air

65

sensitive CaM binding by the avian Cay-channel protein.

The CaM derivatives employed by the two laboratories also differed.
Airey et al. (1993) used a mammalian CaM with a methyl azido benzimidate
crosslinker which attaches at various amino sites on the CaM molecule,
mostly near the C-domain. Yang et al. (1994) used wheat germ CaM which
has a single site for crosslinking in the N-domain. To determine whether the
differing crosslinking results could also be due to differences in crosslinking
efficiency between the N- and C-domain of CaM, another CaM derivative,
a site-specific mutant of chicken CaM which has a Cys in the C-domain of
protein (Q143C) was employed. The benzophenone-4-ma1eimide crosslinker
was conjugated to this location.

The crosslinking results with wheat germ CaM and Q143C CaM show
CaM affinity labeling patterns similar to that reported by Airey et al. (1993)
results when using their buffer condition (Figure 2.4 and 2.5). However, the
autoradiogram (Figure 2.5B) indicates the or-isoform is the predominantly
affinity labeled isoform of the Cay-channel protein when using (Q143C)
[‘Z’I]-Bz-CaM. Comparison of autoradiograms of Figure 2.4 and Figure 2.5
indicates that effects of buffer conditions on affinity labeling of the or subunit
are the same with both forms of CaM. These results further support the:
importance of buffer condition on the affinity labeling patterns.

The crosslinking results with Q143C CaM further suggest that the a
and B isoforms of turkey muscle may interact differently with CaM, since the
B isoform is not affinity-labeled by this chicken mutant CaM derivative
under any of the buffer condition examined (Figure 2.5B). The lack of
crosslinking by the B subunit of the channel protein with Q143C CaM and
reduced crosslinking of the B subunit by the CaM derivative employed by
Airey et al. (1993) suggest that the binding site of CaM for B subunit of

66

Figure 2.5 [Mg’*] and [Ca2+] dependence of affinity labeling of
turkey skeletal muscle heavy SR with chicken (Ql43C) [mll-Bz-
CaM under different buffer conditions. These studies were
conducted using the site-specific mutant of chicken CaM which has a
Cys in the C-domain of protein (Cysl43). Crosslinking experiments
were conducted as indicated in Figure 2.4. (A) Coomassie blue stained
gel. (B) Autoradiogram of dried gel. 1 mM EGTA (lanes 1-3, and 7-
9) or 0.1 mM CaCl2 (lanes 4-6, and 10-12) were included in buffer
with 0, 1, or 10 mM MgC12. Molecular weight markers were indicated
as kDa.

67

12

7891011

456

3

6
1
1

_

— 97.4

i—45
—29

 

10

1100

0

 

mm“
.2

—45
— 29

68

channel protein differs fiom that of the a subunit. The channel protein
binding site may be in a different location on the CaM molecule such that
the crosslinker is not in the pr0per orientation for crosslinking, alternatively
the crosslinkers in the C-domain may sterically block binding of the B
isoform.

These studies indicate that the factors of species and CaM derivative
differences did not cause the observed differences between our previous
studies with pig (Yang et al., 1994) and those of Airey et al. (1993) with
chicken. In addition, in the presence of NaCl at physiological ionic strength,
the CaM affinity labeling of the channel protein was Ca2*-independent
(Figure 2.4) which is consistent with porcine studies (Yang et al., 1994).
These results suggest that differences in affinity labeling patterns between
avian and mammalian channel proteins are affected by crosslinking

conditions.
E. Quantitation of Calmodulin-binding Activity of Cay-channel Protein in
Turkey Skeletal Muscle

Crosslinking studies provide valuable information on identification of
channel proteins which bind CaM. However, crosslinking studies are not
quantitative, and therefore, may yield misleading results on affinity of the
channel protein for CaM. In particular, crosslinking of CaM to target
proteins in the presence of EGTA may occur if weak complexes are formed.
These crosslinking patterns might not be physiologically representative.
Fluorescence studies were initiated to determine whether CaM indeed
strongly binds under the conditions of low Ca2+ concentrations.

Turkey skeletal heavy SR vesicles were titrated into Rh-CaM under
two different conditions: 1 mM EGTA which would represent the case of

resting muscle, or 0.1 mM CaCl2 which would simulate contracting muscle

69
[Ca2+] conditions. In both cases, titration of SR vesicles into Rh-CaM

resulted in an increase in fluorescence anisotropy which is attributable to the
increased molecular mass of the Rh-CaM/Ca’+-channel protein complex
(Figure 2.6). These results support the affinity labeling data in Figure 2.4
which indicate that in the presence of 0.15 M NaCl, binding of CaM to the
channel protein is Cay-insensitive.

Data fiom these titrations (Figure 2.6) were used to determine the
anisotropy values for the free Rh-CaM species (A,) and for the fully bound
Rh-CaM/Ca2*-channel protein complex (Ab) for subsequent experiments to
determine stoichiometry of Rh-CaM binding. The average A, values
obtained from three turkey heavy SR vesicle preparations for each metal ion
condition are 0.2344 (+0.1 mM CaClz) and 0.2234 (+1 mM EGTA). The Af
values obtained from 10 nM samples of Rh-CaM, under these two buffer
conditions in the absence of added SR, were 0.1653 (+0.1 mM CaClz) and
0.1656 (+1 mM EGTA). There was no significance in fluorescence intensity
upon binding of Rh-CaM to the Ca2+-channel protein indicating that there
was no change in quantum yield upon complex formation (q=1.0).
Therefore, the fraction of Rh-CaM bound was calculated using equation 2 of
Materials and Methods.

CaM crosslinking experiments (Figure 2.4) suggested that ionic
strength affects the CaM affinity labeling to the Ca2*-channel protein. To
obtain quantitative data on the influence of ionic strength on binding of CaM
to the turkey skeletal SR Cap-channel protein, a Rh-CaM/SR mixture was
titrated with KCl, and binding was monitored by the increase in anisotropy
(Figure 2.7). The chosen concentrations of SR vesicles (80 ug for the 0.1
mM CaCl2 condition, 200 ug for the 1 mM EGTA) and Rh-CaM (10 nM)
correspond to the mid-points of the titration curves in Figure 2.6. Increased

7O

 

0.27

0.25 '

0.23 "

0.21 -

Anisotropy

0.19 r

 

0.17 "

 

 

1 11111111 1 11114411 1 1111111

1 10 100 1000

 

0.15

[HSR] ualml

Figure-2.6 Titration of Rh-CaM with turkey skeletal heavy SR veSicles
under different conditions. The sample medium contained 10 nM Rh-CaM,
0.3 M sucrose, 0.15 M KCl, and 50 mM Hepes, pH 7.0, and either 1 mM
EGTA (A) or 0.1 mM CaCl2 (a) in a starting volume of 1 mL. The Rh-
CaM sample was titrated with SR vesicles in parallel with a buffer blank
containing the same media minus Rh-CaM. Corrections were made for light
scattering as described under Methods. Points represent the means :1: SE of
three preparations.

71

 

0.23
0.22 "
0.21 "

0.20- A‘QAAAA

Anisotropy

t>

r>

r>

t>
t>

t>

t>

b

b

0.19 - ‘ A
0.18 ‘

0.17 ‘

 

 

016 1 11111111 1 1 1141111 1 Llllll
I

1 10 100 1000

 

[KCI]. m

Figure 2.7 [KCI] dependence of binding of Rh-CaM to turkey skeletal
heavy SR vesicles under different [Ca2+] conditions. The sample buffer
contained heavy SR, 10 nM Rh-CaM, 0.3 M sucrose, 50 mM Hepes, pH 7.0,
and either 1 mM EGTA (A) or 0.1 mM CaCl2 (A) in a starting volume of 1
mL. Points represent the means :1: SE of three preparations. CaM bound
was calculated from measured anisotropy as described under Methods.

72

anisotropy as a function of increased [KCl] reflects increased complex
formation; decreased anisotropy reflects decreased binding. The KCl titration
data clearly show that in the presence of EGTA, the binding of Rh-CaM to
the channel protein was highly ionic strength dependent. Binding is
strongest over the range of physiological ionic strength, ~100-150 mM. At
[KCl]>0.3 M, the anisotropy declined, indicating decreased binding of Rh-
CaM to the Ca2*-channel protein. In the presence of 0.1 mM Ca2+, titration
of KCl into SR/Rh-CaM mixture also indicated a significantly higher affinity
of Rh-CaM for the channel protein. However, in contrast to the EGTA
conditions, binding of Rh-CaM to the channel protein did not significantly
decrease at higher KC] concentration. These results were similar to that
obtained for porcine SR vesicles (Yang et al., 1994), and are consistent with
data obtained in crosslinking studies (Figure 2.7).

Taken together, the results of Figures 2.4-2.7 suggest that the observed
differences in affinity labeling patterns of the Ca2+ channel between Yang et
al. (1994) studies with porcine muscle and those of Airey et al. (1993) with
avian skeletal muscle result from the different buffer conditions of the
experiments.

These experimental conditions were subsequently used to determine
the binding capacity and affinity of the skeletal Cay-channel protein in SR
vesicles for Rh-CaM under both Ca” conditions: 0.1 mM CaClz, and 1 mM
EGTA. Rh-CaM was titrated into a suspension of SR vesicles. Ar and A,
values were used to calculate the fraction of Rh-CaM bound to the Ca2+-
channel protein in SR vesicles for each point in the titration. The fi'action
of Rh-CaM bound was then converted to stoichiometry and affinity.

Results of titrations conducted in the presence of EGTA indicate a

single class of binding sites on the turkey SR Cay-channel proteins for CaM

73

(Figure 2.8 and Table 2.1). Analysis of data using the Enzfitter program
yields a dissociation constant, Kd, of 9.1i1.7 nM and a binding capacity,
Bum, of 56.2:I:4.7 pmol/mg of SR protein. In the presence of 0.1 mM CaClz,
the titration data also suggest a single class of binding site, which has K,I of
7.3:l:1.4 nM, and BM of 84.4:t24.2 pmol/mg of SR protein (Figure 2.9 and
Table 2.1). According to statistical analysis, Bm values of the Cay-channel
protein for CaM shows significant difference (p<0.05) between these two
conditions, whereas Kd values were not significantly different. Ryanodine
binding studies on the turkey heavy SR preparations yielded a BM value of
4.01 pmol/mg of SR protein (Figure 2.3). Since one mole of ryanodine
binds specifically with high affinity per mole of Ca2+ channel protein
tetramer, these data suggest that there are approximately 3.5 CaM molecules
bound per channel protein subunit (14 CaM molecules per channel protein
tetramer) in the presence of EGTA and 5 CaM molecules bound per subunit
(21 CaM molecules per channel tetramer) in the presence of 0.1 mM CaClz.
Crosslinking studies in the presence of EGTA indicated substantially greater
CaM bound to the a subunit than to B (Figure 2.4, lane 7). In the presence
of 0.1 mM Ca”, the or and B subunits appear to be crosslinked in
approximately a 1:1 ratio (Figure 2.4, lane 10), thus suggesting that as Ca2+
concentration is increased, the additional CaM binding occurs at B subunit
(Figure 2.4). These results suggest that CaM differentially regulates Ca2+
release activity by a and B subunits of avian skeletal muscle.

Studies of titrations conducted are indicative of a single class of CaM
binding site on the SR Cal" channel protein for CaM in the presence of
EGTA, and two classes of CaM binding sites in the presence of 0.1 mM
CaCl, (Yang et al., 1994). The stoichiometry of CaM binding to the porcine

skeletal muscle Ca2’-channel protein is approximately 20 mol/mol of tetramer

74

Figure 2.8 Titration of skeletal heavy SR vesicles with Rh-CaM
in the presence of EGTA. (A) Anisotropy plot of titrations of heavy
SR with Rh-CaM . (B) Rh-CaM/SR saturation binding curve. The
inset is a Scatchard plot of Rh-CaM binding to SR vesicles. The
sample medium contained 200 11g of heavy SR, 0.3 M sucrose, 0.15
M KC], 50 mM Hepes, pH 7.0, and 1 mM EGTA in a starting volume
of 1 mL. Points represent the means i SE of four preparations. CaM
bound was calculated from measured anisotropy as described under
Methods. .

V

WWW/mm

75

 

0.2 1

0.20 l"

0.19 "

 

l

1 1 111111

1 11111111

1

 

Jilllll

 

0.17
0.1

1

[MIN

10

100

 

60

50”

30"

1o-

 

 

 

 

 

 

 

1O

 

76

Figure 2.9 Titration of skeletal heavy SR vesicles with Rh-CaM
in the presence of CaCl,. (A) Anisotropy plot of titrations of heavy
SR with Rh-CaM . (B) Rh-CaM/SR saturation binding curve. The
inset is a Scatchard plot of Rh-CaM binding to SR vesicles. The
sample medium contained 80 ug of heavy SR, 0.3 M sucrose, 0.15 M
KCl, 50 mM Hepes, pH 7.0, and 0.1 mM CaCl2 in a starting volume
of 1 mL. Points represent the means :1: SE of four preparations. CaM
bound was calculated from measured anisotropy as described under
Methods. .

momma/mm

77

 

0.20 "

0.18 1'

017 -

 

 

 

0.16 l I 1111111 1 l jllllll l I [ll

°-‘ 1 10 100
ICON]. IN

 

100

 

60'

 

 

 

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20 A l A I l A L

0 10113400001170.0011
“WWI-m
O 1 1 1 1 1 1

O 1 0 2O 30 40 50 60 7O

 

 

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78

Table 2.1 Equilibrium Constants for Rh-CaM Interaction with the
Ca’+-channel Protein in Turkey Skeletal Muscle Heavy SR vesicles"

 

 

 

Condition Kd , nM Bm pmol/mg
.lmM CaCl2 7.3 :1: 1.4 84.4 :1: 24.2'
lmM EGTA 9.1 :t 1.7 56.2 :1: 4.7'

' p<0.05.

" Data were obtained fiom titrations of SR vesicles with Rh-CaM in the
presence of 0.3 M sucrose, 0.15 M KC], 50 mM Hepes, pH 7.0, and
metal ion conditions as listed below. Data are :1:SE of the means of four
preparations each.

79

(5 monol of subunit) in the presence of 1 mM EGTA. In the presence of
0.1 mM CaClz, the Ca”’-channel protein binds with 5 CaM at the high
affinity site, and 16 CaM at the low affinity site (Yang et al., 1994). In
agreement with Yang et al. result, Tripathy et al. (1995) indicated that the
stoichiometry of CaM binding to rabbit skeletal muscle Ca’*-channel protein
with high affinity is 16 mol/mol at $0.1 11M Ca2+ and 4 mol/mol at 100 11M
Ca2+. However, our results of turkey skeletal muscle Ca2+ channel protein
suggest that there is only one class of CaM binding sites on the 01 Ca”
channel protein isoform in the presence of both EGTA and 0.1 mM CaCl2
(Figure 2.8 and Figure 2.9). The additional CaM binding in the presence
of Ca2+ may take place on the B isoform, which is consistent with the

"cardiac-like" nature of this isoform.

80

IV. Conclusions
Like the chicken skeletal muscle Ca” channel protein, the Ca2+ channel

protein of turkey skeletal muscle contains two isoforms, a and B. The Ca”
dependence of [3H]ryanodine binding by turkey skeletal muscle heavy SR
vesicles shows maximum binding at about 10 11M, and Ca’*-channel protein
activity was only partially inhibited at 1 mM Ca2+. This ryanodine binding
inactivation phenomena of the Ca2*-channel protein at high Ca2+
concentrations were attributed to the properties of the B subunit. In addition,
the difference in the Kd values of crude SR at 10 and 300 M Ca2+ suggests
different isoform(s) regulate Ca2+ release at different Ca2+ concentrations.
According to the results of O'Brien et al. (1995), the ryanodine binding
activity of or isoform is predominant at 10 11M Ca2+, whereas the B isoform
has the major channel activity at 100 11M Ca2+. Thus, a ryanodine binding
peak shown at 300 11M Ca2+ in most turkey heavy SR vesicles could result
from the B isoform activity. The result suggested that some turkeys lack
this ryanodine binding peak at 300 uM Ca2+, possibly resulting from some
defects in B isoform CaZI-channel protein. Thus, there may be two different
genetic groups in the experimental turkey.

The stoichiometries of CaM binding to the turkey skeletal muscle Ca2+-
channel protein are approximately 3.5 CaM molecules bound per channel
protein subunit in the presence of EGTA and 5 CaM molecules bound per
Subunit in the presence of 0.1 mM CaClz. Crosslinking studies in the
presence of EGTA indicated substantially greater CaM bound to the a
Subunit than to B. In the presence of 0.1 mM Ca2+, the a and B subunits
appear to be crosslinked in approximately a 1:1 ratio, thus suggesting that as
Ca2+ concentration is increased, the additional CaM binding occurs at B
Subunit. These results suggest that CaM differentially regulates Ca2+ release

 

81
activity by or and B subunits of avian skeletal muscle.

The affinity labeling pattern of both turkey skeletal muscle Ca2+ -
channel protein isoforms with CaM was dependent on buffer conditions and
different kind of CaM. In the presence of the NaCl, the affinity labeling of
the channel with CaM was Ca2"-independent. On the other hand, in the
absence of NaCl, the affinity labeling was Ca2"-dependent. In addition, the
a and B isoforms of turkey skeletal muscle may interact differently with the
C-domain of CaM (chicken mutant CaM: Q143C), since the B isoform was
not affinity-labeled by this CaM derivative.

This study shows that the characteristics of the turkey skeletal Ca2+ -
channel protein are different from mammalian, fish and even chicken Ca2+ -
channel proteins. The unusual properties of turkey Ca2+-channel protein may
result fi'om that two isoforms played different regulation roles in different

conditions.

CHAPTER 3
THE BIOCHEMICAL BASIS FOR PSE QUALITY PROBLEMS
ASSOCIATED WITH TURKEY

I. Introduction
The modern turkey industry has grown rapidly over the past twenty

years to meet consumer demand for lean, inexpensive and convenient meat
products. To meet this demand, the industry has intensely bred for the
efficient growth and heavy muscling. Over the past few years the turkey
processing industry has been experiencing severe meat quality problems
which closely resemble pale, soft, and exudative (PSE) pork. Meat from
these stress-susceptible birds shows a very rapid pH decline resulting in
product which is very exudative, and has softer texture, poorer bind and
higher cooking losses compared to non-stressed birds (Sosnicki, 1993).
Stress factors which increase the incidence of the PSE condition in turkey
include exposure to heat or cold, and loading turkeys into trucks and
transporting them to the slaughter plant (Froning et al., 1978). The striking
Similarity of the factors leading to development of PSE meat in turkey to that
of swine suggests that there may be a genetic basis for this syndrome in

linkeys.
Porcine stress syndrome (PSS) is an inherited skeletal muscle disorder

Which affects 10-20% of the swine population in the US. (Vansickle, 1989).
This syndrome results in substantial economic losses to farmers, owing to

death of animals from stresses of transportation, heat, crowding, etc., before

82

 

83

reaching market (Louis, 1993). A substantial fi‘action of PSE pork is directly
attributable to PSS (Pommier and Houde, 1993). While PSS has been
observed in many swine breeds, the prevalence of this disorder correlates
strongly with genetic selection of leaner, more heavily muscled and faster
growing animals (Zhang et al., 1992).

Biochemical studies indicated that skeletal muscle SR vesicles or
skinned muscle fibers from the PSS-susceptible animal have abnormal Ca2+
regulation (Mickelson et al., 1986, 1988; Endo et al., 1983). These data
suggested that there is a defect in the Ca2+-release channel protein of the
skeletal muscle SR. The ultimate cause of PSS in pigs has been identified
as a point mutation in the skeletal muscle Ca2*-channel protein (Arg‘ls to
Cys) (Fujii et al., 1991).

The similarities in factors which influence formation of PSE meat fiom
turkey with those of PSE pork strongly suggests that genetic selection for
desirable growth traits in both species may have resulted in inadvertent
selection for a mutated form of the SR channel which is associated with
development of undesirable meat quality characteristics.

Our hypothesis is that a subpopulation of commercial turkeys have an
increased frequency of an altered SR CaZI-channel protein, resulting in the
abnormal Caz‘-channel protein activity which is responsible for development
of PSE meat. To test this hypothesis, the biochemical approaches used to
characterize the defect in PSS swine were employed to determine whether
a similar abnormality might exist in turkeys. Ryanodine binding assays were
conducted on SR vesicles prepared from commercial turkeys (as a stress-
susceptible group) and from a genetically unimproved (as a control group)
turkeys to determine whether the Cazl-channel protein is altered in the

population of commercial turkeys. SDS-PAGE and CaM affinity labeling

84

experiments were also used to investigate the basis for differences between

commercial and unimproved turkeys.

85

11. Materials and Methods
A. Materials
1.Turkeys

Two different populations of turkeys were utilized in this study. A
random-bred genetically unimproved group of turkeys (McCartney, 1964)
was used as a control population. These turkeys were obtained fiom the
Ohio Agricultural Experiment Station, Wooster, Ohio. Commercial turkeys
were obtained with the cooperation of BilMar Foods (Zeeland, MI). Turkeys
were killed by intravenous injection of sodium pentobarbital (12.5 mg/ml).
The breast muscles were removed, cut into one-inch cubes, and immediately
frozen in liquid nitrogen. Samples were stored at -80°C.

2. Chemicals

Benzophenone-4-maleimide and tetramethylrhodamine-x-maleimide
were purchased from Molecular Probes (Junction City, OR). Naml and
[3H]ryanodine were obtained from DuPont-NEN (Boston, MA). Ryanodine
was purchased from Calbiochem (La Jolla, CA) or Wako Chemical USA,
Inc. (Richmond, VA). Stains-all dye was obtained from Sigma (St. Louis,
MO).
B. Methods

1. Preparation of Sarcoplasmic Reticulum Vesicles

Skeletal muscle SR was isolated from the breast muscles by the
procedure of Mickelson et al. (1986). Briefly, the frozen muscle cubes were
homogenized in a Waring blender for 60 seconds using 5 vols. (w/v) of 0.1
M NaCl, 5 mM Tris-maleate (pH 6.8). The procedure was modified to
include three proteinase inhibitors (0.1 mM PMSF, 1 rig/ml aprotinin, and
1 rig/ml leupeptin) in the homogenization buffer and in each subsequent step

of the preparation. After centrifugation of the homogenate for 30 min. at

86

3500 x gm, the pellet was discarded, and the resultant supernatant was
centrifuged for 30 min. at 10,000 x gm. This pellet was resuspended with
0.6 M KCl, 5 mM Tris-maleate (pH 6.8) and centrifuged for 40 min. at
180,000 x gum in a Beckman Ti-70 rotor. For preparation of crude total
membrane fraction (crude SR), the pellet was resuspended in 10% sucrose
and then centrifuged at 180,000 x gum; in Ti-70 rotor for 40 min. The pellet
was resuspended to 10% sucrose, quick-fiozen and stored at -80°C. The
yield of crude SR was ~150 mg from 100 g of unimproved or commercial
turkey breast muscles.

For purification of the SR fraction enriched in the Ca2+-channel protein
(heavy SR), the pellet from the 0.6 M KCl extraction was resuspended in
10% sucrose (w/v), 0.4 M KCl, 20 uM CaClz, 5 mM Tris-maleate (pH 6.8)
and placed on discontinuous sucrose gradients (22%, 36%, and 45%)
containing 0.4 M KCl, 20 11M CaClz, 5 mM Tris-maleate (pH 6.8) in all
layers. The tubes were centrifuged for 5 hr at 112,000 x glnllx in a Dupont
Sorvall AH-629 rotor, and the material located at the interface between each
sucrose layer was removed. Fractions were diluted at least 1:2 with 10%
sucrose, and centrifuged at 180,000 x gum in Ti-70 rotor for 40 min. The SR
fraction collected between 22% and 36% sucrose gradient layers was light
SR; the SR fraction obtained between 36% and 45% sucrose gradient layers
was heavy SR. The pellets were resuspended in 10% sucrose, quick-frozen
and stored at -80°C. The yields were typically 17 mg skeletal heavy SR
from 100 g of unimproved turkey muscle and 30 mg skeletal heavy SR
from 100 g of commercial turkey muscle.

2. Ryanodine Binding Assays
The ryanodine binding assays are based on that of Mickelson et al.

(1988). [3H]Ryanodine binding was performed by incubating 0.2 mg of

87

heavy SR protein/ml or 1 mg of crude SR protein/ml for 90 min at 37°C in
media containing 0.25 M KCl, 25 mM PIPES (pH 7.0), 4 or 10 nM [3H]
ryanodine, and a CaClz-EGTA-nitrilotriacetic acid buffer to give a specific
[Ca2+]fm ranging fiom 0.01-1000 1.1M. Free Ca2+ concentrations were
calculated using the computer program of Perrin and Sayce (1967), which is
based on equilibrium concentrations of metal-ligand mixtrues. This
information was used to derive optimal [Ca2+],m for subsequent
[3H]ryanodine binding studies designed to obtain the KI and BM values.
The ryanodine concentration was varied by addition of unlabeled ryanodine.
Samples were filtered with Whatman GF/B filters and washed three times
with 5 ml of ice-cold buffer (0.25 M KCl, 25 mM PIPES, pH 7.0). Specific
ryanodine binding was determined by subtracting the nonspecific binding
obtained in the presence of 100 11M unlabeled ryanodine.

The Kd and B,“m values for ryanodine binding by the SR preparations
were calculated from the fit of bound vs. free ryanodine using Enzfitter
computer program (Biosoft, Cambridge, UK) when ryanodine was varied
fiom 1-500 nM.

3. Calmodulin Purification and Derivatization

Wheat germ calmodulin was prepared as described by Strasburg et al.
(1988). Chicken CaM, site-specifically mutated at glutamine 143 to a
cysteine residue (QI43C), was obtained from Dr. Albert Wang, Boston
Biomedical Research Institute. For crosslinking experiments, both wheat
germ and Q143C chicken CaM were radiolabeled with 12’I and
monofunctionally substituted at Cys 27 of wheat germ CaM or at Cys 143
of chicken CaM with the photo-activatable crosslinker, benzophenone-4-
maleimide (Strasburg et al., 1988). For fluorescence experiments, wheat

germ CaM was labeled at Cys 27 with tetramethylrhodamine-X-maleimide

88

(Strasburg et al., 1988).
4. Calmodulin Crosslinking

Affinity labeling of CaM-binding proteins in turkey skeletal SR
vesicles was performed by incubating in darkness 0.1 M wheat germ or
Q143C [‘”I]-Bz-CaM with 100 11g of heavy SR vesicles using the buffer
conditions of Yang et al. (1994) [20 mM Hepes, pH 7.5, and 0.15 M NaCl].
CaClz, MgClz, and EGTA were included as indicated in the figure legends.

The mixtures were placed in plastic microcentrifuge tubes, the tubes
were covered with plastic wrap, and the samples were illuminated for 5 min
in a Stratalinker 1800 photoreactor (Stratagene Crop., La Jolla, CA) equipped
with lamps of 21m = 254 nm. After photolinking, the samples were
centrifuged in Beckman TL-100 centrifuged at 88,000 x gm,x for 20 min.
The membrane pellets were resuspended in 50 111 water. For electrophoresis,
25 111 dye buffer containing 3.7% SDS, 12 mM EGTA, 30% glycerol,
~0.001% bromphenol blue, and 46 mM Hepes, pH 7.5 was added to each
sample.
5. Electrophoresis

The electrophoresis procedure employed was that described by
Laemmli (1970) with the modification that 4-10% linear acrylamide gradient
gels were used. Slab gels were stained with 0.05% Coomassie blue in 50%
methanol/10% acetic acid and then destained with 50% methanol/10% acetic
acid. The gels were scanned using GS 300 Transmittance Reflectance
Scanning Densitometer (Hoefer Scientific Instruments, San Francisco, CA).
The gels were dried and placed with Kodak Omat XAR-SX-ray film in
autoradiography cassettes equipped with Dupont Lightning Plus intensifying

screens.

89

6. Stains-all Staining

Staining with the cationic carbocyanine dye Stains-all (1-ethyl-2-[3-(1-
ethyl-naphtho [1,2d] thiazolin-2-ylidene)-2-methylpropenyl] naptho [1,2d]
thiazolium bromide) was carried out as described by Campbell et al. (1983).
Slab gel was fixed overnight with 25% isopropyl alcohol and washed
exhaustively in 25% isopropyl alcohol to remove SDS. The gel was then
stained in the dark for at least 48 hours with 0.0025% Stains-all, 25%
isopropyl alcohol, 7.5% forrnamide, and 30 mM Tris-base, pH 8.8. This
staining procedure resulted in blue staining of Cay-binding proteins (such as
calsequestrin) but other proteins stained pink. The slab gel was destained
completely in 25% isopropyl alcohol, and then stained with Coomassie blue.
7. Amino Acid Sequencing

The 75 kDa protein identified by 4-10% SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) was analyzed at the MSU Macromolecular
Structure Facility. The 75 kDa band was removed fi'om the gel, cut to ~1
mm2 pieces, and equilibrated with 100 mM ammonium bicarbonate buffer,
pH 8.0 at 37°C at least 24 hours and adjusted pH to 8.0 with NaOH. After
the final pH reached 8.0, the trypsin was added to reach final concentration
to 4% and incubated at 37°C for 24 hours (Fernandez et al., 1992). The
mixture was fractionated by HPLC using a C,8 column (0.8 11M x 250 mm),
and buffers (0.1% trifluoroacetic acid and 90% CH3CN/10% HZO/0.085%
trifluoroacetic acid) to elute the peptides. The fractions were sequenced by
Sequencer 494 (Perkin Elmer Applied Biosystems Divison, Foster City, CA)
for seven to eleven cycles to obtain partial amino acid sequence compared
with the database sequence (GeneBank, EMBL, and Swiss-pro) to look for

possible candidate proteins to identify the unknown protein.

90
8. Protein Assay
SR protein concennations were determined by the Lowry method
(Lowry et al., 1951) using bovine serum albumin as a standard.
9. Statistical analysis

Comparisons of the mean KI and BM values for the unimproved and
commercial turkey, for the crude and heavy SR, for the crude SR at two

different Ca2+ concentrations were made with Student's t test.

91

III. Results and Discussions

A. Electrophoretic Analysis of Skeletal Muscle Heayy SR Proteins fiom

Unimproved and Commercial Turkeys
The electrophoretic analysis of heavy SR preparations purified from

seven commercial and seven unimproved turkeys is shown in Figure 3.1.
The arrows shown near the top of the gel indicate the two isoforms (or and
B) of the Cay-channel protein fiom both types of turkey skeletal muscles.
In addition, one major band of 75 kDa was observed in five of seven
commercial turkey samples. This protein was apparently present in low
abundance in each sample of unimproved turkey. When present in
commercial turkey samples, this protein was usually the most abundant
protein in the SR preparations (Figure 3.1). The densitometric analysis of
the gel shown in Table 3.1 indicated that the relative amounts of the 75 kDa
protein range were from 19.9-26.1% of the total protein in the five
commercial turkey preparations showing high abundance of this protein. In
the other two preparations the 75 kDa protein comprised 4.5-6.1% of the
total protein. The seven genetically unimproved turkey preparations showed
the 75 kDa protein present in the range of only 09-24% of the total protein
(Table 3.1) The results suggest that there may be two different phenotypes
or groups in commercial turkeys based on the variable amount of the 75 kDa
protein found in these samples.

The relative amounts of both Ca2*-channel protein isoforms (or and B)
in unimproved turkey heavy SR preparations were at least three times as
great as those in the commercial turkey heavy SR (Table 3.1). However, the
ratio of the amounts of or isoform to the B isoform was consistently around
1:1 in both kind turkey heavy SR preparations (Table 3.1). One explanation

for the decreased relative abundance of Cay-channel protein isoforms in

 

92

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Figure 3.1 SDS—polyacrylamide gel of heavy SR preparations from
unimproved and commercial skeletal muscles. Lanes 1-7: skeletal
muscle heavy SR from seven individual genetically unimproved
turkeys; lanes 8-14: skeletal muscle heavy SR fi'om seven individual
commercial turkeys; lane 15: molecular weight markers. The upper
two arrows indicate the Ca2*-channel protein subunits which occur as
two isoforms in turkey. The lower arrow indicates the 75 kDa band.
The molecular weight markers were indicated as kDa.

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commercial turkey preparations could be an artifact of the high abundance

of the 75 kDa protein. However, this was not the case, as indicated by
ryanodine binding analysis (see below).

Mickelson et al. (1988) used SDS-PAGE to compare malignant
hyperthermia-susceptible (MHS) and normal porcine heavy SR preparations.
Unlike turkey SR preparations, the SDS-PAGE patterns of the heavy SR
prepared from normal and MHS porcine skeletal muscles were not different.
In addition, densitometric analysis of the gel demonstrated no significant
difference in the content of the Cay-channel protein between MHS and
normal SR (Mickelson et al., 1986, 1988).

B. Cay-dependence of Ryanodine Binding to the Ca2+-channel Protein fi'gm

Unimproved and Commercial Turkeys
[3H]ryanodine binding assays were applied to determine whether there

are differences in SR Cazl-channel activity between genetically unimproved
and commercial turkeys. If a defect in function is present in the Ca2+-
channel of SR from commercial turkeys, the results will probably be
manifested as an altered affinity for ryanodine, as an altered Ca2+-dependence
for ryanodine binding, or both.

The Ca” dependence of [3H]ryanodine binding of commercial turkey
skeletal muscle heavy SR vesicles is shown in Figure 3.2. Seven turkey
skeletal muscle SR vesicle preparations were examined. Two SR Ca”-
channel protein preparations have higher [3H]ryanodine binding because both
SR preparations contained less 75 kDa protein (Figure 3.1, lanes 10 and 13).
[3H]ryanodine binding was activated with a threshold of approximately 0.2
11M Ca2+ and was maximal between 3 and 30 11M fi'ee Ca2+ (Figure 3.2).
The mean [3H]ryanodine binding at the plateau was 0.24 pmol/mg of heavy
SR protein (Figure 3.3). As with the unimproved turkey SR samples, when

95

 

1.00
0.80 "
0.60 '-

o.4o e ‘ .

Bound. pmololmg 8|!

 

 

 

00° “1'" 1111111 1 11111111 1 11111111 1 1 lllLll

0.1 1 10 100 1000

[0011:th III

Figure 3.2 Ca2+ dependence of [’leyanodine binding to commercial
turkey skeletal muscle heavy SR vesicles. Curves represent results from
seven individual commercial turkey SR vesicle preparations.

l0 05:02:20 -Iflfifll

Fi
an

re]
(C

Bound. pmololmg on

Figure 3.3 Ca2+ dependence of [’H]ryanodine binding to unimproved
and commercial turkey skeletal muscle heavy SR vesicles. Points
represent the means :1: SE of duplicates for each of seven individual unimproved

96

 

1.00

0.80 r'

080'
I

0.40 ‘

 

1 1

10 100

[Calcium]. ul

(0) and commercial (0) heavy SR preparations.

 

1 000

97
free Ca2+ concentration reached 1 mM, the [3H]ryanodine binding of the

commercial turkey preparations was not inactivated. The mean value of
[3H]ryanodine binding at 1 mM Ca2+ was 0.17 pmol/mg of heavy SR protein,
corresponding to a 25% inhibition compared to 0.22 pmol/mg at 10 11M free
Ca2+ (Figure 3.3). Thus, the Ca’2+ dependence of [3H]ryanodine binding
showed the similar tendency in both unimproved and commercial turkey
skeletal muscle heavy SR (Figure 2.2, Figure 3.2, and Figure 3.3). At the
same time, the maximal [3H]ryanodine binding of the commercial turkey
heavy SR was about 36% of that of the unimproved turkey (0.22 pmol/mg
vs. 0.60 pmol/mg)(Figure 3.3). According to densitometric analysis (Table
3.1), the total amount of the Cay-channel protein in unimproved turkey
heavy SR preparations was ~3-fold greater than that from commercial turkey.
Therefore, after normalizing the ryanodine binding values of commercial
turkey, the Ca2+ dependence of {3H]ryanodine binding curves were not
significantly different between commercial and unimproved turkey based on
the [3H]ryanodine binding values.

The Ca2+ dependence of [3H]ryanodine binding to the isolated heavy
SR from turkeys are in sharp contrast to that observed for porcine SR. The
Ca2I-dependence of [3H]ryanodine binding fi'om both normal and MHS pigs
follows a bell-shaped curve, but [3H]ryanodine binding by MHS SR is
consistently greater than that of normal SR at various ryanodine
concentrations (Mickelson et al., 1988). The [3H]ryanodine binding of
rabbit skeletal muscle heavy SR is also a broad bell-shaped curve, which
shows complete inactivation at 1 mM Ca2+ (O'Brien et al., 1995). These
results with turkey SR preparations showed Ca2+ dependence of
[3H]ryanodine binding curve which extends over a greater range of [Ca2+]

and shows limited inactivation property at high Ca2+ concentration (1 mM)

98

in both commercial and unimproved turkey heavy SR (Figure 2.2 and Figure
3.2). O'Brien et al. (1995) reported that the B isoform of the Ca2*-channel
protein from fish is more insensitive to inactivation by millimolar Ca2+ than
the or isoform. Thus, the difference in Ca2+ dependence of [3H]ryanodine
binding between porcine and turkey SR probably results fi'om the fact that
the Cay-channel protein of turkey skeletal muscle consists of two isoforms
(both a and B), whereas porcine skeletal muscle only one isoform which
frmctionally resembles the a Ca2*-channel protein isoform. In addition, our
data showed no difference in [3H]ryanodine binding to commercial and
unimproved turkey SR at 4 nM ryanodine after normalizing the data, except
for a more pronounced ryanodine binding peak at 300 M Ca2+ in most
samples of unimproved turkey SR (Figure 3.3). The physiological
significance of this peak is as yet unclear.
C. Ryanodine-dependence of Ryanodine Binding to SR Vesicles fi'om
Unimproved and Commercial Turkeys

As noted in the previous section, frmctional differences in channel
protein activity may be reflected by altered channel protein affinity for
ryanodine. [3H]ryanodine binding activity of commercial turkey skeletal
heavy (Figure 3.4A) and crude (Figure 3.5A) SR vesicles was measured in
the presence of 10 11M Ca2+ over a range of ryanodine concentrations from
0-200 nM, the ryanodine binding parameters were calculated using the
Enzfitter program, and Scatchard plots were constructed (Figure 3.4B). For
the seven commercial turkey preparations, an average K,i of 1222159 nM
and a BM of 1.10:t0.06 pmol/mg of heavy SR protein were obtained (Table
3.2). Comparing [3H]ryanodine binding parameters of the unimproved and
commercial turkey SR (Table 3.2), both KI and Bum of the commercial
turkey heavy SR were significantly different from those of the unimproved

99

Figure 3.4 Ryanodine dependence of [’H]ryanodine binding to
unimproved and commercial turkey skeletal muscle heavy SR
vesicles at 10 11M Ca". (A) Specific [3H]ryanodine binding by
unimproved (O) and commercial (0) turkey heavy SR. (B) Scatchard
plot of [3H]ryanodine binding by unimproved (O) and commercial (O)
turkey heavy SR. Points represent the means i SE of duplicates for
each of seven different heavy SR preparations.

100

 

 

 

 

 

 

 

l " 1 l J

O 50 100 150 200 250 300
Pro. W III

 

0.20

0.16

0.12

0.08

0.04

 

 

 

.9.

1 1 1 1 l

 

0.00 1
0.0 05 1.0 15 20 25 so :15 4o 45

mmmmm

101

Figure 3.5 Ryanodine dependence of [’H]ryanodine binding to
unimproved and commercial turkey skeletal muscle crude SR
vesicles at 10 11M Ca". (A) Specific [3H]ryanodine binding by
unimproved (0) and commercial (0 ) turkey crude SR. (B) Scatchard
plot of [3H]ryanodine binding by unimproved (0) and commercial (0 )
turkey crude SR. Points represent the means i SE of duplicates for
each of four different crude SR preparations.

WWW"

0.60

0.50

0.40

0.20

0.10

102

 

 

 

 

 

 

 

120

 

0.04

0.03

0.02

0.0 1

0.00

0.00

 

 

 

 

0.30

Baum-MM“!!!

0.60

103

Table 3.2 Equilibrium Constants of [3H]Ryanodine Binding of

Crude and Heavy SR from Unimproved and Commercial Turkeys
at 10 11M or 300 11M [Ca’*]

 

 

K,l , nM BM, pmol/mg
Unimproved
10 11M [Ca2+]
Heavy SR‘ 20.5 :1: 7.6‘ 4.01 :1: 0.17"
Crude SR” 19.0 :1: 4.3‘I 0.52 :1: 0.13f
300 1.1M [Ca2+]
Crude SR" 25.1 :1: 2.6" 0.58 i 0.22f
Commercial
10 1.1M [Ca2+]
Heavy SR' 12.2 :1: 59° 1.10 :1: 0.06“
Crude SR" 8.9 :1: 5.1c 0.29 :l: 0.12ll
300 uM [Ca2+]
Crude SR“ 15.7 i 3.7cl 0.34 i 0.06“

 

“bf" Different letters in column indicate significant differences (p<0.05).
“'3‘" Different letters in column indicate significant differences (p<0.05).

' n=7.
" n=4.

104

turkey heavy SR. For commercial turkey heavy SR the Kd=12.215.9
compared to 20517.6 nM for unimproved turkey (p<0.05). The Ca”-
channei protein content or Bm=1. 1010.06 for commercial turkey heavy SR
vs. 4.011017 pmol/mg for unimproved turkey (p<0.0005). The differences
in affinity of channel proteins of populations of unimproved vs. commercial
turkeys suggest that there are functional differences in one or both channel
isoforms. The differences in channel protein content further suggest
functional differences in Ca2+ regulation in the muscle cell.

The difference in BMx between commercial and unimproved turkey
heavy SR could result from artifactual differences in distributions of the
Caz’fl-charmel protein in SR membrane. To address this question,
[3H]ryanodine binding from total SR membrane preparations (crude SR)
between commercial and unimproved turkeys were compared.

The [3H]ryanodine binding to commercial turkey skeletal muscle crude
SR vesicles yielded a Kd of 8,915.1 nM and a Brm of 0.29:1:0.12 pmol/mg
at 10 11M Ca2+ (Figure 3.5 and Table 3.2). The results indicated that the
overall Cay-channel protein content of commercial turkey crude SR was still
significantly lower than that of the unimproved turkey crude SR (02910.12
vs. 05210.13 pmol/mg for crude SR, p<0.05). These results suggest the

difference in B between commercial and unimproved turkey heavy SR

does not result from the different distribution, but rather different amounts
of the Ca2‘-channel protein in SR membranes. At the same time, even after
normalizing the BM value of commercial turkey heavy SR (3 times), the
binding capacity of commercial turkey heavy SR is still lower than that of
unimproved turkey heavy SR.

The consistent Kd values between crude and heavy SR in both turkey

indicate that the affinity of Cay-channel protein for ryanodine is not affected

105

by other SR membrane proteins which might be lost upon fiactionation of
heavy SR (Table 3.2). In addition, the Cay-channel protein binding affinities
of the commercial turkey heavy and crude SR were significantly higher than
those of unimproved turkey heavy and crude SR (Kd=12.215.9 vs. 20.517 .6
nM for heavy SR; 8915.1 vs. 19014.3 nM for crude SR)(Table 3.2).
Therefore, the result indicates that there could be alterations of the
commercial turkey Ca2*-channel protein activity and quantity.

Studies on the porcine MH defect suggest that alterations in Ca2+-
channel protein content as well as activity may play a role in the defect.
Mickelson et al. (1994) indicated that crude membrane preparations fi'om 28-
day-old homozygous MHS pigs exhibited only 75% of the [3H]ryanodine
binding capacity of preparations of crude SR isolated from normal pigs.
Likewise, our results showed that crude SR from a commercial turkey
preparation displayed only 56% of [3H]ryanodine binding of crude SR
isolated from unimproved turkeys. However, in contrast to our observations
with heavy SR, [3H]ryanodine binding by porcine heavy SR is not different
between MHS and normal pigs (Mickelson et al., 1994). Since heavy SR
preparations are highly enriched in junctional membranes, these results
suggest that there may be differences in the localization of Cay-channel
proteins between the two populations.

Because of the presence of the additional ryanodine binding peak at
300 11M Ca”, ryanodine binding assays were conducted at this [Ca2+] to
determine whether functional differences in channel activity might emerge
at Ca2+ concentrations corresponding to that of contracting muscle. At 300
11M Ca2+, the [3H]ryanodine binding by commercial turkey crude SR vesicles
has a Kd of 15.7137 nM and B of 03410.06 pmol/mg (Figure 3.6 and
Table 3.2). As with unimproved turkey, the Bm values of the commercial

MIX

106

Figure 3.6 Ryanodine dependence of [’leyanodine binding to
unimproved and commercial turkey skeletal muscle crude SR
vesicles at 300 11M Ca". (A) Specific [3H]ryanodine binding by
unimproved (V) and commercial (V) turkey crude SR. (B) Scatchard
plot of [3H]ryanodine binding by unimproved (v) and commercial (V)
turkey crude SR. Points represent the means :1: SE of duplicates for
each of four different crude SR preparations.

“Mancunian“

107

 

 

 

 

 

 

 

 

1'me

 

0.04

0.03 '-

0.02

0.0 1

 

 

 

 

0.00
0.00

0. 1 O 0.20 0.30 0.40 0.50

mammalian:

0.60

108

turkey SR were not significantly different between 10 11M and 300 11M Ca2+
treatments in crude SR vesicles. However, at 300 11M Ca2+, the binding
affinity of the Cay-channel protein of the unimproved turkey for ryanodine
was significantly lower than that at 10 1.1M Ca2+ concentrations (p<0.05).
The data showed the relationships among those parameters of the commercial
turkey SR are similar with those of the unimproved turkey SR (Table 3.2).
The difference in ryanodine binding affinity between 10 11M and 300 11M
Ca2+ concentrations in both turkeys indicates that different Ca2+-channel
protein isoforms are responsible for ryanodine binding at different Ca2+
concentrations. At 10 11M Ca2+, both a and B isoforms are likely activated
in different ratios; at 300 11M Ca2+, only B isoform is likely activated because
01 isoform is inhibited by high Ca2+ concentrations (O'Brien et al., 1995).
These results suggest that there may be functional differences between B
isoforms Ca2'-channel protein of the commercial and unimproved turkeys.

D. Calmodulin Affinity Labeling of SR Vesicles from Unimproved and

Commercial Turkeys
Previous results using CaM derivatized with the affinity label in the C-

domain (Q143C CaM) indicated affinity labeling of only the a-iSOfOI‘m of the
channel protein (Figure 2.58). As a beginning step to determine whether
CaM regulation of the Ca2’-channel protein isoforms might be altered in a
population of commercial turkeys, the question was asked "do the two
commercial turkey Ca"-channel protein isoforms have the same CaM affinity
labeling pattern as the unimproved turkey Cay-channel protein isoforms?"
Results of Q143C CaM affinity labeling of commercial turkey SR are
indicated in the autoradiogram (Figure 3.7B) of the gel electrophoretogram
(Figure 3.7A). In contrast to the results fi'om unimproved turkey SR, the
commercial turkey SR shows binding activity of both commercial turkey

109

Figure 3.7 [Mg”'] and [Ca1+] dependence of affinity labeling of
commercial turkey skeletal muscle heavy SR with chicken (0143C)
[”51]-Bz-CaM. These studies were conducted using the site-specific
mutant of chicken CaM which has a Cys in the C-domain of protein
(Cysl43). [‘”I]-Bz-CaM was incubated with commercial skeletal
muscle heavy SR vesicles (lanes 1-6 and 7-12 represent two different
commercial turkey heavy SR preparations) in 20 mM Hepes (pH
7.5)/0.15 M NaCl plus following components: lanes 1 and 7, 2 mM
EGTA; lanes 2 and 8, 2 mM EGTA and 1 mM MgC12; lanes 3 and 9,
2 mM EGTA and 10 mM MgC12; lanes 4 and 10, 0.1 mM CaClz;
lanes 5 and 11, 0.1 mM CaCl2 and 1 mM MgC12; lanes 6 and 12, 0.1
mM CaCl2 and 10 mM MgC12. Molecular weight markers were
indicated as kDa. (A) Coomassie blue stained gel. (B)
Autoradiogram of dried gel.

l

2345

 

110

6789101112

—116
—97.4

—66

—45
—29

— 116
—97.4
— 66

—45
—29

1 1 1
isoforms for the Q143C [‘2’I]-Bz-CaM. These results suggested that the C-

domain of CaM may interact differently with the B isoform compared to the
unimmoved turkey skeletal muscle Ca2*-channel protein. Thus, the results
imply that there are some fimctional differences associated with CaM
regulation of the B isoform between unimproved and commercial turkeys.
The overall affinity labeling pattern of commercial turkey preparations
using both wheat germ and Q143C CaM showed additional proteins binding
CaM. The autoradiogram showed the CaZI-channel protein of the
commercial turkey was no longer the main CaM receptor (Figure 3.7B and
Figure 3.8). Intense crosslinking bands were identified in the autoradiograms
(Figure 3.7B and Figure 3.8), whereas molecular weights were calculated as
111 kDa and 94 kDa. After subtracting the molecular weight of CaM (17
kDa), these CaM receptors in commercial turkey heavy SR preparations are
approximately 94 kDa and 77 kDa proteins. The latter protein could be the
75 kDa protein which has been found in high abundance in the commercial
turkey heavy SR preparations previously (Figure 3.1). The 111 kDa complex
could be derived from the 75 kDa protein crosslinked with two CaM
molecules. In support of this argument, these intense CaM crosslinking
bands of 111 kDa and 77 kDa did not appear in the commercial turkey SR
preparation which contains low 75 kDa protein abundance (data not shown).
Alternatively this protein could be triadin, a 95 kDa SR protein (Kim et al.,
1990); or glycogen phosphorylase, a 97 kDa protein which can be
phosphorylated in the presence of C a” and CaM (Molla et al., 1985). These
results suggest that the differences in composition distribution of heavy SR
vesicles between commercial and unimproved turkey could change the CaM

regulation for the Car-channel protein.

112

123456789101112

.33: - ._ rac'PFS.’
y y as. 3:
— 205
‘
— 116
— 66
__ 45

M -29

Figure 3.8 [Mg’*] and [Ca’*] dependence of affinity labeling
autoradiogram of commercial turkey skeletal muscle heavy SR
with wheat germ [”sll-Bz-CaM under different buffer conditions.
These experiments were conducted using wheat germ CaM with the
benzophenone-maleimide cross-linker at Cys-27. 0.1 M (lanes 1-6)
and 0.5 11M [‘z’l]-Bz-CaM (lanes 7-12) were incubated with
commercial skeletal muscle heavy SR vesicles in 20 mM Hepes (pH
7.5)/0.15 M NaCl plus following components: lanes 1 and 7, 2 mM
EGTA; lanes 2 and 8, 2 mM EGTA and 1 mM MgC12; lanes 3 and 9,
2 mM EGTA and 10 mM MgC12; lanes 4 and 10, 0.1 mM CaCIZ;
lanes 5 and 11, 0.1 mM CaCl2 and 1 mM MgC12; lanes 6 and 12, 0.1
mM CaCl2 and 10 mM MgC12. Molecular weight markers were
indicated as kDa.

113

E. Partial Characterization of 75 kDa Protein of Commercial Turkey Skeletal
Muscle Heafl SR

The presence of the 75 kDa protein in such great abundance in most
commercial turkey SR preparations could be an important indicator
associated with the meat quality problems. The identity of this protein on
the basis of molecular weight alone is unknown. Chadwick et a1. (1988)
showed that a 71 kDa protein crosslinked with the Cay-channel protein of
terminal cistemae in rabbit skeletal muscle. However, they did not identify
this protein which was present in low abundance. Another candidate to be
considered for analysis was calsequestrin, a Ca2+-binding protein in normally
present high abundance in SR preparations. Calsequestrin exhibits varying
mobility on SDS-PAGE (44,000-63,500) depending on the type of gel used, '
and the tissue source. Because of its abundance and variable mobility on
gels, the cationic carbocyanin dye "Stains-all" was used to determine whether
the 75 kDa protein could be calsequestrin which, because of its very acidic
amino acid composition, stains dark blue or purple. In addition, Stains-all
dye will stain the other acidic Cap-binding proteins, such as calmodulin,
troponin C, and S-lOO, dark blue or purple, while most other proteins with
red or pink.(Campbell et al., 1983).
l. Stains-all Staining Gel

Ten heavy SR preparations, purified from three unimproved and seven
commercial turkeys, were analyzed by SDS-PAGE and staining with
Coomassie blue (Figure 3.9A) and Stains-all (Figure 3.98). In Figure 3.9B
(lanes 4-10), the 75 kDa protein is clearly stained pink rather than blue.
However, most proteins were hard to see in the Stains-all gel, because of the
pink background (Figure 3.98). Further destaining tended to decolorize

protein as well as background. There was one blue band clearly observed

114

Figure 3.9 Coomassie blue and Stains-all stained SDS-
polyacrylamide gel of heavy SR preparations from unimproved
and commercial skeletal muscles. Lanes 1-3: 3 unimproved turkey
skeletal muscle heavy SR; lanes 4-10: 7 commercial turkey skeletal
muscle heavy SR; lane ll: molecular weight markers. (A) Coomassie
blue stained gel. (B) Stains-all stained gel. The upper two arrows
indicate the Ca2‘-channel protein subunits which occur as two isoforms
in turkey. .The lower arrow indicates the 75 kDa band.

115

1234567891011

— 116
— 97.4
:66

-—45

 

1234 567891011

-— 116
— 97.4

:66

—45

 

116

at 157 kDa and one dark blue band at 45 kDa in unimproved and
commercial turkey heavy SR preparations (Figure 3.93). The former could
be the 160-kDa glycoprotein associated with the DHP receptor (Campbell et
al., 1983). The 45 kDa band is probably calsequestrin because its apparent
molecular weight (45,000) is consistent with that reported for mammalian
skeletal muscle (Campbell et al., 1983). These results indicate that the 75
kDa protein in commercial turkey heavy SR preparations is not calsequestrin,
nor is it an acidic Cay-binding protein, or glycoprotein.

2. Amino Acid Sequencing Analysis

Amino acid sequence analysis was used in an effort to identify the 75
kDa protein. Attempts to determine the N-terminal sequence of the 75 kDa
protein were unsuccessful because the N-terminus of the 75 kDa is blocked
as is the case with most muscle proteins. To obtain partial sequence
information, the 75 kDa protein was subjected to limited digestion with
trypsin, and then peptide fragments were purified by capillary HPLC. The
amino acid sequence of one purified trypsin-digested fragment was identified
as "Asp-Phe-Glu-lle-Val-Pro-Gly-Ser—Gly-Lys".

After matching the peptide using computer database sequences and
matching candidates to a molecular size of approximately 75 kDa, the
candidates which emerged were guanylate cyclase (83.3% identify in 6
amino acids overlap), and anthranilate synthase (75.0% identify in 8 amino
acids overlap).

Guanylate cyclase is a 73 kDa enzyme which catalyzes the generation
of cyclic 3’,5'-guanosine monophosphate (cGMP) from guanosine
triphosphate (GTP). cGMP serves as a second messenger which activates a
cGMP-dependent protein kinase. Recently, Galione et al. (1993) proposed

cyclic ADP-ribose (cADPR), which is an endogenous activator of Ca2+-

117

induced Ca2+ release, was enhanced by cGMP. However, cADPR has no
effect on the type 1 Ca2*-channel protein of skeletal muscle (on isoform) so
its messenger function is restricted to type 2 cardiac Cay-channel protein (B
isoform) (Bern'dge, 1993). It seems unlikely that there would be so much
guanylate cyclase in SR.

Anthranilate synthase is a ~65 kDa enzyme which involved with the
biosynthesis of tryptophan. It converts chorismate to anthranilate, the first
step in the reaction sequence leading on to tryptophan production. However,
the tryptophan synthesis pathway is only present in microorganisms and
plants, the enzymes that synthesize essential amino acids have apparently
been lost early in animal evolution. Thus, the identity and function of the

75 kDa protein are unknown.

118

IV. Conclusions

The commercial turkey population which likely includes a stress-
susceptible sub-population and the genetically unimproved control ttu'key
skeletal muscle SR Ca2*-channel activities were very different in ryanodine
dependence of [3H]ryanodine binding. The commercial turkey heavy SR had
higher binding affinity (Kd=12.2 versus 20.5 nM) and lower binding capacity
(Bm=l.10 versus 4.01 pmol/mg) than unimproved turkey. The differences
in binding affinity and capacity were also observed in crude SR from both
turkeys. The results suggest that there is an altered Cay-channel protein
content and altered activity of the Ca2*-channel protein in at least some of
the commercial turkey population, which may be related to the incidence of
PSE meat.

The contents of the SR Ca’*-channel protein in heavy SR preparations
between commercial and unimproved turkey were different. The content of
both a and B isoforms in the commercial turkey skeletal muscle heavy SR
(0.8-1.4% total protein) was lower than that of the unimproved turkey heavy
SR (1.6-7.0% total protein). In addition, the SDS-PAGE indicated that there
was a 75 kDa protein which is the predominant SR protein in most but not
all commercial turkey SR samples examined. This protein was in very low
abundance in unimproved turkey SR preparations. The identity and function
of this 75 kDa protein, which binds CaM, is still unknown.

The Ca” dependence of [3H]ryanodine binding in both commercial and
unimproved turkey heavy SR displayed no characteristic difi‘erences. Avian
Cap-channel protein has both a and [3 isoforms, whereas mammalian only
has a isoform. Therefore, unlike mammalian muscle, the avian Cay-channel
protein activity was not inhibited very much at high Ca2+ concentration. This

behavior is attributable to the B isoform of the Ca2*-channel protein (O'Brien

119

et al., 1995). However, both the Ca2+ dependence and ryanodine
dependence of the [3H]ryanodine binding of the Cay-channel protein SR
indicated the Cay-channel protein from commercial turkey have different
channel activity and content compared to unimproved turkey when measured
at 300 pM Ca2+ concentration.

The crosslinking studies showed differences in the CaM affinity
labeling of the Cay-channel protein B isoform between commercial and
unimproved turkeys. Q143C [‘”I]-Bz-CaM binds to the B isoform of the
commercial turkey Ca2*-channel protein, but the B isoform of unimproved
turkey does not show crosslinking with this CaM derivative. Results with
wheat germ CaM indicate that the B subunit does bind CaM. These results
suggest that there could be a defect in the commercial turkey SR Ca”-
channel protein B isoform which is associated with the meat quality problems

with commercial turkeys.

CHAPTER 4
CALMODULIN INTERACTION WITH THE PURIFIED CARDIAC
Can-CHANNEL PROTEIN (B-LIKE ISOFORM)

I. Introduction

The contraction and relaxation of cardiac muscle depends on the
cytosolic level of Ca” ions. Ca2+ release fi'om the sarcoplasmic reticulum
(SR) via the Caz‘lchannel protein contributes to muscle contraction; the Ca2+
uptake into the SR gives rise to muscle relaxation via the Ca21-ATPase.
Although the Ca2+ uptake process has been described in considerable detail,
the molecular mechanisms and regulations involved in Ca2+ release are, by
comparison, poorly understood.

In the inotropic effect in the heart, myocardial contractility is enhanced
though accelerated Caz’ uptake via phosphorylation of phospholamban,
decreased aflinity of tr0ponin C for Ca2+ resulting from phosphorylation of
troponin I, and enhanced Caz‘ activation of Cay-release via phosphorylation
of the DHR receptor. The precise role of the SR Cay-channel protein in the
inotropic effect of the heart, and the precise mechanisms governing its
regulation are still unclear.

Numerous molecular mechanisms modulate the SR Ca2+-channel
protein activity. Cazfirelease is enhanced by micromolar concentrations of
cytoplasmic Caz‘ (Cf-induced Cay-release), and by adenine nucleotides,
caffeine, and nanomolar concentrations of ryanodine (Meissner et al., 1986).
In contrast, the Ca2+ channel protein activity is inhibited by Mg”, calmodulin

120

121

(CaM), ruthenium red and micromolar concentrations of ryanodine (Smith
et al., 1985; Meissner and Henderson, 1987). CaM may modulate the
cardiac Cay-channel protein activity through two different ways: by directly
binding to the Ca2*-channel protein and inhibiting Cay-release (Meissner,
1986; Smith et al., 1989) or by CaM-dependent phosphorylation of the Ca2+-
channel protein which enhances channel activity by increasing the open
probability of the channel (Witcher et al., 1991).

The studies of Chapters 2 and 3 suggest that the B isoform of the Ca2+-
channel protein in turkey skeletal muscle could play an important role in
determining the PSE-like meat quality problem in commercial turkeys.
Therefore, a better understanding of the biochemical properties of the B
isoform may help solve the PSE problems in turkeys. Immunological
techniques identified epitopes in the amphibian or and B skeletal isoforms
common to the mammalian skeletal and cardiac forms of the Ca2+-channel
protein, respectively (Lai et al., 1992). Thus, the interaction of the cardiac
Ca2*-channel protein with CaM is an indirect way to understand the
biochemical properties of the B isoform from skeletal muscle. The objective
of this study is to define the stoichiometry and affinity of binding of CaM

to the cardiac Cazfichannel protein under different physiological conditions.

122

11. Materials and Methods
A. Materials:
1. Purified Canine Cardiac Ca2+ -channel Proteins

Purified cardiac Cay-channel protein preparations from canine were
provided by Dr. Jones at the Department of Pharmacology and Toxicology,
and the Department of Medicine at Indiana University. Purified cardiac
Cay-channel protein was in medium containing 1 M NaCl, 0.2 mM CaCl,,
20 mM MOPS, pH 7.2, 0.5% CHAPS, 50 ug/mL Pefabloc, and 10-25%
sucrose.
2. Chemicals

BenZOphenone-4-maleimide, tetramethylrhodamine-x-maleimide, and
CaM-dependent protein kinase II were purchased fiom Molecular Probes
(Junction City, OR). Na‘z’l and [3H]ryanodine were obtained from DuPont-
NEN (Boston, MA). Ryanodine was purchased from Calbiochem (La Jolla,
CA).
B. Methods
l. Calmodulin Purification and Derivatization

Wheat germ calmodulin was prepared as described by Strasburg et al.
(1988). For crosslinking experiments, wheat germ CaM were radiolabeled
with ”51 and monofunctionally substituted at Cys 27 of wheat germ CaM
with the photo-activatable crosslinker, benzophenone-4-maleimide (Strasburg
et al., 1988). For fluorescence experiments, wheat germ CaM was labeled
at Cys 27 with tetramethylrhodamine-X-maleimide (Strasburg et al., 1988).
2. Calmodulin Crosslinking

Affinity labeling of CaM-binding proteins in purified canine cardiac
Cazi-channel proteins was performed by incubating in darkness 0.1 uM
wheat germ ['z’l]-Bz-CaM with 80 pl (~O.9 pg) of purified cardiac Ca2+-

123
channel proteins using the buffer conditions of Yang et al. (1994) [20 mM
Hepes, pH 7.0, and 0.15 M NaCl] (final volume=140 pl). CaClz, MgClz, and
EGTA were included as indicated in the figure legends.

The mixtures were placed in plastic microcentrifuge tubes, the tubes
were covered with a plastic wrap film, and the samples were illuminated for
5 min in a Stratalinker 1800 photoreactor (Stratagene Crop, La Jolla, CA)
equipped with lamps of A“; 254 nm. For electrophoresis, 25 pl sample
buffer containing 3.7% SDS, 12 mM EGTA, 30% glycerol, ~0.001%
bromphenol blue, and 46 mM Hepes, pH 7.5 was added to each sample.

3. Electrophoresis

The electrophoresis procedure employed was that described by
Laemmli (1970) with the modification that 4-10% linear acrylamide gradient
gels or 5% mini-gels were used. The gels were stained with 0.05%
Coomassie blue in 50% methanol/10% acetic acid and then destained with
50% methanol/10% acetic acid. The gels were dried and placed with Kodak
Omat XAR-SX-ray film in autoradiography cassettes equipped with Dupont
Lightning Plus intensifying screens.

4. Ryanodine Binding Assay

The ryanodine binding assays are based on that of Mickelson et al.
(1988). [3H]ryanodine binding was performed for 90 min at 37°C in media
containing 25 pl purified canine cardiac Cay-channel protein, 1 M KCl, 100
mM PIPES (pH 7 .0), 10 nM [3H]ryanodine, 65 nM unlabeled ryanodine and
20 pM CaClZ. This information was used to determine maximal ryanodine
binding activity of the purified Cali-channel protein. Subsequently, samples
were filtered with Whatman GF/B filters and washed three times with 5 ml
of ice-cold buffer (0.25 M KCl, 25 mM PIPES, pH 7.0). Specific ryanodine
binding was determined by subtracting the nonspecific binding obtained in

124
presence of 100 pM unlabeled ryanodine.

5. Fluorescence Anisotropy Measurements

Fluorescence anisotropy was used to characterize CaM/calcium-channel
protein interaction in SR vesicles. The principle underlying fluorescence
anisotropy is that the rapid Brownian tumbling of a small molecule is slowed
upon binding to a larger molecule to form a complex. If the smaller
molecule is fluorescent, the altered mobility of this species resulting from
complex formation can be detected by measuring the change in
depolarization of fluorescent light emitted upon excitation by polarized light
(Lakowicz, 1983).

Fluorescence measurements were performed using an SLM 4800
spectrofluorometer equipped with two detectors in T-format detector.
Samples were held in a thermostated cell block maintained at 22°C. The
excitation wavelength of Rh-CaM was 532 nm, monochromator slits were set
at 4 nm, and emitted light was isolated using Schott 06-570 filters. During
titrations, samples were allowed to equilibrate for 4 min after each addition
of SR or Rh-CaM. All samples were measured in buffer containing 10%
sucrose, 0.1 mM CaCl,, 2 mM MgCl,, 1 mM NaF, 50 mM Hepes, pH 7.0,
and 0.15 M NaCl and proteinase inhibitors (1 pg/ml aprotinin, 1 pg/ml
leupeptin, and 0.1 mM PMSF).

All fluorescence measurements were performed using semi-micro,
quartz fluorescence cuvettes (4 mm x 10 mm). Prior to fluorescence
experiments, the cells were rinsed with 1 mg/ml bovine serum albumin to
minimize Rh-CaM adsorption to the walls of the cuvette. Following this
treatment, the measured anisotropy value for Rh-CaM was independent of
concentration over the range of 1 nM to 1000 nM, indicating that there was
negligible Rh-CaM adsorption to the cuvettes (Yang et al., 1994).

125
The anisotropy of ligand (Rh-CaM) is directly proportional to the
fraction of ligand bound to receptor (Cf-channel protein). Thus, if Af is the
anisotropy of free Rh-CaM and Ab is the anisotropy of fully bound ligand,
then the fraction bound, f,, is determined from:

(Am-'Af)

f”: Ann-Q) +q(Ab) -Af (1)

 

Where Am is the measured anisotropy for a given ligand concentration and
q, the change in quantum yield, is the ratio of fluorescent intensity of bound
species over that of three species. If the change in quantum yield is
negligible upon binding of ligand, then the equation (1) reduces to:

- (Am—At)

'_<A,-A,) <2)

fb
The fraction of ligand bound, and the concentrations of bound and free Rh-
CaM are readily calculated.

The anisotrow of unbound Rh-CaM, Ar, was measured in the absence
of SR or purified Cab-channel protein. The anisotropy of fully bound
species, Ab, was obtained by titration of Rh-CaM with SR vesicles or
purified Car-channel proteins, following by curve-fitting using the Enzfitter
computer program (Biosoft, Cambridge, UK) for a single class of ligand
binding site. Corrections for light scattering and background fluorescence

were made by application of the equation:

A=f1A1 +f2A2 (3 )

where A, A1 and A2 are the anisotropies of sample, the blank, and the

corrected sample, respectively. The fractional contributions, fl and f2, of

126

these species were calculated from the intensities measured with the
excitation monochromator in the vertical position and the emission
monochromator at 55°. The corrected sample anisotropy, therefore, is that

value in the absence of background interference.

127

III. Results and Discussions
A. Calmodulin Binding Activig of the Purified Cardiac Ca2+-Qhannel Protein

Ryanodine-activity was measured to determine biological activity of
purified Ca’*-channel protein and to determine channel protein content in the
preparations. The SDS-PAGE of the cardiac purified Cay-channel protein
preparation (Figure 4.1A) showed high purity of the Ca2"-channel protein.
There are two major bands: the upper one is the Ca2*-channel protein and the
lower one has been identified as the degradation fi'agment from the Ca2+-
channel protein. In addition, there is a faint band at ~100 kDa which is
probably Cay-ATPase (Figure 4.1A).

Purified Ca2*-channel protein from canine cardiac muscle preparations
was incubated with the affinity-labeling derivative [‘2‘I]-Bz-CaM to confirm
binding activity of the channel protein for CaM and to demonstrate that no
other CaM-binding protein were present in the purified preparations. The
autoradiogram (Figure 4.1B) of the gel electrophoretogram (Figure 4.1A)
indicated that the major complex formed was a doublet of Mr > 450,000
which corresponds to CaM plus the channel protein subunit (Seiler et al.,
1984). The amount of complex obtained was much greater in the presence
of Ca2+ than in the presence of EGTA at each Mg” concentrations examined
(Figure 4.1B), suggesting that CaM-binding to cardiac Ca’*-channel protein
is Ca2*-dependent.

These results are similar to those obtained by Seiler et al. (1984) and
showed that the azido-['2’1]CaM labeling of the canine cardiac Cay-channel
protein in SR vesicles was largely dependent upon the presence of Ca2+. In
contrast, Takasago et al. (1991) using [”51] Denny-Jaffe-labeled—CaM with
similar buffer conditions showed that ['”I]CaM bound to the canine cardiac

Cay-channel protein preferentially in the absence of Ca2+. The differences

r°+

 

Figure 4.1 . [Mg’*] and [Ca2+] dependence of affinity labeling of the
purified cardiac Ca’*-channel protein with wheat germ ["sll-Bz-
CaM under different buffer conditions. These experiments were
conducted using wheat germ CaM with the benzophenone—maleimide
cross-linker at Cys-27, and using buffer conditions: 20 mM Hepes, pH
7.0, and 0.15 M NaCl. (A) Coomassie blue stained gel. (B)
Autoradiogram of dried gel. The lower band has been idenitified as
a proteolytic degradation product of the upper band.

129
in the CaM affinity labeling patterns between Takasago et al. (1991) results

and ours could result from differences in the CaM derivative employed.

The CaM affinity labeling pattern of the cardiac Cazi-channel protein
differs from that of the skeletal muscle Ca2+-channel protein isoform.
Crosslinking studies using porcine skeletal muscle SR vesicles suggested that
CaM-binding is Ca2+-independent (Yang et al., 1994). Those results were
confirmed by fluorescence analysis of binding (Figure 4.1). The result
indicates that CaM affinity labeling binding with or isoform of Ca2+-channel
protein is more insensitive to Ca”; the CaM affinity labeling binding of or
isoform is greater in the presence of EGTA than that in the presence of
CaCl2 (Yang et al., 1994). These results suggest that CaM differentially
regulates Ca2+ release activity by a and B isoforms.
B. Stoichiomeg and Affinity of Rh-CaM with the Purified Cardiac Ca2+-
channel Protein

Previous results of SR cardiac Ca’+-channel protein showed that there
was not CaM binding activity at all in the presence of EGTA by
fluorescence anisotropy measurement (Strasburg et al., 1993). In agreement
with previous results, the purified cardiac Ca2*-channel protein showed little
affinity labeling with CaM in the presence of EGTA in both crosslinking
experiments (Figure 4.1) and fluorescence measurement (Figure 4.2).
Therefore, to quantify CaM binding activity under conditions, the simulating
contracting muscle buffer condition were chosen which included CaCl2 plus
MgCl2 for the fluorescence anisotropy study.

Purified cardiac Caz‘-channel proteins were titrated into Rh-CaM under
buffer condition containing 0.1 mM CaCl2 plus 2 mM MgC12. In each
preparation, titration of the purified channel protein into Rh-CaM resulted

in a large increase in fluorescence anisotropy attributable to the increased

 

130

 

 

 

 

0.24
0.22 '
h
D.
O
b
‘6 0.20 -
.2
0.18 -
0.16 J J rrirrrL r I 1111411 1 11111111 A
1 10 too 1000 3000

Calcium Channel Protein. no

Figure 4.2 Titration of Rh-CaM with the purified cardiac Ca’+-channel
protein. The sample medium contained 10 nM Rh-CaM, 0.3 M sucrose,
0.15 M NaCl, and 50 mM Hepes, pH 7.0, plus either 0.1 mM CaCl2 and 0.2
mM MgC12(O) or 2 mM EGTA (0) in a starting volume of 1 mL. The Rh-
CaM sample was titrated with the purified cardiac Cay-channel protein in
parallel with a buffer blank containing the same media minus Rh-CaM.
Corrections were made for light scattering as described under Methods.

131

molecular mass of the Rh-CaM/Ca’*-channel protein complex (Figure 4.2).
Data from these titrations (Figure 4.2) were used to determine the anisotropy
values for free Rh-CaM (Ar) and for the full bound Rh-CaWCa’*-channel
protein complex (Ab). Normally these values are obtained from the limits of
the titration curves. However, values for Ab could be underestimated in
these experiments because the binding curves somewhat decreased, probably
because of light scattering, after adding certain amount purified Ca2*-channel
protein instead of maintain a saturation state. Therefore, Al, values were
calculated by extrapolation using the Enzfitter program applied for a single
ligand-binding site on Rh-CaM. The A, values for two preparation averaged
0.2561. The mean value of the Af from two preparations was 0.1727. There
was no significant change in fluorescence intensity upon binding of Rh-CaM
to the channel protein (q=1.0); therefore, the fraction of Rh-CaM bound was
calculated using eq 2.

The Ar and AI, values form the basis for subsequent study to estimate
the affinity constant and stoichiometry of CaM/Cardiac Ca’*-channel protein
interaction. Purified cardiac Cap-channel protein preparations were titrated
with Rh-CaM, fluorescence anisotropy was measured (Figure 4.3A), and the
data were analyzed using the non-linear regression program Enzfitter (Figure
4.38). Scatchard plot of the data showed two classes of ligand-binding sites
on the cardiac Ca2’-channel protein for CaM in the presence of CaCl2 plus
MgCl2 (Figure 4.38). The high-affinity class of sites has Kdl=0.6li0.36 nM
and Bml=831i293 pmol/mg, and the results of low-affinity binding class
site show Kd2=7 .19i0.95 nM and Bmm=5135i246 pmol/mg (Table 4.1).
The data suggest that there are ~2 CaM high-affinity binding sites per tetramer
Ca2*-channel protein, and ~12 CaM low-affinity binding sites per tetramer in

cardiac Cay-channel protein.

132

Figure 4.3 Titration of the purified cardiac Ca’*-channel protein
with Rh-CaM in the presence of CaCl2 and MgClz. (A) Anisotropy
plot of titrations of Ca2‘-channel proteins with Rh-CaM . (B) Rh-
CaM/Ca"-channel protein saturation binding curve. The inset is a
Scatchard plot of Rh-CaM binding to Ca’*-channel proteins. The
sample medium contained 1.8 ng (average) of the Cay-channel
protein, 0.3 M sucrose, 0.15 M NaCl, 50 mM Hepes, pH 7.0, 0.1 mM
CaCl,2 and .0.2 mM MgCl2 in a starting volume of 1.2 mL. Points
represent the means of two preparations. CaM bound was calculated
from measured anisotropy as described under Methods.

 

 

trottir
mm

at is!
, The
ham
,1 at
Port
‘ulattd

Bot-id Rh- CaM. pmol/Ina

133

 

0.25

0.10 '

0.21 "

0.10 '-

 

l

I rrrrrl

1 1 11111

L1

1

 

llllll

 

0.1 7
0.1

fill-Call. IN

10

100

 

0000
5000
4000
0000

 

1000

 

 

 

 

 

 

 

Fro. Rh-CIM. I'M

 

134

Table 4.1 Equilibrium Constants for Rh-CaM Interaction with the
Purified Cardiac Ca”'-channel Protein in the presence of .1 mM
CaCl2 + 2 mM MgCl,‘

 

 

Kd, nM Bum, pmol/mg CaM/tetramer
High-affinity site 0.61 i 0.36 831 :1: 293 ~2
(Kdl)
Low-affinity site 7.19 :i: 0.95 5135 :l: 246 ~12
(K42)

 

' Data were obtained from titrations of purified cardiac Ca2+-channel
proteins with Rh-CaM in the presence of 0.3 M sucrose, 1 mM NaF, 0.15
M NaCl, 50 mM Hepes, pH 7.0, 0.1 mM CaCl2 and 0.2 mM MgC12_

Data are the means of two preparations.

 

135

Our previous studies on CaM-binding to Sr vesicles, however, only
showed a single class of binding sites on the SR Ca”’-channel protein for
CaM with a Kd of 13 nM and a Bm of 55 pmol/mg (Strasburg etal., 1993).
The data indicate that there are 3 CaM binding sites per channel protein
subunit (12 CaM binding sites per tetramer) (Strasburg et al., 1993). Thus,
the results with purified cardiac Cay-channel protein were consistent with the
previous data for the SR Ca2+-channel protein in vesicles. Differences in
affinity with that observed previously may result from differences in use of
SR vesicles vs. solubilized protein.

Porcine skeletal muscle SR Ca2*-channel protein (or-like isoform) also
has two classes of CaM binding sites (Yang et al., 1994). In the presence
of CaCl2 and MgClz, there are l CaM high affinity (Kd,=0.l nM) and 7 CaM
low affinity binding sites (Ka=l7 nM) in Cay-channel protein tetramer
(Yang et al., 1994). However, in contrast to cardiac muscle Ca2"-channel
protein, the skeletal isoform binds CaM even at low Ca2+ (Tripathy et al.,
1995). Under these conditions CaM activates the skeletal Cay-channel
protein for Car-release, whereas CaM could not execute this function with
the cardiac isoform since there is no binding at low Caz". Under contractile

conditions,.however, CaM inactivates both isoforms.

 

 

 

136

IV. Conclusions

Physiological differences between the or and B Ca2+-channel proteins
have different Ca2*-sensitivity to Ca2*-induced Ca2+ release, and to Ca”-
inhibition (O'Brien et al., 1995). Due to the differences in biochemical
properties between or and B Ca’+-channel proteins, O'Brien et al. (1995)
proposed that a isoforms are directly coupled with the T-tubule DHP
receptor. Activation of or isoform channels by voltage changes would result
in the release of Ca2+ into the sarcoplasm and thereby activate a neighboring
"slave" channel, the B isoform Ca’*-channel protein. These results show that
CaM interaction with the mammalian skeletal (or-like isoform) and cardiac
(B-like isoform) Ca2*-channel protein differs in Ca”'-sensitivity. CaM only
bound to the cardiac muscle Ca”'-channel protein in the presence of Ca2+,
whereas CaM bound to the mammalian skeletal muscle Ca2+-channel protein
in the presence or absence of Ca2+. These results suggest that Ca2*-induced
Ca2*-release of the B isoform may be inhibited at a rate limited by direct
binding of CaM. Compared with regulation of the or isoform, the CaM
inhibitory action in the presence of Ca2+ may be more important for the B
Ca2+-channel protein because it is directly inhibited by high Ca2+
concentrations, while the B isoform remains.

In mammalian skeletal muscle, CaM bound to the Ca2+-channel protein
in both muscle resting and contractile states (Yang et al., 1994). Binding of
CaM in the presence of low Ca2+ may activate the channel protein (Tripathy
et al., 1995), whereas in the presence of Ca2+, CaM inhibits channel activity.
However, in the presence of EGTA, the cardiac muscle Ca’*-channel protein
did not interact with CaM molecules, suggesting increasing cytoplasmic Ca2+
is the only way to activate the cardiac Cay-channel protein, and that the

inhibitory function of CaM for the cardiac Cay-channel protein is its primary

137
function in the heart.

CONCLUSIONS

In muscle excitation-contraction coupling, depolarization of the cell
triggers release of Ca2+ fi'om the terminal cistemal SR, that fraction of SR
which is coupled to the T-tubule via the Ca’+-channel protein. Although this
protein plays an important role in this process, the mechanisms involved in
regulation of Ca2+ release and the relationship of abnormal Ca2+ regulation
to meat quality are still poorly understood.

PSE meat quality problems, which have caused substantial economic
losses to the swine industry for many years, have become increasingly
prevalent and severe in the turkey industry. Porcine stress syndrome (PSS)
is responsible for a significant fiaction of PSE pork; the basis of this
inheritable disorder has been identified as a mutation in the gene for the
Ca2*-channel protein in pigs. The similarity of the PSE meat quality
problems and of factors which influence the prevalence of PSE meat from
pigs and turkeys suggests that a subpopulation of commercial turkeys could
have a defect in the Ca2'-channel protein resulting in abnormal Caz“
regulation.

In first study, turkey SR preparations were Optimized for subsequent
studies to define Ca2‘-dependence of ryanodine binding, ryanodine binding
affinity, channel protein content and calmodulin (CaM) binding properties of
the Ca2*-channel protein from a control population of turkeys. Avian skeletal
muscle has two Cap-channel protein isoforms ((1 and B) in contrast to

mammalian skeletal muscle which only has one isoform. The Ca”-

138

139
dependence of ryanodine binding in turkey SR reflects difierences from

mammalian muscle in Ca2+ regulation, including a broad Ca’*-dependence
of Ca2*-induced Ca2+ release and lack of Ca2+-inhibition at Ca2+
concentrations of muscle contraction. CaM binding to avian or and B Ca2+-
channel protein isoforms resembles the binding to the mammalian skeletal
and cardiac muscle Ca2+-channel protein, respectively.

The second study indicated that the biochemical properties of the Ca2+-
channel protein from commercial turkey SR vesicles show differences
compared to those of genetically unimproved turkey. SR preparations fi'om
seven commercial turkey show an increased affinity of the Ca’+-channel
protein for ryanodine and a decreased channel protein content compared to
SR from seven unimproved turkeys. The altered affinity of the Ca2"-channel
protein for ryanodine suggests that there is a defect in the SR Cay-channel
protein in some commercial turkeys. Differences in channel protein content
suggest that may be some factors which down-regulate gene expression of
the Cay-channel protein. The differences in CaM affinity labeling to the B
Ca2*-channel protein isoform from commercial and unimproved turkeys
suggest that the defect may be specific to the B isoform of commercial
turkey.

The presence of the 75 kDa protein in most commercial turkey SR
preparations but not genetically unimproved turkey suggests that this protein
could be associated with the PSE meat quality in commercial turkeys. Our
results suggest that there is a genetic defect in the Ca2+-channel protein in a
significant fraction of the commercial turkey population. However, definitive
information awaits correlation of changes in channel protein structure with
incidence of PSE meat.

In third study, the CaM binding properties of the purified mammalian

140

cardiac Ca2*-channel protein which resemble the avian B isoform were
investigated. Affinity labeling and fluorescence experiments indicate that the
cardiac isoform binds four CaM per subunit, but does so only in the presence
of Ca2+. This is in contrast to the skeletal muscle isoform which binds five
CaM per subunit, independently of [Ca2+]. Results from these studies lend
support to previous observations that the a subunit of the avian channel
protein is regulated in a similar fashion to mammalian skeletal muscle,
whereas the B subunit is regulated in similar fashion to mammalian cardiac
muscle. The result provides further evidence that or and B isoforms are

regulated differently by CaM in different muscle conditions.

FUTURE RESEARCH

In this study, the SR Ca’*-channel protein from commercial turkeys
showed an increased binding affinity for ryanodine and a decreased channel
protein content in SR membranes. These results suggest that there is a
defect in SR Ca2*-channel protein in some commercial turkeys. However,
the results do not directly identify the specific defect which occurs in the
gene or at the protein level, nor which isoform(s) are specifically responsible
for these biochemical property changes. In order to answer these questions,
the Cay-channel protein will need to be purified from commercial and
genetically unimproved turkeys and the biochemical properties (especially
ryanodine binding properties) of the Cay-channel proteins will need to be
compared. If the ryanodine binding properties of the purified Ca2+-channel
protein isoforms are different between commercial and unimproved turkeys,
these differences would offer more direct evidence of a defect in the Ca2+-
channel protein of a subpopulation of the commercial turkey. Otherwise,
there could-be an unidentified component associated the Cay-channel protein
in commercial turkey which is responsible for the defect. If the ryanodine
binding activities of the purified Ca2*-channel protein are not different
between two kinds of turkeys, this could be because an unidentified
component is removed by purification.

Secondly, if a defect is present in the Cap-channel protein in

commercial turkey skeletal muscle, which Cay-channel protein isoform(s) are

141

142

responsible for the defect? Our results suggest that the B isoform may be
a possible candidate. To clarify this question, both or and B isoforms will
need to be purified from commercial and unimproved turkey and the
biochemical properties will be compared. If a biochemical defect is
identified in the channel protein isoform, sequencing of cDNA should
indentify the specific defect. A simple genetic test could be developed to
screen breeding stock for the presence of the defect gene.

The identity and fimction of the 75 kDa protein in some commercial
turkeys are unknown and could be associated with the formation of PSE
meat. Thus, this protein must be purified and characterized with respect to
amino acid composition and sequence to identify this protein. If this protein
cannot be identified as a known SR protein, then antibodies should be raised

against this protein for localization and characterization of function.

REFERENCES

LIST OF REFERENCES

Adams, BA, and Beam, K.G. 1990. Muscular Dysgenesis in Mice: A
Model System for Studying Excitation-Contraction Coupling. FASEB.
J. 4: 2809-2816.

Adams, B. A., Tanabe, T., Mikami, A., Numa, S., and Beam, K. G. 1990.
Intramembrane Charge Movement Restored in Dysgenic Skeletal
Muscle by Injection of Dihydropyridine Receptor cDNAs. Nature. 346:
569-572.

Agnew, W. S. 1987. Dual Roles for DHP Receptors in Excitation -
Contraction Coupling. Nature. 328: 297.

Agnew, W. S. 1988. Proteins that Bridge the Gap. Nature. 334: 299-300.
Agnew, W. S. 1989. Cloning of the SR Foot. Nature. 339: 422-423.

Airey, J. A., Beck, C. F., Murakami, K., Tanksley, S. J., Deerinck, T.J.,
Ellisman, M. H., and Sutko, J. L. 1990. Identification and
Localization of Two Triad Junctional Foot Protein Isoforms in Mature
Avian Fast Twitch Skeletal Muscle. J. Biol. Chem. 265: 14187-
14194.

Airey, J.A., Grinsell, M.M., Jones, L.R., Sutko, J.L., and Witcher, D. .1993.
Three Ryanodine Receptor lsofonns Exist in Avian Striated Muscles.
Biochemistry. 32: 5739-5745.

Babu, Y.S., Bugg, CE, and Cook, W.J. 1988. Structure of Calmodulin
Refined at 2.2 A Resolution. J. Mol. Biol. 204: 191-204.

Babu, Y.S., Sack, J .S., Greenhough, T.J., Bugg, C.E., Means, A.R.,and Cook,

W.J. 1985. Three-dimensional Structure of Calmodulin. Nature. 315:
37-40. -

143

144

Ball, SP, and Johnson, K]. 1993. The Genetics of Malignant Hyperthermia.
J. Med. Genet. 30: 89-93.

Beam, K. G., Adams, B. A., Niidome, T., Numa, S., and Tanabe, T. 1992.
Function of a Truncated Dihydropyridine Receptor as Both Voltage
Sensor and Calcium Channel. Nature. 360: 169-171.

Berridge, M.J. 1993. A Tale of Two Messengers. Nature. 365: 388-389.

Block, B.A., Imagawa, T., Campbell, K.P., and Franzini-Armstrong, C. 1988.
Structural Evidence for Direct Interaction Between the Molecular
Components of the Transverse Tubule/Sarcoplasmic Reticulum
Junction in Skeletal Muscle. J. Cell. Biol. 107: 2587-2600.

Bull, R., Marengo, J.J., Suarez-151a, B.A., Donoso, P., Sutko, J.L., and
Hidalgo, C. 1989. Activation of Calcium Channels in Sarcoplasmic
Reticulum from Frog Muscle by Nanomolar Concentrations of
Ryanodine. Biophys. J. 56: 749-756.

Campbell, K.P., Leung, AT, and Sharp, AH. 1988. The Biochemistry and
Molecular Biology of the DihydrOpyridine-sensitive Calcium Channel.
TINS. 11: 425-430.

Campbell, K.P., MacLennan, DH, and Jorgensen, AD. 1983. Staining of
the Ca2*-binding Proteins, Calsequestrin, Calmodulin, Troponin C, and
S-100, with the Cationic Carbocyanine Dye "Stains-all". J. Biol.
Chem. 258: 11267-11273.

Carl, S. L., Felix, K., Caswell, A.H., Brandt, N.R., Brunschwig, J.-P.,
Meissner, G., and Ferguson, DC. 1995. Immunolocalization of
Triadin, DHP receptors, and Ryanodine Receptors in Adult and
Developing Skeletal Muscle of Rats. Muscle and Nerve. 18: 1232-
1243.

Caswell, AH, and Brandt, N. 1989. Does Muscle Activation Occur by
Direct Mechanical Coupling of Transverse Tubules to Sarcoplasmic
Reticulum? TIBS. 14: 161-165.

Caswell, A. H, Brandt, N. R., Brunschwig, J.-P., and Purkerson, S. 1991.
Localization and Partial Characterization of the Oligomeric Disulfide-
Linked Molecular Weight 95000 Protein (Triadin) Which Binds the

 

 

145

Ryanodine and Dihydropyridine Receptors in Skeletal Muscle Triadic
Vesicles. Biochemistry. 30: 7507-7513.

Catterall, W. A. 1988. Structure and Function of Voltage-Sensitive Ion
Channels. Science. 242: 50-61.

Catterall, W. A. 1991. Excitation-Contraction Coupling in Vertebrate
Skeletal Muscle: A Tale of Two Calcium Channels. Cell. 64: 871-874.

Chadwick, C.C., Inui, M. and Fleischer, S. 1988. Identification and
Purification of a Transverse Tubule Coupling Protein Which Binds to
the Ryanodine Receptor of Terminal Cistemae at the Triad Junction
in Skeletal Muscle. J. Biol. Chem. 263: 10872-10877.

Cheah, K.S., Cheah, A.M., Crosland, A.R., Casey, J.C., and Webb, AJ.
1984. Relationship between Ca Release, Sarcoplasmic Ca, Glycolysis
and Meat Quality in Halothane-sensitive and Halothane-insensitive
Pigs. Meat Sci. 10: 117-130.

Chen, S.R., Zhang, W.L., and MacLennan, DH. 1992. Characterization of
a Ca Binding and Regulatory Site in the Ca2+ release channel
(Ryanodine Receptor) of Rabbit Skeletal Muscle Sarcoplasmic
Reticulum. J. Biol. Chem. 267: 23318-23326.

Chrystal], BB. 1970. Macroscopic, Microscopic and Physiochemical Studies
of Influence of Heating on Muscle Tissues and Proteins. Ph.D.
Dissertation, Univ. of Missouri.

Chu, A., Diaz-Munoz, M., Hawkes, M.J., Brush, K., and Hamilton, S.L
1990. Ryanodine as A Probe for the Functional State of the Skeletal
Muscle Sarcoplasmic Reticulum Calcium Release Channel. Mol.
Phannacol. 37: 735-741.

Cooper, C.L., Vandaele, S., Barhanin, J., Fosset, M., Lazdunski, M., and
Hosey, MM. 1987. Purification and Characterization of the
Dihydropyridine-sensitive Voltage-denpendent Calcium Channel from
Cardiac Tissue. J. Biol. Chem. 262: 509-512.

Coronado, R., Morrissette, J., Sukhareva, N., and Vaughan, D. M. 1994.
Structure and Function of Ryanodine Receptors. Am. J. Physiol. 266

146
(Cell Physiol.) 35: C1485-C1504.

DeJongh, K.S., Merrick, D.K., and Catterall, W.A. 1989. Subunits of
Purified Calcium Channel: A 212-kDa Form of or, and Partial Amino
Acid Sequence of a Phosphorylation Site of an Independent B Subunit.
Proc. Nat]. Acad. Sci. USA 86: 8585-8589.

Endo, M. 1977. Calcium Release from the Sarcoplasmic Reticulum.
Physiol. Rev. 37: 71-108.

Endo, M., Yagi, S., Ishizuka, T., Horiuti, K., Koga, Y., and Amaha, K.
1983. Changes in the Ca2*-induced Ca2+ Release Mechanism in
Sarcoplasmic Reticulum fi'om A Patient with Malignant Hyperthermia.
Biomed. Res. 4: 83-92.

Fabiato, A. 1983. Calcium-induced Release of Calcium from the Cardiac
Sarcoplasmic Reticulum. Am. J. Physiol. 245: C1-C14.

Fernandez, J., DeMott, M., Atherton, D., and Mische, SM. 1992. Internal
Protein Sequence Analysis: Enzymatic Digestion for Less Than 10 pg
of Protein Bound to Polyvinylidene Difluoride or Nitrocellulose
Membranes. Anal. Biochem. 201: 255-264.

Fil], M., Coronado, R., Mickelson, J. R., Vilven, J., Ma, J ., Jacobson, B. A.,
and Louis, C. F. 1990. Abnonna] Ryanodine Receptor Channels in
Malignant Hyperthermia. Biophys. J. 50: 471-475.

Fleischer, S., and Inui, M. 1989. Biochemistry and Biophysics of
Excitation-Contraction Coupling. Annu. Rev. Biophys. Biophys Chem.
18: 333-364.

Fleischer, S., Ogunbunmi, E.M., Dixon, M.C., and Fleer, B.A.M. 1985.
Localization of Ca Release Channels with Ryanodine in Junctional
Terminal C istemae of Sarcoplasmic Reticulum of F ast Skeletal Muscle.
Proc. Nat]. Acad. Sci. USA. 82: 7256-7259.

Franzini-Arrnstrong, C. 1970. Studies of the Triad : 1. Structure of the
Junction in Frog Twitch Fibers. J. Cell. Biol. 47: 488-499.

Franzini-Armstrong, C. 1980. Structure of SarcOplasmic Reticulum.
Fed. Proc. 39: 2403-2409

147

Froning, G.W, Babjik, A.S., Mather, RB. 1978. The Effect of Preslaughter
Temperature, Stress, Struggle, and Anesthetization on Color and
Textural Characteristics of Turkey Muscle. Poultry Sci. 57: 630-633.

Fujii, J. , Ostu, K. , Zorzato, F., Leon, S. D., Khanna, V. K., Weiler, J. E.,
O'Brien, P. J ., and MacLennan, D. H. 1991. Identification of
Mutation in Porcine Ryanodine Receptor Associated with Malignant
Hyperthermia. Science. 253: 448-451.

Galione, A., White, A., Willmott, N., Turner, M. and Potter, B.V. 1993.
cGMP Mobilizes Intracellular Ca in Sea Urchin Egg by Stimulating
Cyclic ADP-ribose Synthesis. Nature. 365 : 456-459.

Grand, R.J.A., Shenolikar, S., and Cohen, P. 1981. The Amino Acid
Sequence of the 8 Subunit (Calmodulin) of Rabbit Skeletal Muscle
Phosphorylase Kinase. Eur. J. Biochem. 113: 359-367.

Hain, J., Nath. S., Mayrleitner, M., Fleischer, S., and Schindler, H. 1994.
Phosphorylation Modulates the Function of Calcium Release Channel
of Sarcoplasmic Reticulum from Skeletal Muscle. Biophys. J. 67:
1823-1833.

Hain, J ., Schindler, H., Nath, S., and Fleischer, S. 1993. Phosphorylation
of the Skeletal Muscle Calcium Release Channel Removes Block by
Magnesium Ions. Biophys. J. 64: A151.

Hakamata, Y., Nakai, J., Takeshima, H., and Imoto, K. 1992. Primary
Structure and Distribution of a Novel Ryanodine Receptor/Calcium
Release Channel from Rabbit Brain. FEBS. 312: 229-235.

Hall, G.M., Lucke, J.N., and Lister, D. 1980. Malignant Hyperthermia:
Pearls Out of Swine. Brit. J. Anaesth. 52: 165-171.

Harbitz, 1., Chowdhary, B., Thomas, P.D., Davies, W., Kaufinann, V., Kran,
S., Gustavsson, 1., Christensen, K., and Hauge, JG. 1990. Assignment
of the Porcine Calcium Release Channel Gene, A Candidate for the
Malignent Hypertherima Locus, to 6p11—)q21 Segment of
Chromosome 6. Genomics. 8: 243-248.

Harrison, G.G. 1979. Porcine Malignant Hyperthermia. Int]. Anaesth.
Clinics. 17:25-61.

148

Herrmann-Frank, A., and Varsanyi, M. 1993. Enhancement of Ca2+ Release
Channel Activity by Phosphorylation of the Skeletal Muscle Ryanodine
Receptor. FEBS Lett. 332: 237-242.

Hymel, L., Inui, M., Schindler, H., and Fleischer, S. 1988. Reconstitution
of Purified Cardiac Muscle Calcium Release Channel (Ryanodine
Receptor) in Planar Bilayers. Biochem. Biophys. Res. Commun.
152: 308-314.

Ikemoto, N. A., Antonin, B., and Meszaros, LG. 1985. Rapid Flow
Chemical Quench Studies of Calcium Release fi'om Isolated
Sacroplasmic Reticulum. J. Biol. Chem. 260: 14096-14100.

Imagawa, T., Smith, J .S., Coronado, R., and Campbell, KR 1987. Purified
Ryanodine Receptor from Skeletal Muscle Sarcoplasmic Reticulum Is
the Ca2+-permeable Pore of the Calcium Release Channel. J. Biol.
Chem. 262: 16636-16643.

Inui, M., and Fleischer, S. 1988. Purification of Ca2+ Release Channel
(Ryanodine Receptor) fi'om Heart and Skeletal Muscle Sacroplasmic
Reticulum. Meth. Enzymol. 1572490-505.

Inui, M., Saito, A., and Fleischer, S. 1987. Purification of the Ryanodine
Receptor and Identity with Feet Structure of Junctional Terminal
Citemae of Sarcoplasmic Reticulum from Fast Skeletal Muscle. J.
Biol. Chem. 262: 1740-1747.

Jenden, D.J., and Fairhurst, AS. 1969. The Pharmacology of Ryanodine.
Pharmacol. Rev. 21(1): 1-25.

Johnson, K. 1993. Malignant Hyperthermia Hots Up. Human Molecular
Genetics. 2: 849.

Judge, M.D., Aberle, E.D., Forrest, J.C., Hedrick, B.H., and Merkel, RA.
1989. Structure and Composition of Muscle and Associated Tissues.
Chapter 2. In "Principles of Meat Science." p.13 and 23. Kendall/Hunt
Publishing Company. Dubuque, IA.

Kim, K. C., Caswell, A. H., Talvenheimo, J. A., and Brandt, N. R. 1990.
Isolation of a Terminal Cistema Protein Which May Link the
Dihydropyridine Receptor to Junctional Foot Protein in Skeletal

149
Muscle. Biochemistry. 29: 9281-9289.

Kirino, Y., Osakabe, M., and Shimizu, H. 1983. Ca2*-induced Ca2+ Release
from Fragmented Sarcoplasmic Reticulum: Cay-dependent Passive
Ca2+ Efflux. J. Biochem. 94: 1111-1118.

Klee, CB, and Vanaman, TC. 1982. Calmodulin. Advances in Protein
Chemistry. 35: 213-321.

Knudsen, C. M., Stang, K.K., Jorgensen, A.O., and Campbell, K.P. 1993a.
Biochemical Characterization and Ultrastructnral Localization of Major
Junctional Sarcoplasmic Reticulum Glycoprotein (Triadin). J. Biol.
Chem. 268: 12637-12645.

Knudson, C. M., Stang, K.K., Moomaw, C.R., Slaughter, CA, and
Campbell, K.P. 1993b. Primary Structure and Topological Analysis
of a Skeletal Muscle-sepcific Junctional Sarcoplasmic Reticulum
Glycoprotein (Triadin). J. Biol. Chem. 268: 12646-12654.

Kuwajima, G., Futatsugi, A., Niinobe, M., Nakanishi, S., and Mikoshiba, K.
1992. Two Types of Ryanodine Receptors in Mouse Brain: Skeletal
Muscle Type Exclusively in Purkinje Cells and Cardiac Muscle Type
in Various Neurons. Neuron. 9: 1133-1142.

Laemmli, UK. 1970. Cleavage of the Structural Proteins During the
Assembly of the Head of Bacteriophage T4. Nature. 227: 680-685.

Lai, F. A., Erickson, H. P. , Rousseau, E. , Liu, O-Y., and Meissner, G.
1988. Purification and Reconstitution of the Calcium Release Channel
from Skeletal Muscle. Nature. 331: 315-319.

Lai, F.A., Lin. Q.-Y., Xu, L., El-Hashem, A., Kramarcy, N.R., Sealock, R.,
and Meissner, G. 1992. Amphibian Ryanodine Receptor Isoforms Are
Related to Those of Mammalian Skeletal or Cardiac Muscle. Am. J.
Physiol. 263 (Cell Physiol. 32): C365-C372.

Lai, F.A., Misra, M., Xu, L., Smith, HA, and Messner, G. 1989. The
Ryanodine Receptor-Ca2+ Release Channel Complex of Skeletal
Muscle Sarcoplasmic Reticulum. J. Biol. Chem. 264 : 16776-16785.

Lakowicz, J .R. 1983. Principles of Fluorescence Spectroscopy. Plenum

150
Press, NY.

Leung, A. T., Imagawa, T., Block, B., Franzini-Armstrong, C., and
Campbell, K. P. 1988. Biochemical and Ultrastructural
Characterization of the 1,4-Dihydropyridine Receptor fiom Rabbit
Skeletal Muscle. J. Biol. Chem. 263: 994-1001.

Lopez, J.R., Alamo, L.A., Jones, D.E., Papp, L., Allen, P.D., Gergely, J.,
and Sreter, FA. 1986. [Ca2+], in Muscles of Malignant Hyperthermia
Susceptible Pigs Determined in vivo with Ca2+ Selective
Microelectrodes. Muscle and Nerve. 9: 85-86.

Louis, CF. 1993. Porcine Stress Syndrome: Biochemical and Genetic Basis
of this Inherited Syndrome of Skeletal Muscle. Reciprocal Meat
Conference Proceedings. 46: 89-96.

Lowry, O.H., Rosenbrough, N.J., Farr, AL, and Randall, R]. 1951. Protein
Measurement with the F 01in Phenol Reagent. J. Biol. Chem. 193: 265-
275.

Lukas, T.J., Inverson, D.B., Schleicher, M., and Watterson, D.M. 1984.
Structural Characterization of a Higher Plant Calmodulin. Plant
Physiol. 75: 788-795.

Martonosi, A.N. 1984. Mechanisms of Ca2+ Release From Sacroplasmic
Reticulum of Skeletal Muscle. Physiol. Rev. 64: 1240-1319.

Mayrleitner, M., Chandler, R., Schindler, H., and Fleischer, S. 1995.
Phosphorylation with Protein Kinases Modulate Calcium Loading of
Terminal Cistemae of Sarcoplasmic Reticulum from Skeletal Muscle.
Cell Calcium. 18: 197-206.

McCartney, MG. 1964. A Randombred Control Population of Turkeys.
Poultry Sci. 43: 739-744.

McPherson, P. S. and Campbell, K. P. 1993. The Ryanodine Receptor/
Ca2+ release Channel. J. Biol. Chem. 268: 13765-13768.

Meissner, G. 1975. Isolation and Characterization of Two Types of
Sarcoplasmic Reticulum Vesicles. Biochim. Biophys. Acta. 389: 51-
68.

151

Meissner, G. 1986a. Evidence of a Role for Calmodulin in the Regulation
of Calcium Release from Skeletal Muscle Sarcoplasmic Reticulum.
Biochemistry. 25: 244-251.

Meissner, G. l986b. Ryanodine Activation and Inhibition of the Ca2+
Release Channel of Sarcoplasmic Reticulum. J. Biol. Chem. 261:
6300-6306.

Meissner, G. 1994. Ryanodine Receptor/ Ca2+ Release Channels and Their
Regulation by Endogenous Effectors. Ann. Rev. Physiol. 56: 485-
508.

Meissner, G., Darling, E., and Eveleth, J. 1986. Kinetics of Rapid Ca”,
Mg”, and Adenine Nucleotides. Biochemistry. 25: 236-244.

Meissner, G. and Henderson, J.S. 1987. Rapid Calcium Release from
Cardiac Sarcoplasmic Reticulum Vesicles is Dependent on Ca2+ and
is Modulated by Mg”, Adenine Nucleotide, and Calmodulin. J. Biol.
Chem. 262: 3065-3073.

Michalak, M., Dupraz., P., and Shoshan-Barmatz, V. 1988. Ryanodine
Binding to Sarcoplasmic Reticulum Membrane; Comparison between
Cardiac and Skeletal Muscle. Biochim. Biophys. Acta. 939:
587-594.

Mickelson, J .R., Ervasti, J .M., Litterer, L.A., Campbell, K.P., and Louis, CF.
1994. Skeletal Muscle Junctional Membrane Protein Content in Pigs
withDifferent Ryanodine Receptor Genotypes. Am. J. Physiol. 267:
C282-C292.

Mickelson, J .R., Gallant, E.M., Litterer, L.A., Johnson, K.M., Rempel, W.E.,
and Louis, CF. 1988. Abnormal Sarcoplasmic Reticulum Ryanodine
Receptor in Malignant Hyperthermia. J. Biol. Chem. 263: 9310-
9315.

Mickelson, J .R., Gallant, E.M., Rempel, W.E., Johnson, K.M., Litterer, L.A.,
Jacobson, B.A., and Louis, CF. 1989. Effects of the Halothane-
sensitivity Gene on Sarcoplasmic Reticulum Function. Am. J. Physio].
257: C787-C794.

Mickelson, J.R., Ross, J.A., Reed, B.K., and Louis, CF. 1986. Enhanced

152

Ca2+-induced Calcium Release by Isolated Sarcoplasmic Reticulum
Vesicles from Malignant Hyperthermia Susceptible Pig Muscle.
Biochim. Biophys. Acta. 862: 318-328.

Molla, A., Capony, JP, and Demaille, JG. 1985. Cardiac Sarcoplasmic-
reticulum Calmodulin-binding Proteins. Biochem. J. 226: 859-865.

Murayama, T. and Ogawa, Y. 1992. Purification and Characterization of
Two Ryanodine-Binding Proteon Isoforms fi'om Sarcoplasmic
Reticulum of Bullfrog Muscle. J. Biol. 112: 514-522.

Nagasaki, K., and Kasai, M. 1983. Fast Release of Calcium from
Sarcoplasmic Reticulum Vesicles Monitored by Chlortetracycline
Fluorescence. J. Biochem. 94: 1101-1109.

O'Brien, J., Meissner, G., and Block, BA. 1993. The fastest contracting
skeletal muscles of Non-mammalian Vertebrates Express Only One
Isoform of the Ryanodine Receptor. Biophys. J. 65: 2418-2427.

O'Brien, J., Valdivia, H. H., and Block, B. A. 1995. Physiological
Difference Between the or and B Ryanodine Receptors of Fish Skeletal
Muscle. Biophysical J. 68: 471- 482.

Ogawa, Y. 1994. Role of Ryanodine Receptor. Criti. Rev. Biochem.
Molec. Bio]. 29: 229—274.

Ogawa, Y., and Harafuji, H. 1990. Effect of Temperature on
[3H]Ryanodine Binding to Sarcoplasmic Reticulum from Bullfrog
Skeletal Muscle. J. Biochem. 107: 887-893.

Olivares, E. B., Tanksley, S. J., Airey, J. A., Beck, C. F., Onyang, Y.
Deerinck, T. J., Ellisman, M. H., and Sutko, J. L. 1991
Nonmammalian Vertebrate Skeletal Muscles Express Two Triad
Junctional Foot Protein Isoforms. Biophys. J. 59: 1153-1163.

O'Neil, K.T., and DeGrado, W.F. 1990. How Calmodulin Binds Its Targets:
Sequence Independent Recognition of Amphiphilic or-Helices. TIBS.
15: 59-64.

Otsn, K., Willard, H. F., Khanna, V. K., Zorzato, F., Green, N. M. and
MacLennan, D. H. 1990. Molecular Cloning of cDNA Encoding the

153

Ca2+ Release Channel (Ryanodine Receptor) of Rabbit Cardiac Muscle
Sarcoplasmic Reticulum. J. Biol. Chem. 265 : 13472-13483.

Onyang, Y., Deerinck, T.J., Walton, P.D., Airey, J.A., Sutko, J.L., and
Ellisman, M.H. 1993. Distribution of Ryanodine Receptors in the
Chicken Central Nervous System. Brain Res. 620: 269-280.

Perrin, DD, and Sayce, LG. 1967. Computer Calculation of Equilibrium
Concentartions in Mixtures of Metal Ions and Complexing Species.
Talanta. 14: 833-842.

Pessah, I.N., Francini, A.O., Scales, D.J., Waterhouse, AL, and Casida, J.E.
1986. Calcium-Ryanodine Receptor Complex: Solubilization and
Partial Characterization from Skeletal Muscle Junctional Sarcoplasmic
Reticulum Vesicles. J. Biol. Chem. 261: 8643-8648.

Pessah, I.N., Stambuk, R.A., and Casida, J.E. 1987. Ca2*-activated
Ryanodine Binding: Mechanisms of Sensitivity and Intensity
Modulation by Mg”, Caffeine, and Adenine Nucleotides. Mol.
Pharmacol. 31:232-238.

Pessah, I.N., Waterhouse, AL, and Casida, J.E. 1985. The Calcium-
Ryandone Receptor Complex of Skeletal and Cardiac Muscle.
Biochem. Biophys. Res. Commun. 1282449-456.

Pietrobon, D., Prod'hom, B., and Hess, P. 1988. Comformational Change
Associated with Ion Permeation in L-type Calcium Channels. Nature.
333: .373-376.

Plank, B., Wyskovsky, W., Hohenegger, M., Hellman, G., and Suko, J.
1988. Inhibition of Calcium Release fi'om Skeletal Muscle
Sarcoplasmic Reticulum by Calmodulin. Biochim. Biophys. Acta. 938:
79-88. _

Pommier, SA, and Houde, A. 1993. Effect of the Genotype for Malignant
Hyperthermia as Determined by a Restriction Endonuclease Assays on
Quality Characteristics of Commercial Pork Loins. J. Anim. Sci. 71:
420-425.

Powell, J .A. 1990. Muscular Dysgenesis: A Model System for Study in
Skeletal Muscle Development. FASEB. 4: 1798-2808.

154

Radermacher, M., Rao, V., Grassncci, R., Frank, J., Trimerman, A.P.,
Fleischer, S., and Wagenknecht, T. 1994. Cryo-Electron Microscopy
and Three-Dimensional Reconstruction of the Calcium Release
Channel/Ryanodine Receptor from Skeletal Muscle. J. Cell. Biol. 127:
411-423.

Rao, s.r., WU, s., Stayshnr, K.A., Ling, K.Y., Kung, c., and.
Sundaralingam, M. 1993. Structure of Paramecium tetraurelia
Calmodulin at 1.8 A Resolution. Protein Science. I: 436-447.

Reik, T.R., Rempel, W.E., McGrath, OJ, and Addis, RB. 1983. Further
Evidence on the Inheritance of Halothane Reaction in Pigs. J. Anim.
Sci. 57: 826-831.

Rios, E., Karhanek, M., Ma, J., and Gonzalez, A. 1993. An Allosteric
Model of the Molecular Interactions of Excitation-Contraction
Coupling in Skeletal Muscle. J. Gen. Physiol. 102: 449-481.

Rios, E., Pizarro, G., and Stefani, E. 1992. Charge Movement and the
Nature of Signal Transduction in Skeletal Muscle Excitation-Contration
Coupling. Annu. Rev. Physiol. 54: 109-133.

Rousseau, E., and Meissner, G. 1989. Single Cardiac Sarcoplasmic
Reticulum Ca-release Channel: Activation by Caffeine. Am. J.
Physiol. 256: H328-H333.

Sasagawa, T., Ericsson, L.H., Walsh, K.A., Schreiber, W.E., Fischer, B.H.,
and Titani, K. 1982. Complete Amino Acid Sequence of Human
Brain Calmodulin. Biochemistry. 21: 2565-2569.

Schneider, M.F., and Chandler, W.K. 1973. Voltage-dependent Charge
Movement in Skeletal Muscle: A Possible Step in Excitation-
Contraction Coupling. Nature. 242: 244-246.

Seifert, J.,and Casida, J .E. 1986. Ca2*-dependent Ryanodine Binding Site:
Soluble Preparation from Rabbit Cardiac Sacroplasmic Reticulum.
Biochem. Biophys. Acta. 861: 399-405.

Seiler. S., Wegener, A. D., Whang, D.D., Hathaway, DR, and Jones, LR.
1984. High Molecular Weight Protein in Cardiac and Skeletal Muscle
Junctional Sarcoplasmic Reticulum Vesicles Bind Calmodulin, Are

155

Phosphorylated, and Are Degraded by Ca2+-activated Protease. J. Biol.
Chem. 259: 8550-8557.

Serysheva, I. I., Orlova, B.V., Chin, W., Sherman, M.B., Hamilton, S.L., and
Heel, M.V. 1995. Electron Cryomicroscopy and Angular
Reconstitution Used to Visualize the Skeletal Muscle Calcium Release
Channel. Nature Structural Biology. 2: 18-24.

Simpson, SP, and Webb, A.J. 1989. Growth and Carcass Performance of
British Landrace Pigs Heterozygous at the Halothane Locus. Anim.
Prod. 49: 503-509.

Smith, J .S., Coronado, R. and Meissner, G. 1985. Sarcoplasmic Reticulum
Contains Adenine Nucleotide-activated Calcium Channel. Nature. 316:
446-449.

Smith, J.S., Coronado, R. and Meissner, G. 1986a. Single-channel Calcium
and Barium Currents of Large and Small Conductance from
Sarc0plasmic Reticulum. Biophys. J. 50: 921-928.

Smith, J.S., Coronado, R. and Meissner, G. 1986b. Single Channel
Measurements of the Calcium Release Channel from Skeletal Muscle
Sarcoplasmic Reticulum: Activation by Ca2+ and ATP and Modulation
by Mg”. J. Gen. Physiol. 88: 573-588.

Smith, J.S., Imagawa, T., Ma, J., Fill, M., Campbell, K.P. 1988. Purified
Ryanodine Receptor from Rabbit Skeletal Muscle Is the Calcium-
release Channel of Sacroplasmic Reticulum. J. Gen. Physiol. 92: 1-
26.

Smith, J.S., Rousseau, E., and Meissner, G. 1989. Calmodulin Modulation
of Single Sarcoplasmic Reticulum Ca2*-release Channel from Cardiac
and Skeletal Muscle. Circulation Research. 64: 352-359.

Sosnicki, AA. 1993. Focal Myonecrosis Effects in Turkey Muscle Tissue.
Reciprocal Meat Conference Proceedings. 46: 97-102.

Sosnicki, A.A., Cassens, R.G., McIntyre, D.R., Vimini, R.J., and Greaser,
ML. 1988. Characterization of Hypercontracted Fibers in Skeletal
Muscle of Domestic Turkey. Food Microstructure. 7: 147-152.

156

Sosnicki, A.A., Cassens, R.G., Vimini, R.J., and Greaser, M.L. 1991a.
Physiology and Reproduction: Distribution of Capillaries in Normal
and Ischemic Turkey Skeletal Muscle. Poultry Science. 70: 343-348.

Sosnicki, A.A., Cassens, R.G., Vimini, R.J., and Greaser, M.L. 1991b.
Histopathological and Ultrastructural Alterations of Turkey Skeletal
Muscle. Poultry Science. 70: 349-357.

Sosnicki, AA. and Wilson, B.W. 1991. Pathology of Turkey Skeletal
Muscle: Implications for the Poultry Industry. Food Structure. 10:
317-326.

Sosnicki, AA. and Wilson, B.W. 1992. Relationship of Focal Myopathy of
Turkey Skeletal Muscle to Meat Quality. World's Poultry Congress:
Proceeding 19th World's Poultry Congress: Amsterdam. 3: 43-47.

Strand, M. A., Louis, C. F., and Mickelson, J. R. 1993. Phosphorylation of
the Porcine Skeletal and Cardiac Muscle Sarc0plasmic Reticulum
Ryanodine Receptor. Biochim. Biophys. Acta. 1175: 319-326.

Strasburg, G.M., Hogan, M., Birrnachu, W., Thomas, DD, and Louis, CF.
1988. Site-specific Derivatives of Wheat Germ Calmodulin. J. Biol.
Chem. 263: 542-548.

Strasburg, G.M., Reedy, M.M., Wang, LI, and Jones, LR. 1993.
Calmodulin-dependent Phosphorylation of the Cardiac SR Calcium
Channel Inhibits Calmodulin Binding. Biophys. J. 64: A10.

Suko, J., Maurer-Fogy, 1., Plank, B., Bertel, O., Wyskovsky, W.,
Hohenegger, M., and Hellman, G. 1993. Phosphorylation of Serine
2843 in Ryanodine Receptor-calcium Release Channel of Skeletal
Muscle by cAMP-, cGMP-, and CaM-dependent Protein Kinase.
Biochim. Biophys. Acta. 1175: 193-206.

Takagi, T., Nemoto, T., Konishi, K., Yazawa, M., and Yagi, K. 1980. The
Amino Acid Sequence of the Calmodulin Obtained from Sea Anemone

(Metridium senile) Muscle. Biochem. Biophys. Res. Comm. 96:
377-38].

Takasago, T., Imagawa, T., Furukawa, K., Ogurusu, T., and Shigekawa, M.
1991. Regulation of Cardiac Ryanodine Receptor by Protein Kinase-

157
dependent Phosphorylation. J. Biochem. 109: 163-170.

Takeshima, H., Nishimura, S., Matsumoto, T., Ishida, H., Kangawa, K.,
Minamino, N., Matsuo, H., Ueda, M., Hanaoka, M., Hirose, T., and
Numa, S. 1989. Primary Structure and Expression of Complementary
DNA of Skeletal Muscle Ryanodine Receptor. Nature. 339: 439-445.

Takeshima, H, Nishimura, S., Nishi, M., Ikeda, M., and Sugimoto, T. 1993.
A Brain-specific Transcript fiom the 3'-terminal Region of the Skeletal
Muscle Ryanodine Receptor Gene. FEBS Lett. 322: 105-110.

Tanabe, T., Beam, K.G., Adams, B. A., Niidome, T., and Numa, S. 1990.
Regions of the Skeletal Muscle Dihydropyridine Receptor Critical for
Excitation-Contraction Coupling. Nature. 346: 567 -569.

Taylor, D.A., Sack, J.S., Maune, J.F., Beckingham, K., and Quiochot, FA.
1991. Structure of A Recombinant Calmodulin from Drgsophila
melanogaster Refined at 2.2 A Resolution. J. Biol. Chem. 266: 21375-
21380.

Toda, H., Yazawa, M., Kondo, K., Honma, T., Narita, K., and Yagi., K.
1981. Amino Acid Sequence of Calmodulin from Scallop
(Parinopecren) Adductor Muscle. J. Biochem. 90: 1493-1505.

Toda, H., Yazawa, M., Sakiyama, F., and Yagi, K. 1985. Amino Acid
Sequence of Calmodulin from Wheat Germ. J. Biochem. 98: 833-842.

Tripathy, A., Xu, L., Mann, G., and Meissner, G. 1995. Calmodulin
Activation and Inhibition of Skeletal Muscle Ca2+ Release Channel
(Ryanodine Receptor). Biophys. J. 69: 106-119.

Vansickle, J. 1989. PSE Problems in US. Pork. National Hog Farmer.
October, 15.

Vergara, J., Tsien, R. Y., and Delay, M. 1985. Inositol 1,4,5-trisphophate:
A Possible Chemical Link in Excitation-Contraction Coupling in
Muscle. Proc. Nat]. Acad. Sci. USA. 82: 6352-6356.

Wang, J., and Best, PM. 1992. Inactivation of the Sarcoplasmic Reticulum
Calcium Channel by Protein Kinase. Nature. 359: 739-741.