.
é . i: ..,
.33.: (ST
1:43.:

5 . 41,.

s F. V
. 5...? . .. 3.
. .35" ._Wd.uuur4
, 2.. ......,
3mm... .3 . . ‘
433...»...9
..
.. .2 .
J J}. .7.) a
"N ha .

. 51
a; 3.; 1i...

xhuww
. a v A .
3.333.335.3333?
. y n “hi: K
2. 31...:
. .
t

v’.

3

ﬁrm
1.. .31 t . 2.4....
L. I

E ‘.
{hi

i

.
3 ‘
5‘ .a/
«.65.;

‘2'

._.,
_.n

.
,J
. 3.5“ u. 15...;-
v‘.1u ‘ . 1'4
bunt.

h t:<; :. <.
1;... . 2.: y

35,.“
V 3»
5:»?!

x 3. .

\9 z: 1.?
3791.}

 

 

 

 

 

 

 

 

 

.7...
, ..9
3.3:;

:7

wk IA . 3 . ,3

33 3333333 33 w . 3333

.r.‘

 

 

 

LIBRARY
Michigan State
University

 

 

 

This is to certify that the
thesis entitled
Sarcoplasmic Reticulum Ca2+-channel Protein
Function and Regulation Differences in Random-

Bred and Commercial Turkey Populations

presented by

Haiyan Zhang

has been accepted towards fulﬁllment
of the requirements for

Master degree in Food Science

 

faZzMw

Major professor/

Date 11/28/2000

 

0-7639 MS U is an Afﬁrmative Action/Equal Opportunity Institution

 

 

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE ‘ DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

11m chIRCJDateOuepGS—p.“

SARCOPLASMIC RETICULUM Cay-CHANNEL PROTEIN FUNCTION AND
REGULATION DIFFERENCES IN RANDOM-BRED AND COMMERCIAL
TURKEY POPULATIONS
By

Haiyan Zhang

A THESIS

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

MASTER OF SCIENCE
Department of Food Science and Human Nutrition

East Lansing, Michigan
2000

ABSTRACT

SARCOPLASMIC RETICULUM Cay-CHANNEL PROTEIN FUNCTION
AND REGULATION DIFFERENCES IN RANDOME-BRED AND
COMIVIERCIAL TURKEY POPULATIONS

BY

Haiyan Zhang

Pale, soﬁ and exudative (PSE) meat is a major meat quality problem in the turkey
processing industry. Its causes are likely rooted in both genetics and management. The
purpose of this study is to test the hypothesis that a defect in one or both sarcoplasmic
reticulum Ca2+ channel isoforms and/or altered Ca2+ channel regulation in a commercial
turkey subpopulation contributes to the incidence of PSE meat. [3H]Ryanodine binding
assays were optimized for turkey skeletal muscle, and were used to compare two groups
of commercial turkeys with random-bred control turkeys. Group I commercial turkeys
(n=17) exhibited a higher (p < 0.05) affinity for ryanodine and lower (p < 0.05) Ca2+
channel protein content compared to that from random-bred turkeys (n =8) (Kd=8.4 vs
16.0 nM, Bmax=5.9 vs 12.5 pmol/mg), suggesting altered ﬁanction of Ca2+ channel in this
commercial group. Group 11 commercial turkeys exhibited similar ryanodine binding
constants to that from random-bred and signiﬁcantly different from that of group I. These
results suggest that there is one commercial subpopulation that possesses similar Ca2+
channel ﬁinction to random-bred control turkeys. [3H]PN200-110 binding assays yielded
a coupling ratio of one dihydropyridine receptor molecule per ryanodine receptor. There
was no signiﬁcant diﬁ‘erence in coupling ratio between commercial and random-bred

turkeys.

ACKNOWLEDGEMENTS

I would like to give my special thanks to my major academic advisor, Dr. Gale M.
Strasburg, for his knowledge, support and understanding during my study. He is an

excellent teacher, good friend.

I am grateful to my committee members: Dr. Alden M. Booren, Matthew E
Doumit, for their assistance and guidance. Thanks are also due to Dr. Peter Saama, for all
the statistical consultant work. My appreciation extended to the friends and graduate
students that I have been working with, for their help and encouragement.

I am extremely grateﬁil for the love and support from my dear parents, Chunxu

Zhang, Shuxia Zhao, and my sister, Hongyan Zhang.

iii

TABLE OF CONTENTS

List of Tables ...................................................................................... vi
List of Figures .................................................................................... vii
Introduction ........................................................................................... 1
Chapter 1. Literature Review ....................................................................... 3
I. Skeletal Muscle Structure ............................................................... 3
II. Excitation-Contraction Coupling ...................................................... 5
III. Cay-channel Protein and Isoforms .................................................. 8
A. SR Ca2+ channel or Ryanodine receptor ...................................... 8
B. Modulator Regulation of Cay-release Channel Protein (RyR). . . . .. 13
1. Calcium (Ca2+) ........................................................ 13
2. Magnesium (Mg2+) .................................................... 14
3. Adenine Nucleotides .................................................. 15
4. Ryanodine .............................................................. 16
5. Caffeine ................................................................ 19

C. Dihydropyridine receptor (DI-IPR) or L-type Ca2+ channel. . . . . . 19
IV. Protein-protein Interaction Between Ryanodine Receptor (RyR) and

Dihydropyridine Receptor (DI-IPR) in Skeletal Muscle ....................................... 23
V. Cay-channel related meat quality problem: Malignant Hyperthermia (MH),
Porcine Stress Syndrome (PSS), Central Core Disease (CCD) ............................... 27
A. Malignant Hyperthermia, Porcine Stress Syndrome (PSS)...............27
B. Central Core Disease (CCD) ................................................. 31
Chapter 2. Modiﬁcation of experimental condition for ryanodine binding assay of turkey
skeletal muscle ....................................................................................... 34
I. Introduction .............................................................................. 34
11. Materials and Methods ................................................................. 36
1. Materials ........................................................................ 36
2. Methods ......................................................................... 36
III. Results and Discussions ............................................................... 39
IV. Conclusions ............................................................................. 47
Chapter 3. Ca2+-channel protein activity and regulation associated with turkey PSE meat
quality problems ..................................................................................... 48
I. Introduction .............................................................................. 48
11. Materials and Methods .................................................................. 51
1. Materials ........................................................................ 51
2. Methods .......................................................................... 51
III. Results and Discussions ............................................................... 55
IV. Conclusions .............................................................................. 68

iv

Conclusions ........................................................................................... 70
Future Research ..................................................................................... 72

List of References ................................................................................... 74

LIST OF TABLES

Table 1.1 Mutations in Skeletal Muscle Cay-release Channel Protein (Rle) in Human
MH/CCD .................................................................................. 33

Table 2.1 Equilibrium Constants of Ryanodine Binding of Heavy Sarcoplasmic
Reticulum Fractions from Two Turkey Breast Muscle Samples ................... 40

Table 3.1 Equilibrium Constants of Individual [3H]Ryanodine Binding of Heavy SR from
Random-bred and Commercial Turkeys (group I and II) ........................... 58

Table 3.2 Equilibrium Constants of Individual [3H]Ryanodine Binding of Crude SR from
Random-bred and Commercial Turkeys (group II) ................................. 59

Table 3.3 Equilibrium Constant Means of [3H]Ryanodine Binding of Crude and Heavy
SR from Random-bred and Commercial Turkeys (group I, II) .................... 60

Table 3.4 Ratio of Dihydropyridine and Ryanodine Receptors between Random-bred and
Commercial Turkey Skeletal Muscle Crude SR Vesicles .......................... 65

vi

LIST OF FIGURES

Figure 1.1 Diagrammatic representation of mammalian myoﬁbers freed of their
endomysium ............................................................................ 4

Figure 1.2 A two component model for the calcium release process in non-mammalian

vertebrate skeletal muscle ............................................................. 12
Figure 1.3 Ryanodine structure showing positions of tritium labels (9,21). .. 17
Figure 1.4 Model of the subunit structure of the dihydropyridine receptor ................. 21
Figure 1.5 The structure of four homologous repeats of the DI-IPR 0L1 subunit... ... ......22

Figure 1.6 Diagram of the spatial relationships between the junctional complex
components ............................................................................. 25

Figure 3.1 Ryanodine dependence of ryanodine binding to random-bred and commercial
representative turkey skeletal muscle heavy sarcoplasmic reticulum vesicles

(HSR) at 10 IIM Ca2+ .................................................................. 56

vii

INTRODUCTION

The modern turkey industry has grown rapidly during the last 30 years to meet
consumers’ demand for lean and inexpensive turkey meat products. To meet this demand,
breeders have intensively selected for heavy muscling and rapid growth of turkeys. Over
the past three decades, the market age of turkeys has shortened from 26—28 weeks to 14-
16 weeks, while the market weight has increased from about 20 lb to about 35 lb.
However, the improvement in size and grth efﬁciency has come with a cost: increased
incidence of meat quality defects including pale, soft, exudative (PSE) meat. This meat
has soft texture, lighter color, decreased water holding capacity and poor cooking yield.
Stresses like heat or cold, transportation, crowding, etc, could increase the incidence of
the PSE in turkey (Froning et al., 1978).

The PSE problem in turkey seems to be associated with a rapid onset of rigor
mortis that is similar to the development of the PSE condition that occurs in pig muscle
aﬁer death (Greaser, 1986; Sosnicki, 1995). Pigs with the inherited disorder called
Porcine Stress Syndrome (PSS) are more likely than non-stress-susceptible pigs to yield
PSE pork. Porcine Stress Syndrome (PSS) is associated with the intensive selection for
leanness and rapid growth. This syndrome causes substantial economic losses to farmers,
owing to death of animals from stresses of transportation, heat, crowding, etc, before
reaching market (Louis, 1993). After slaughter, PSS-susceptible pigs exhibit increased
glycolytic rates during the conversion of muscle to meat, leading to higher carcass
temperatures in combination with acidic pH that favors protein denaturation. This results
in decreased protein solubility and poor water holding capacity. Fujii et al., (1991)

reported that a single point mutation in the porcine gene for the skeletal muscle

sarcoplasmic reticulum Ca2+ channel was correlated with PSS in ﬁve major breeds of
lean, heavily muscled swine. This Cay-release channel protein regulates Ca2+ release
from sarcoplasmic reticulum, thus triggering muscle contraction and glycolysis. The
mutation in PSS-susceptible pigs results in higher channel protein activity, which is
responsible for the accelerated glycolysis and heat production.

Based on the numerous similarities between development of PSE pork and PSE
turkey, we hypothesize that there is a Cay-channel defect in function and/or regulation
existing in a subpopulation of commercial turkeys, which is responsible for the PSE meat
quality problem. Preliminary studies by Wang et al. (1999) suggested that there could be
genetic differences in ryanodine receptor between commercial turkeys and a genetically
unimproved, random-bred control population. However, their assay conditions, which
were adapted from procedures developed for porcine muscle, yielded signiﬁcance only
when comparing a large number of animals from the two turkey lines. Variation of assay
results for individual birds made it impossible to use this assay for identiﬁcation of
differences in channel protein activity among individual birds. Therefore, the ﬁrst aim of
this study was to optimize ryanodine binding procedures for turkey skeletal muscle SR in
order to do individual comparisons. The second aim was to use this assay to determine
whether there exists a subpopulation of commercial turkeys with altered channel protein
activity which could predispose these turkeys to the production of PSE meat. Finally,
analysis of the coupling ratio of the voltage sensor (dihydropyridine receptor protein) to
Cay-channel was conducted to determine whether difference in coupling ratio could

account for the increased incidence of PSE meat in commercial turkeys.

Chapter 1
LITERATURE REVIEW

1. Skeletal Muscle Structure

Skeletal muscle is made up of a group of muscle ﬁbers surrounded and supported
by the connective tissue proteins, collagen and elastin. Skeletal muscle ﬁbers vary from 1
to 40 mm in length and from 10 to 100 um in diameter (Leeson and Leeson, 1970). The
muscle ﬁber or cell is the fundamental unit of muscle and in addition to the typical
cellular organelles - nulei, mitochondria, etc. - a substantial volume of the cell consists of
specialized contractile organelles called myoﬁbrils. The membrane surrounding the
myoﬁber is called the sarcolemma (Figure 1.1). The sarcolemma penetrates the myoﬁber,
forming a network of transverse tubules (T-tubules). Together, the sarcolemma and the T-
tubules play a key role in muscle contraction by transmitting the motor nerve impulse as
a membrane depolarization from the neuromuscular junction along the length of the ﬁber.
The T-tubules transmit the depolarization signal into the interior of the ﬁber to the
sarcoplasmic reticulum (SR).

The SR is a specialized form of endoplasmic reticulum which consists of a series
of enclosed membrane vesicles lying adjacent to both the T-tubules and the myoﬁbrils.
The SR serves as a reservoir of Ca2+, which is released into the sarcoplasm when a
muscle contraction stimulus is received. When the stimulus stops, Ca2+ is pumped back
into SR, and the muscle relaxes.

SR can be separated into two fractions, based on differences in density, using

sucrose gradient centriﬁigation. Heavy SR (HSR) is enriched in junctional membrane

   
  
 
 
   
  

  

Capillary Nerve Branch

Nucleus

Mitochondrion

ll

1

it‘ll

it‘l' 11' ”'
f Ail \1

Transverse
tubule

Myofilaments .ﬂ \

c: '-. " . . ‘
\ h" Terminal Sarcoplasmic
cistemae reticulum

Myotibril Triad Sarcolemma

Figure 1.1 Diagrammatic representation of mammalian myoﬁbers freed of
their endomysium, demonstrating both A) the arrangement of their blood
and nervous supply and B) the arrangement of the specialized membranous
systems in relation to the protein scaffold (adapted from Strasburg and
Byrem, 2000).

vesicles (including the Ca2+-channel protein and calsequestrin), triads, terminal cisternae,
etc. The light SR fraction (LSR) is mainly longitudinal tubule membrane vesicles
enriched in the Ca2+ pump protein (Meissner, 1975; Franzini-Armstrong, 1980).

II. Excitation-Contraction Coupling

When an electrochemical signal from the motor nerve stimulates the plasma
membrane of the skeletal muscle, the voltage sensor of the T-tubule membrane is
activated and the signal is transmitted to the SR. Ca2+ is released from the SR to the
sarcoplasm, causing the sarcoplasmic Ca2+ concentration to rise from less than 10'7 M at
the rest to more than 10" M. Some of this Ca2+ binds to troponin C (TnC), causing a
conformational change that alters the position of the troponin-tropomyosin complex in
the thin ﬁlament, permitting actin and myosin to interact, and thus, leading to muscle
contraction (lot and Potter, 1987). The sarcoplasmic Ca2+ concentration is lowered by
action of the CaATPase pump (Martonosi 1984). The process whereby depolarization of
the sarcolemma is translated into muscle contraction is called Excitation-Contraction
coupling (E-C coupling) (Franzini-Armstrong and Protasi, 1997).

There are three distinct E-C Coupling mechanisms found in muscle: 1) Ca2+-
induced Ca2+ release, i.e., the inﬂux of Ca2+ through channels in the T-tubular membrane
induces the release of Ca2+ from the SR. 2) Second messenger mechanism. The inositol
1,4,5-trisphosphate (lnsP3) released from the T-tubular membrane acts as a second
messenger to induce the release of Ca2+ from the SR. 3) Mechanical coupling induced
Ca2+ release. The dihydropyridine receptor (DI-IPR), which serves as the voltage sensor
on the T-tubule membrane, physically transmits the signal to the Cay-release channel or

ryanodine receptor (RyR) in the SR through protein-protein interaction.

The various muscle types exhibit different mechanisms of E-C coupling. It is
generally accepted that the mechanism of cardiac E-C coupling is Calcium-Induced
Calcium Release (CICR). The ﬁrst step in the ﬂow of information following electrical
excitation in heart muscle is Ca2+ inﬂux through voltage-dependent Ca2+ channels in the
T-tubule. Ryanodine receptor (RyR) Ca2+ channels are positioned in junctional SR
elements within short distances (~20 nm) of voltage-dependent Ca2+ channels (DPIPR) in
the T-tubules of ventricular heart cells. Ryanodine receptors (RyRs) are activated by local
Ca2+ which enters the cell through voltage-dependent Ca2+ channels (“local control”)
(Cannell et al., 1995; Cheng et al., 1996; Lopez-Lopez et al., 1995). Local Ca2+ in the
subsarcolemmal junctional space is likely to reach relatively high concentrations (> 10
uM), corresponding to levels required for signiﬁcant RyR channel activation. Ca2+
released through these activated RyR channels acts as an ampliﬁer for timber SR Ca2+
release. The SR ultimately contributes 90% of the Ca2+ needed for contraction of heart
muscle (Jaggar et al., 2000). Thus, local Ca2+ control of Ca2+ release can explain the
graded increases in Ca2+ release through RyR channels in response to Ca2+ inﬂux in
cardiac muscle (Cannell et al., 1995; Lopez-Lopez et al., 1995; Soeller and Cannell,
1997)

The mechanism of EC coupling in skeletal muscle, however, was believed to
differ ﬁ'om cardiac muscle because most DI-IPRs are non-functional as Ca2+ channels in
skeletal muscle (Schwartz et al., 1985). Vergara et al. (1985) and Volpe et al. (1985)
observed an activation of force after the application of InsP3 to skinned skeletal muscle
ﬁbers. Both groups regarded this substance as a second messenger in the coupling

process. They suggested that the action potential initiates InsP3’s release from the

sarc0plasmie face of the sarcolemma. InsP3 would diffuse across the gap and bind to a
receptor at the SR causing the release of Ca2+ from the terminal cistemae. However, a
skeletal muscle InsP3 receptor has not yet been identiﬁed unambiguously (Melzer et al.,
1995). Also the Ca2+ release initiated by Inng is hormonally activated while Ca2+ release
from skeletal muscle is activated by depolarization (Caswell and Brandt, 1989).

Since the inﬂux of Ca2+ and the release of InsP3 apparently play no direct role in
the E-C coupling process in skeletal muscle, the third possible mechanism, i.e., the
voltage-dependent Ca2+ release, becomes the center of interest. This model involves
protein-protein interactions between the Ca2+ release channel of the skeletal muscle SR or
ryanodine receptor (Rle) and the DHPR which leads to Ca2+ release from the SR
(Meissner and Lu, 1995).

DHPR-mediated opening of Ca2+ release channels and Ca2+-induced Ca2+ release
channel opening have both been proposed to result in elementary Cay-release events
(sparks) that originate at the frog skeletal muscle triad and lead to ﬁrrther Ca2+ release
(Klein et al., 1996). Tetracaine, which inhibits Ca2+-induced Ca2+ release, eliminated all
elementary Ca2+ release events, suggesting that Cay-induced Ca2+ release, and not
depolarization, is the mechanism for elementary Ca2+ release events. The
phenylalkylamine D600, a well-known inhibitor of the membrane voltage sensor,
suppressed elementary Ca2+ release events from SR and the peak of depolarization-
induced Ca2+ release (Shirokova and Rios, 1997). The restoration of elementary Ca2+
release events and the peak of release by high voltage suggest that, in skeletal muscle,
depolarization-induced Ca2+ release is the trigger for Ca2+-induced Ca2+ release (Leong

and MacLennan, 1998). Recent studies of rat muscle ﬁbers suggest that localized Ca2+

release does not exist in mammalian muscle, so that elementary events of Cami-induced
Ca2+ release cannot be a trigger for E-C coupling in mammalian muscle (Shirokova and
Rios, 1998).

Upon repolarization, the DHPR undergoes a conformational change which is
believed to be transferred to RyR] to terminate Ca2+ release (Suda 1995). Rle has also
been shown to enhance DHPR channel activity during E-C coupling (Nakai et al, 1996;
Yamazawa et al, 1996) through a process called retrograde signaling. This leads further
support to the mechanical coupling model of skeletal muscle E-C coupling.

III. Ca2+-Channel Protein and Isoforms
A. SR Ca2+ Channel or Ryanodine Receptor

The SR Cay-channel protein is an oligomeric protein complex located in the SR
junction with the T-tubule, and constitutes about 2-3% of the total SR protein. This
channel protein is also called the ryanodine receptor (RyR), because it binds the neutral
plant alkaloid ryanodine with high affinity. The RyR complex includes four identical
monomers with a calculated molecular mass of 565 kDa (Takeshima, et al. 1989). In
addition, each RyR tetramer binds four molecules of calmodulin, four molecules of FK-
506 binding protein and perhaps others. There are three isoforms of RyR. Rle is mainly
found in skeletal muscle, RyR2 is present in cardiac muscle, and RyR], RyR2 and a third
isoforrn, RyR3, are found in brain as well as other non-muscle tissue. The three isoforms
of RyR are remarkably similar in primary structure, consisting of ~ 5000 amino acids
with a membrane—spanning hydrophobic domain at the carboxyl-terminal end and a large

hydrophilic domain at the amino-terminal end (Takeshima, 1993).

Electron microscopic images of puriﬁed RyRs show a quatrefoil structure of 22-
28 nm x 22-28 nm x 8-12 nm as is true of the junctional foot proteins seen in
micrographs of muscle triads (Franzini-Annstrong and Jorgensen, 1994; Franzini-
Armstrong and Protasi, 1997; Wagenknecht and Radermacher, 1997; Jeyakumar et al.,
1998; Saito et al., 1988; Orlova et al., 1996). In the Rle structure, the hydrophobic
domains of the four subunits comprise the baseplate which spans the SR membranes and
forms the channel (Lai, et al. 1988, Wagenknecht, et al. 1989). The hydrophilic domains
form the cytoplasmic foot structure which bridges the gap between T-tubule and the
terminal cistema of the SR membrane. Each of the four subunits apparently contributes
equally to the formation of the foot and the channel region, and the complete tetramer is
necessary for ryanodine binding and channel activity (Lai et al., 1988). There are no
striking differences in general appearance among image reconstructions of the RyR
isoforms (Ogawa et al., 1999).

The protein-staining pattern of mammalian skeletal muscle SR on SDS-PAGE
shows a single band of RyR in contrast to closely spaced RyR double bands in gels of SR
from ﬁ'og skeletal muscle and chicken (Olivares et al., 1991; Murayama and Ogawa,
1992). Now it is generally accepted that there is only one ryanodine receptor isoform
(Rle) expressed in mammalian skeletal muscle, whereas there are two isoforms (or- and
B-RyR) in most non-mammalian skeletal muscles, e.g. those of ﬁsh, chicken, bullfrog,
and turkey. Airey et al. (1990) ﬁrst identiﬁed the existence of two isoforms (or and [3) in
chicken pectoral muscles. In the chicken breast muscle cells, equal amounts of both
isoforms are found in microsomes, but the a—isofonn is more abundant in microsomes

from chicken thigh muscle. Therefore, the two isoforms do not necessarily exist in equal

amounts (Ogawa, 1994). Lizard and snake skeletal muscles show a single band of or-
RyR, while turtle, crocodile, ﬁsh, and avian show or- and B-RyRs (O’Brien et al., 1993).
Certain specialized muscles such as extraocular and swimbladder muscles from ﬁshes,
which contract very rapidly, show only a-RyR while their body skeletal muscles have
both or- and B-RyR (Ogawa et al., 1999).

Initial studies on comparisons of or and B isoforms of non-mammalian skeletal
muscle with the RyR isoforms of mammals suggested that the or isoform was most
homologous to RyR] and that the B-RyR might correspond to RyR2 (Lai et al., 1992;
Bull and Marengo, 1993). Airey et al. (1993a) identiﬁed a third distinct isoform in avian
cardiac muscle, and cross reactivity was observed on Western blot analysis with anti-B-
RyR antibody of microsomes from bullfrog cardiac muscle. Sequences of the cloned
cDNAs showed that or- and B-RyR of frog skeletal muscle are most homologous to
mammalian RyR] and RyR3, respectively (Oyamada et al., 1994).

Despite the structural similarity and amino acid homology, the or and B isoforms
exhibit signiﬁcant ﬁJnctional differences. Both [3H]ryanodine binding and single channel
conductance studies indicate that the or isoform has a biphasic response to Ca2+ (O’Brien
et al., 1995). The channel is activated at micromolar Ca2+, with peak activation in the 1-
10 M range, and is subsequently inhibited by millimolar Ca2+. This is strikingly similar
to Rle from mammalian skeletal muscle. In addition to these physiological similarities,
the or isoform of ﬁsh and other vertebrates is the speciﬁc target of Imperatoxin A, a
scorpion toxin that selectively activates [3H]ryanodine binding and channel activity of the
mammalian skeletal muscle RyR isoform (Valdivia et al., 1994). O’Brien et al. (1995)

also indicated the similarities between the ﬁsh skeletal muscle [3 isoform and the cardiac

10

muscle isoform from both mammals and ﬁsh. They are all relatively insensitive to
inactivation by millimolar Cay when studied either by [3H]ryanodine binding (Pessah et
al., 1985; Michalak et al., 1988; Chu et al., 1993) or single-channel recording (Rousseau
et al., 1986; Chu et al., 1993). Furthermore, both the B isoform and the mammalian
cardiac RyR are insensitive to Imperatoxin A (Valdivia et al., 1994).

There appears to be a physiological distinction in the role of or and B isoforms in
muscle. A lethal mutation, crooked neck dwarf mutant, exists in chicks in which skeletal
muscle cells lack a-RyR, whereas the B isoform is expressed normally. There is no
response to electrical stimulation of the muscle, nor is Cay-induced Ca2+ release
observed, but Ca2+ is released with the application of caffeine (Airey et al., 1993b, c;
Ivanenko et al., 1995). Likewise in the Rle-knockout dyspedic mice, myotubes do not
respond to electrical stimulation (Takeshima et al., 1994). These studies suggest that or-
RyR is critically involved in the E-C coupling of the skeletal muscle, as is the case of
Rle in mammalian skeletal muscle (Ogawa et al., 1999).

O’Brien et al. (1995) proposed a two component model for the calcium release
process in non-mammalian vertebrate skeletal muscle. In this model, voltage-sensitive
channels are gated by voltage change across the T-tubular membrane, presumably via the
DHPR, whereas the other SR Ca2+ release channels are presumed to operate by Ca“-
induced Ca2+ release. Activation of one channel by voltage would result in the release of
Ca2+ and thereby activate a neighboring “slave” channel (Figure 1.2). The or RyR isoform
is likely to be in RyR isoform directly coupled to DHPR, and the B isoform is a logical

candidate to be the calcium-coupled RyR isoform because it has sequence and

11

Two-component model of calcium release

T-tubule

 

 

 

o
...........
' o

 

 

 

B RYR

2
Ca'

 

SR Lumen

 

 

 

L

Figure 1.2 A two component model for the calcium release process in non-
mammalian vertebrate skeletal muscle (adapted from O’Brien et al., 1995)

12

functional similarity to the cardiac and nonmuscle RyR, which operate by Cay-induced
Ca2+ release.

The cytosolic domain of Rle contains binding sites for various activating
ligands including calcium (uM), ATP, calmodulin (which binds both in the absence and
presence of calcium), caffeine and ryanodine (nM), and inactivating ligands such as
calcium (> 100 uM) and magnesium in mM concentrations (Meissner, 1994, Coronado et
al. 1994). Ryanodine modulates Rle in a biphasic manner; nM concentrations can
activate while mM concentrations can inactivate the channel. RyRs in the open state can
bind [3H]ryanodine speciﬁcally and with high affinity (Bull et al., 1989; Lai et al., 1989;
Chu et al., 1990).

B. Modulator Regulation of the Cay-release Channel Protein (RyR)
1. Calcium (Ca2+)

Physiologically, one of the most important substances that modulates the gating of
the RyR is Ca2+ (Ebashi, 1991; Fleischer and Inui, 1989; Meissner, 1994; Coronado et al.,
1994). Ca2+ has a two-fold action on the RyR; it can activate the receptor at
concentrations lower than 0.1 mM and inhibits the receptor at concentrations above 0.1
mM. In the absence of other regulatory ligands like Mg2+ and ATP, a bell-shaped Ca2+
activation curve of Ca2+ ef’ﬂux ﬁom heavy SR vesicles is observed, with Ca2+ efﬂux
being maximal in the micromolar Ca2+ concentration range (Fill et al., 1990; Kirino et al.,
1983; Meissner et al., 1986a; Moutin and Dupont, 1988; Nagasaki and Kasai, 1983).
Such a curve suggests that the Cay-release channel possesses high-afﬁnity activating and
low-aﬁ'rnity inhibitory Ca2+ binding sites (K0,, values of ~ 1 UM and ~1 mM,

respectively; Pessah et al., 1985; Michalak et al., 1988).

13

Many studies of RyR functional activity have been performed using the single
channel, planar lipid bilayer technique. A single RyR channel is transferred into a planar
lipid bilayer and ion conductance across the bilayer, via the channel, is recorded as a
change in current. Smith et al. (1986b) reported that the channel’s fraction of open time
(Po) is close to zero with nanomolar free Ca2+ on the cis (SR cytoplasmic) side of the
bilayer. The addition of micromolar Ca2+ to the cis chamber activates the channel and
induces rapid channel openings and closings occurring as single events and bursts that
could last from less than one to many milliseconds.

[3H]ryanodine binding measurements also suggest a bimodal regulation of RyR]
by Ca”, and further indicate that the afﬁnity and cooperativity of interaction between
high- and low- affinity [3H]ryanodine binding sites are dependent on Ca2+ concentration
and ionic strength (Chu et al., 1990; Lai et al., 1989; McGrew et al., 1989; Meissner and
El-Hashem, 1992; Mickelson et al., 1990; Pessah et al., 1987).

2. Magnesium (Mg2+)

By measuring the ”Ca2+ efflux from SR vesicles under various conditions, it was
demonstrated that millimolar concentrations of Mg2+ inhibit Ca2+ release (Meissner,
1984; Nagasaki and Kasai, 1983). Likewise, in single channel recordings, cis 4 mM Mg2+
partially inhibits the Can-channel activity by decreasing open state probability of the
channel (Lai et al., 1988). I

Pessah et al. (1987) indicated that Mg2+ at physiological levels inhibits
[3H]ryanodine binding by decreasing both the afﬁnity and the number of ryanodine-
binding sites by altering the apparent afﬁnity of activator site for Ca”. Mg“ also

signiﬁcantly retards the association kinetics of ligand binding.

14

The multiple mechanisms proposed for the Mg2+ inhibitory effect are: 1)
competitive displacement of Ca2+ from its activating high-afﬁnity site (Pessah et al.,
1987); 2) binding of Mg2+ to the inhibitory low-afﬁnity Ca2+ binding site (Kirino et al.,
1983; Meissner and El-Hashem, 1992); 3) Mg2+ could sterically block the Cay-channel
as it binds to a site near the conduction pathway in agreement with its known high
permeability (Smith et al., 1986)

3. Adenine Nucleotides

ATP enhances Ca2+ release from isolated SR (Meissner, 1984; Smith et al., 1985),
increases ryanodine binding (Campbell et al., 1987; Liu et al., 1989; Hymel et al., 1988;
Michalak et al., 1988; Lai et al., 1988; Zarka and Shoshan-Barmatz, 1993), and activates
single Ca2+ release channels incorporated into planar bilayers (Smith et al., 1985; Lai et
al., 1988; Hymel et al., 1988; Ashley, 1989).

Pessah et al. (1987) reported that 1 mM AMP-PCP (the non-hydrolysable analog
of ATP), in the presence of 1 mM Mg“, nearly triples the ryanodine-binding site afﬁnity
while increasing the total ryanodine binding by 25—fold. Other adenine nucleotides
exhibit the same properties in modulating the [3H]ryanodine-binding site with a potency
order of AMP-PCP > CAMP > ADP~adenosine > AMP. They proposed that adenine
nucleotides inﬂuence the kinetics of channel opening once Ca2+ is bound to the activator
site, either by increasing the efﬁciency of the gating mechanism or by increasing the
duration of the open state of the channel.

By using the planar lipid bilayer method, Smith et al. (1986b) reported that
millimolar ATP increases the frequency of open events and decreases the duration of

closed events (P0 = 0.6) in the absence of Ca2+. Millimolar ATP and micromolar Ca”

15

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 (Po > 0.99).

Meissner et al. (1997) indicated that by using AMP, the apparent afﬁnity of the
Ca2+ inactivation sites for Ca2+ is lowered by greater than 3-fold, whereas there is an
increase (< 2-fold) in the Ca2+ affinity of the channel activation sites. AMP does not
substantially increase the afﬁnity of the Ca2+ activation sites for the competing cations
(Mg2+ and monovalent). Thus, the primary effect of AMP is to decrease the Ca2+ afﬁnity
of the low-afﬁnity channel inactivation sites.

While the speciﬁc involvement of an adenine nucleotide binding site in the
potentiation of Ca2+ release channel activity is well established, the mechanism by which
ATP produces its stimulatory effect remains unclear (Shoshan-Barmatz and Ashley,
1998). Other adenine nucleotides (AMP-PCP, ADP, AMP, cAMP, adenosine, and
adenine) potentiate Ca2+ release (Meissner, 1984) and ryanodine binding (Michalak et al.,
1988; Pessah et al., 1987; Zarka and shoshan-Barmatz, 1993), suggesting that activation
involves binding to an effector site rather than covalent modiﬁcation of the channel
protein by phosphorylation.

4. Ryanodine

Ryanodine was ﬁrst characterized after isolation and crystallization from the
ground stem wood and root of Ryania speciosa Vahl by Rogers et al. (1948). Ryanodine
is a neutral plant alkaloid that is soluble in water and alcohol. The structure of ryanodine
is shown in Figure 1.3. Bianchi (1968) ﬁrst suggested that ryanodine interferes with the
release and uptake of Ca2+ by the sarcoplasmic reticulum (SR). Meissner (1986b)

demonstrated that the effect of ryanodine on isolated SR vesicles is either to stimulate or

16

    
 

HO rum".

.....tl’

HO ——
* \\“‘«

CH3

Figure 1.3 Ryanodine structure showing positions of tritium labels (9,21).

17

to inhibit Ca2+ efﬂux, depending on the experimental conditions. Vesicles are rendered
permeable to 45Ca2’ at a ryanodine concentration of 0.0] uM when diluted into a medium
containing the two Ca2+ release channel inhibitors, Mg“ and ruthenium red. At ryanodine
concentrations greater than 10 MM, 45Ca2” efﬂux is inhibited in channel-activating (5 uM
ea”) or -inhibiting (10 mM Mg2+ plus 10 M ruthenium red) media.

In both skeletal and cardiac muscle SR, high-affinity sites and a low-afﬁnity
[3H]ryanodine-binding sites have been identiﬁed. At nM concentrations, ryanodine only
binds to the open state of the Cay-channel, locking it irreversibly into an open
subconductance state. At IIM concentrations, ryanodine binding results in closure of the
channel. It is generally accepted that there is only a single high-afﬁnity site for
[3H]ryanodine per RyR tetramer (Lai et al., 1988). The ratio of high-afﬁnity (K. = 8 nM)
to low-afﬁnity (K; = 5 uM) binding sites has been reported to be 1:3 (Shoshan-Barmatz
and Ashley, 1998). A model based on allosteric negative cooperativity between the four
possible ryanodine binding sites in the homotetrameric RyR suggests that binding of
ryanodine to one of the four available sites (with high afﬁnity) triggers conversion of the
other sites in the tetramer to low-afﬁnity binding sites (Pessah and Zimany, 1991). In
addition, the [Ca2+-RyR] complex is very stable, and ryanodine only dissociates very
slowly, with a tin of about 14 hours (Pessah et al., 1987). This supports the suggestion
that the receptor undergoes a conformational change on binding Ca2+ and/or ryanodine so
that ryanodine becomes physically occluded and cannot diffuse freely off its binding site
(Shoshan-Barmatz and Ashley, 1998).

Because ryanodine binds to the open Ca2+ channel and keeps it open, and because

the binding afﬁnity is high and speciﬁc, the analysis of ryanodine binding to the channel

18

protein can provide important information about the Cay-channels functional state, its
modulation by various compounds, and its distribution in various tissue (Shoshan-
Barmatz and Ashley, 1998). In particular, ryanodine binding experiments may be used to
identify difference in channel protein activity when a mutation has occurred (see section
V).
5. Caffeine

The plant alkaloid 1,3,7-trimethylxanthine (caffeine) has been used extensively in
studies of Ca2+ release (Stephenson, 1981; Sorenson et al., 1986; Endo, 1977; Palade,
1987; Fabiato, 1983; Meissner and Henderson, 1987). Pessah et al. (1987) showed that
caffeine (20 mM) in the presence of 1 mM Mg2+ decreases the threshold for Ca2+
activation of [3H]ryanodine binding by ~20-fold and increases the apparent afﬁnity of the
activator site for Ca2+ by 17- to 27-fold. Caffeine, therefore, appears to act on a domain
which allosterically modulates the sensitivity of the Ca2+ activator site for Ca2+, and
reversing Mg2+ inhibition by selectively increasing the apparent afﬁnity of the Ca2+
binding site for Ca2+. Chu et al. (1990) reported that caffeine enhances the ryanodine
association rate of the Ca2’-channel protein without a change in the dissociation rate.

Caffeine increases the mean open times of the channels without changing their
unit conductances. The mechanism of this activation is shown to involve both a Ca“-
dependent increase in the frequency and duration of openings and a Cay-dependent
activation at high caffeine concentrations (Rousseau et al., 1988).
C. Dihydropyridine Receptor (DHPR) or L-type Ca2+ Channel

The skeletal muscle DHPR is a L-type Ca2+ channel, a member of a family of

voltage-gated ion channels that includes Na’, Ca2+ and K+ channels (De Waard et al,

19

1995; Catterall, 1996), and is localized to the junctional T-tubule membrane. It is called
dihydropyridine receptor (DHPR) because it has very high binding afﬁnity for drugs that
share the common basic structure of dihydropyridine. Examples of these drugs include
nitrendipine, PN200-110 (iSOpropyl 4-(2,1,3-benzoxadiazol-4-yl)-l,4-dihypro-2,6-
dimethyl-methyoxy-carbonylpyridine-3-carboxylate) and azidopine. Biochemical
evidence ﬁrst indicated that DHPRs were located in T-tubules of adult skeletal muscle
(Fosset et al., 1983). In skeletal muscle, DI-IPRs are a complex of ﬁve subunits or] (185
kDa), (12 (143 kDa), B (54 kDa), y (30 kDa) and 6 (26 kDa) (Figure 1.4). The Oil subunit
is the receptor for Ca2+ antagonists and can itself function as a voltage-gated ion channel
(Lacerda et al. 1991; Mikami et al. 1989), while the other subunits modulate or] channel
activity. The structure of the or] subunit has four homologous transmembrane ‘repeats’
(Figure 1.5), loop I, II, III, IV, each with 6 hydrophobic segments (Sl-S6), which are
thought to span the T-tubular membrane. The positively charged segment 4 (S4) of each
repeat contains positively charged (Arg or Lys) residues in every third or fourth position,
and is regarded as the voltage-sensing element (Melzer, et al. 1995). In Ca2+ channels, the
III-IV loop has not been implicated in voltage-dependent inactivation, but S6 in domain I
and the cytoplasmic loop linking domains I and II are involved in the inactivation of Ca2+
channels (Zhang et al., 1994). The cytoplasmic loop linking domain I and II interacts
with the B subunit and the binding of the B subunit may modulate the inactivation process
in the or] subunit (Pragnell et al., 1994). The C-terminal region contains important
elements for the regulation of Ca2+ channel activity (Wei et al., 1994). The skeletal orl

subunit loop linking domains 11 and III is involved in skeletal muscle E-C coupling

20

WWW

. s “2 ml will ll
. ll‘l‘llllllllllll lUJ

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1.4 Model of the subunit structure of the dihydropyridine receptor
(adapted from Catterall, 1988). P, sites of cAMP—dependent protein

phosphorylation ; w, N-linked glycosylation.

3.

 

I-II loop

II-III loop

Figure 1.5 The structure of four homologous repeats of the DHPR a; subunit
(adapted from Pragnell et al., 1994).

22

(Tanabe et al., 1990), and a mutation in the loop linking domain III and IV has been
linked to malignant hyperthermia (Monnier et al., 1997), a condition resulting from
abnormal Ca2+ release. A mutation in the DHPR III-IV loop is unlikely to alter Ca2+
conductance, since this loop does not form part of the channel pore and has not been
identiﬁed as being important for either activation of inactivation of DHPR (Catterall,
1996), but could modify the coupling between the DHPR and ryanodine receptors by
delaying or inhibiting closure of Rle upon repolarization (Suda, 1995).

The B hairpins, which form the aqueous pore, control the ion selectivity (Melzer,
et al. 1995). The association of the B subunit with 0:1 increases the efﬁciency of coupling
between voltage sensing and pore opening (Olcese et al. 1994) and increases the current
amplitude substantially (Wei et al. 1991). Gregg et al. (1996) demonstrated that Bt-null
mice which lack the primary B subunit in skeletal muscle die at birth from asphyxia. Also
normal action potential in Bl-null myotubes fails to elicit a Ca2+ transient. L-type Ca2+
current is decreased 10- to 20-fold in Bl-null cells compared with Iittermate controls.
Immunohistochemistry of cultured myotubes shows that not only is the B1 subunit absent,
but also the on subunit is undetectable. These ﬁndings suggest that the B1 subunit may not
only play an important role in the transport or insertion of the at, subunit into the
membrane, but also may be vital for the targeting of muscle DHPR complex to the T-
tubule/SR junction (Ogawa et al., 1999).

IV. Protein- rotein Interaction Between R anodine Rece tor

Dihydropyridine Receptor (DHPR) in Skeletal Muscle

The ﬁrst biochemical evidence of direct interaction between proteins in the T-

tubule membrane and the sarcoplasmic reticulum membrane was the observation that

23

12”I-labeled T-tubules, when mixed with sarcoplasmic reticulum, transferred 12"1 to a
protein doublet of molecular mass 325 and 300 kDa, which are major proteolytic
fragments of Rle (Cadwell and Caswell, 1982). RyR] and DHPR have been
coimmunoprecipitated from triad vesicles solubilized in CHAPS, by antibodies directed
against DHPR. Immunoprecipitation with antibodies against Rle also coprecipitates
both Rle and DHPR, providing the evidence for binding between Rle and DHPR
(Marty et al., 1994). Also, the activities of RyR] and DHPR are reciprocally coupled,
such that presence of the Rle stimulates the skeletal muscle DHPR a1 subunit and vice
versa (Nakai et al., 1996). Yano et al. (1995) showed that by using a ﬂuorescent
conformational probe, a conformational change of the Ca2+ release channel and Ca2+
release are stimulated by depolarization. Nimodipine, an antagonist of the voltage sensor,
inhibits both the conformational change and Ca2+ release, providing evidence for an
interaction between the voltage sensor and the Ca2+ release channel that leads to the
activation of channel opening (Rios and Brurn, 1987).

Block etal. (1988) showed that in the swim-bladder muscle of toadﬁsh, DHPRs
are clustered in groups of four and positioned in exact correspondence with Rle
tetramers so that each DHPR on the T-tubule membrane is located immediately opposite
to one of the Rle subunits (Figure 1.6). Image analysis has determined that DI-IPRs are
associated with Rle tetramers. The same disposition of DHPRs and RyRs is observed
in cultured human muscle, mouse, rat and chicken myotubes (Franzini-Armstrong and
Jorgensen, 1994; Franzini-Armstrong and Kish, 1995). Because the tetrad is composed of
four DHPRs and there are two RyRs for every tetrad, the alternate RyR association

pattern would predict a 2:1 ratio of DHPR to RyR (Franzini-Armstrong and Kish, 1995).

24

k“ r-\.\\‘.,\_I.-

 

Figure 1.6 Diagram of the spatial relationships between the junctional
complex components (adapted from Block et al., 1988). Four large shaded
spheres represent each foot; small white spheres are the intramembranous
portion of the foot protein; black discs represent the position of the junctional
T tubule tetrads. In the diagram, tetrads interact with alternate feet.

25

However, various ratios from 0.6 to 2.], determined by biochemical analysis, have been
reported for skeletal muscle (Sutko and Airey, 1996).

Two mouse models show that the interaction between Rle and DHPR is
essential for the E-C coupling. One is the dysgenic mouse model, in which the or; subunit
of DHPR is knocked out. The muscles of these mice do not contract because of a lack of
E-C coupling and they develop poorly (Powell and Fambrough, 1973; Klaus et al. 1983).
The other is the Rle-knockout or dyspedic mouse model in which E-C coupling is
blocked (Takeshima et al. 1994). The triad junction does not have foot structures present
in the dyspedic mouse (F ranzini-Armstrong, 1999).

Protasi et al. (1998) indicated that DHPRs do not associate into tetrads in the
absence of Rle and that tetrads are restored after “rescue” by Rle cDNA. These
results provide strong support of two current hypotheses on junctional architecture. One
is that DHPRs are linked to RyRs in skeletal muscle and this linkage is responsible for
the formation of tetrads (Block et al., 1988; Takekura et al., 1994; Protasi et al., 1997).
The other is that the random disposition of cardiac muscle DHPRs within junctional
domains is the result of the absence of a link to the cardiac RyRs (Sun et al., 1995).

The interaction of RyR] and DHPR is also important for the inactivation of Ca2+
release. Early studies showed that the Ca2+ release that occurs following depolarization is
turned off by subsequent repolarization (Melzer et al., 1984, 1987; Schneider and Simon,
1988; Rios and Pizarro, 1991). The Ca2+ release ﬂux starts to decay within a second after
ﬁber repolarization, suggesting that there is a direct mechanical interaction between the
voltage sensor and the release channel similar to the activation of Ca2+ release following

depolarization (Rios and Pizarro, 1991).

26

In cardiac muscle cells, ﬁ'eeze-fracture electron micrograph images show that the
pattern of DHPRs disposition differs from that in skeletal muscle cell. Franzini-
Arrnstrong (1999) indicated that the DHPRs are in proximity of the feet, but do not seem
to be directly linked to them. Thus, DHPR-RyR2 interaction may be indirect, via a
transmitter (e.g., calcium). Sun et al. (1995) and Protasi et al. (1996) also reported that
the DHPR is clustered in large intramembrane particles, but they do not form tetrads
despite the presence of RyR2. The direct interaction between DHPR and RyR2 is

probably missing in the cardiac muscle.

V. Can-channel Related Meat Quality Problem: Malignant Hyperthermia (Mg),
Porcine Stress Syndrome (PSS), Central Core Disease (CCD)

A. Malignant Hyperthermia and Porcine Stress Syndrome

Malignant hyperthermia (MH) has been recognized in humans since 1960 as an
inherited disorder of skeletal muscle characterized by an accelerated muscle metabolism,
contracture development, and rapidly rising temperature in response to certain anesthetics
(Gronert, 1986; Ellis and Heffron, 1985). The syndrome occurs in about one out of
15,000 anesthetized children and one out of 50,000 anesthetized adult humans (ording,
1996; Britt, 1970; ording, 1985). Malignant hyperthermia can cause neurological, liver,
or kidney damage, and is frequently fatal.

Also in the early 1960’s, it was noted that certain strains of pigs showed a high
frequency of susceptibility to stresses such as heat, crowding, transport, etc. Frequently,
these pigs would exhibit an episode of MH similar to that in human MH. The similarity
between the two diseases is further suggested by the observation that halothane also

triggers MB in the stress-susceptible pigs. Malignant hyperthermia episodes in pigs are

27

usually fatal. Major economic losses in the pork processing industry result from the
development of pale, soft and exudative (PSE) meat that arises from postmortem
manifestation of MB in stress susceptible animals (O’Brien, 1987). Today, it is widely
accepted that a signiﬁcant fraction of pork PSE problem is related to the postmorterm
hypennetabolic condition induced by stress. At the time of slaughter stress induces in
these pigs a tremendous increase in aerobic and anaerobic metabolism, causing the
production of heat, C02 and lactic acid and the contraction of skeletal muscle. High
temperature in combination with rapid pH drop favors the protein denaturation during the
conversion of muscle to meat. This results in the loss of protein water-holding-capacity
and solubility, two main protein characteristics that determine the quality and
acceptability of processed meat products (Cassens, 1975; Strasburg and Byrem, 2000).
The gene associated with this syndrome is sometimes called halothane or HAL gene,
stress gene, or PSS gene (Hall et al., 1980; Harrison, 1979). Since two alleles of this gene
are presented in mammals, there are three porcine genotypes: homozygous normal (NN),
heterozygous (Nn) and homozygous MH-susceptible (MHS) HAL gene (nn). The
homozygous nn gene 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 than homozygous NN pigs
(Zhang et al., 1992).

Studies seeking to deﬁne the primary defect in MI-IS muscle have utilized the
porcine model of the syndrome which, both clinically and biochemically, very closely
resembles the human syndrome in MHS muscle. Analysis of Ca2+ release from isolated

SR fractions, skinned muscle ﬁbers, or muscle ﬁber bundles, suggested a defect

28

associates with the Cay-induced calcium release mechanism of SR or with its control
(Mickelson et al., 1988).

A defect in the Ca2+-release mechanism was originally proposed to be the cause
of MH because MH muscle is hypersensitive to halothane and caffeine (Endo, 1977).
While MI-I SR seems to have normal Ca2+ uptake, several groups reported abnormal Ca2+
release from MHS SR vesicles (Endo et al. 1983; Takagi et a1. 1983; Nelson 1983;
Ohnishi 1987; Kim et al. 1984; Mickelson et al., 1986). Mickelson et al. (1988) used a
[3H]ryanodine binding approach to examine the SR Ca2+-channel protein function in
PSS-susceptible pigs. Their results suggested that the ryanodine receptor of isolated MHS
porcine heavy SR exhibits an altered Ca2+ dependence of [3H]ryanodine binding at the
inhibitory Ca” binding site as well as a lower Kd for ryanodine (92 vs 265 nM) when
compared to normal porcine SR. The channel protein content of the normal and MHS
[3H]ryanodine receptor in heavy SR vesicles (9.3-12.6 pmol/mg) is not signiﬁcantly
different. They concluded that since the SR ryanodine receptor is a Ca2+ release channel
as well as a component intimately involved in transverse tubule-SR communication,
abnormalities in the skeletal muscle ryanodine receptor may be responsible for the
abnormal SR Ca2+ release and contractile properties demonstrated by MH muscle. The
landmark paper published by Fujii et al. (1991) reported the ultimate cause of PSS in
pigs is a mutation in the skeletal muscle Cay-channel protein at the residue 615, in which
arginine is substituted by a cysteine. This discovery has led to the development of a rapid
screening test for the presence of the undesirable alleles in breeding stock.

Like the pork industry, the modern turkey industry has successfully bred for rapid

growth and heavy muscling in birds to meet consumers’ demand for lean and inexpensive

29

turkey meat products. However, in recent years, difﬁculties have emerged in further
processing of turkey breast muscle. Current problems with the soft texture, poor
cohesiveness, and poor juiciness of processed turkey breast muscle result in multi-
million-dollar yearly losses to the turkey industry (Foegeding, 1992). Previous studies
suggested that several similarities exist between the development of PSE meat from
swine and defective muscle quality in domestic turkeys (Sosnicki and Wilson, 1991,
1992; Sosnicki, 1993a,b). Heat, stress and struggling prior to slaughter can trigger
acceleration of postmortem glycolysis in both pork (Mitchell and Heffron, 1982) and
turkeys (Froning, 1978; McKee and Sams, 1997), and cause rapid pH drop. The
combination of high temperature with quick pH decline favors protein denaturation
during the conversion of muscle to meat. Pietrzak et al. (1997) showed that the fast
postmortem glycolysis group (PSE group) is characterized by lower muscle ATP, higher
lactate levels, lower water holding capacity, lower cooking yield, and lighter color
compared with the slow glycolysis group (control group). Numerous similarities between
development of PSE pork and PSE turkey strongly suggest that the genetic selection for
rapid growth rate could result in inadvertent selection for a mutation of the Cay-release
channel protein in skeletal muscle and subsequent undesirable meat quality problem.

The biochemical approaches used to study the Ca2+ release mechanism in stress
susceptible pigs were facilitated by the availability of genetically deﬁned normal and MH
pigs. Utilization of the same approach in turkeys is hampered by the fact that there are no
phenotypic markers to identify normal and abnormal populations. Wang et al. (1999)
assumed that the frequency of a mutation would be greater in modern commercial line of

turkeys compared to a genetically unimproved line. Sarcoplasmic reticulum was isolated

30

from these two lines for comparison of ryanodine binding properties. The SR fractions,
enriched in the Cay-channel protein, from commercial turkeys exhibit a higher mean
afﬁnity for ryanodine when compared to that ﬁom unimproved turkeys (Kd = 12.2 vs 20.5
nM). A four-fold difference (p < 0.05) in mean Cay-channel protein content or Bmax
(1.10 vs 4.01 pmol/mg) is observed between commercial and genetically unimproved
turkey SR fractions. This suggests that there is an alteration in RyR activity in either or
both isoforms of the commercial turkeys compared to controls.
B. Central Core Disease

Central Core Disease (CCD) is a rare, nonprogressive myopathy, presenting in
human infancy, which is characterized by hypotonia and proximal muscle weakness. It is
inherited as an autosomal dominant trait. An important feature of CCD is its close
association with susceptibility to MI-I (Denborough et al., 1973; Brownell, 1988; Eng et
al., 1978; Frank et al., 1980). Diagnosis of CCD is by histologic examination of skeletal
muscle biopsy specimens. Typical CCD samples show amorphous central cores in type 1
ﬁbers. Identiﬁcation of cores is facilitated by staining for oxidative enzyme activity. As
the cores are depleted of mitochondria, they appear as negative areas within the normal
enzyme activity areas of the surrounding muscle ﬁbers (Ogawa, 1994). There are
numerous reports indicating a gene mutation of the Rle in the MH/CCD disorder
(Table 1.1).

Mills (2000) observed central core ﬁber damage in commercial turkey leg
muscles. She suggested that this could indicate mitochondrial abnormalities or alterations
in the distribution of mitochondria due to the large ﬁber sizes, both of which would affect

muscle metabolism and possibly meat quality. The percentage of ﬁbers exhibiting

31

damage in the breast muscles is signiﬁcantly greater in the commercial strain and
increases linearly with age. Muscle damage only occurs at low levels in the traditional
strain with no apparent relationship to age. The commercial birds also show a
signiﬁcantly greater hyperthermia than the traditional birds with a mean rise of 4°C
compared to only 2°C in the traditional line (p < 0.05). It is possible that these central
core ﬁbers have arisen as a result of a mutation in either or both of the ryanodine

receptors in commercial turkeys in similar fashion to human with central core disease.

32

 

 

32 .3 o 3.5 3333 _ coo 3.3-momma: 98er
£32 .3 so 3:352 3.3 m: mam-$33-32 <3.» 20
£332 .3 3 9.3.32 33 53 35333-32 5:8

32 .33 353m 3353 _ :2 38-33-32 3838

32 .33 25.8 33:3 _ m2 maﬁa-ma some

32 .33 283m 3333 _ m: soc-23-32 .3835

82 .3 3 mean 3.33.3 a 80 mm: 35-33-33-30 £25

32 .3 so 3:383 3.3 m: 32-33-30 3383.5
32 .33 3.35 3333 c m: mes-88-5 0280
332 .3 so 33532 3.33.3 _ :2 32-835 :88
332 .3 so mecca: 33 m: “oz-$8-; .3880
£92 .3 3 33532 3333 _ 80 £2 33-83-32 $3335
332 .3 so mecca: 3.3 32 35-32 N32 3338
32 .3 so 23:0 33 m: comic-ma haze
82 .3 3 33:6 3% m: 36-3632 .8336
82 .3 so 3:383 3333 _ :2 cos-$332 :86
£32 .3 co 23:0 3:33 _ coo Mm: com-23-5 omen 2

82 .3 3 23.6 333-3 _ 80 32-8%: oooﬂo
332 .3 s 23.5 33 m: 32-33.35 586

No.2 .3 a 33:6 .33 E3 32-33-30 3530

82 .3 s 23:0 .33 80 £2 35-32-32 .355

32 .3 3 3:3 33.33 _ :2 32-2-38 one;

and 55:38.3 8285 533335 cocoa—0:2

 

88" ..a .o sou-333 so... ceases

nevi: Sea: 5 53: 538.. 322335 23...: .3285 s 2.3.3:: 3 2.3.3

33

Chapter 2

Modification of experimental conditions for ryanodine binding assay of

turkey skeletal muscle

1. Introduction

In skeletal muscle, intracellular Ca2+ channels release Ca2+ from the sarcoplasmic
reticulum (SR) to the sarcoplasm in response to a depolarization potential, which causes
muscle contraction. The SR Cay-release channel is also known as the ryanodine receptor
(RyR) because the channel protein can bind the plant alkaloid ryanodine with very high
afﬁnity.

The activity of the mammalian skeletal muscle ryanodine receptor isoform
(Rle) can be modulated by several physiological and pharmacological factors: uM
Ca2+, mM adenine nuleotides, caffeine, high salt concentration, and nM ryanodine
stimulate the channel activity, whereas mM Mg“, ruthenium red, mM Ca2+ and uM
ryanodine inhibit the activity (Nagasaki and Kasai, 1983; Ikemoto et al., 1985; Meissner
et al., 1986b; Smith et al., 1985; 1986a; b). RyR] exists as a tetramer of four identical
subunits. There is one high aﬂinity ryanodine binding site per tetramer, and 3-4 low
affinity sites. Ryanodine binds to the open state of the channel, and this characteristic has
been exploited as a tool to study functional activity of Rle under various conditions.

Ryanodine binding has previously been used as a tool to probe the ﬁJnctional
activity of the calcium release mechanism in normal and stress-susceptible pigs. A
substantial fraction of meat from these stress-susceptible pigs exhibits a quality defect
referred to as pale, soft and exuative (PSE) meat. Ryanodine binding studies indicated

that SR ﬁ'om stress-susceptible pigs displays higher afﬁnity ryanodine binding activity,

34

which suggests that there is a mutation in the ryanodine receptor. Fujii et al. (1991)
identiﬁed a mutation of arginine to cysteine at residue 615 which leads to increased
channel activity and is associated with PSE meat. Wang et al. (1999) analyzed the
ryanodine binding activity of genetically unimproved and commercial lines of turkeys.
They concluded that there is a highly signiﬁcant difference (p < 0.05) in ryanodine
receptor activity between these two lines. However, because of variation in the assay
results, statistical signiﬁcance could only be achieved by averaging of several individuals
from each of the two populations. This variation makes it impossible to use ryanodine
binding activity as a screening tool to identify individual turkeys with altered channel
proteins. One reason for this could be a result of differences between the mammalian and
avian skeletal muscle E-C coupling mechanisms. Therefore, the objective of this portion
of the study was to identify buffer conditions that effectively distinguish individual
differences of ryanodine receptor activities in avian muscle. In this set of experiments,
the concentrations of sucrose, salt and adenine nucleotide, factors which increase the
open state probability and affinity of the channel for ryanodine, were varied to deﬁne the
optimal ryanodine binding conditions for an eventual correlation of individual ryanodine

binding affinity with meat quality.

35

11. Materials and Methods
A. Materials
1. Skeletal Muscle

Breast muscle samples from two Nicholas turkeys were provided by the Tom Otto
turkey farm (Middleville, Ml), a commercial producer. Pectoralis major muscles were cut
into one-inch cubes and immediately frozen in liquid nitrogen and stored at -80°C.
2. Chemicals

Ryanodine was purchased from Wako Chemical USA, Inc. (Richmond, VA),
radioactive ryanodine ([3H]Ryanodine) was purchased from New England Nuclear Life
Science (Boston, MA). Phenylmethylsulfonyl ﬂuoride (PMSF), aprotinin, benzamidine,
pepstatin, leupeptin were purchased from Sigma (St. Louis, MO).
B. Methods

1. Preparation of Heavy Sarcoplasmic Reticulum Vesicles (HSR)

Skeletal muscle SR was isolated from turkey breast muscle samples by the
following procedure adapted from Mickelson et al. (1986) and Wang et al. (1999) with
minor modiﬁcations. Frozen muscle cubes (200 g) were thawed at 4°C for 20 min,
incubated with 5 volumes of Buffer I (0.1 M NaCl, 5 mM Tris-maleate, pH 7.0) for 5
min, and homogenized in a Waring blender for 2 min with intervals of 15 sec. on and 15
sec. off. After 30 min centriﬁigation at 3300 x gm“, the supernatant was collected by
ﬁltering through a l6-layer cheesecloth. The pellet was discarded, and the supernatant
was centrifuged for 30 min at 16,000 x gum. This pellet was collected, resuspended with
20 ml Buffer II (0.6 M KCl, 5 mM Tris-maleate, pH 7.0), and gently homogenized in a

Dounce homogenizer. The homogenized suspension was centrifuged for 40 min. at

36

150,000 x gm“ using a Beckman Ti-70 rotor. The pellet was collected and suspended in
about 5-7 ml 10% sucrose solution, and gently homogenized in a Dounce homogenizer.
The crude SR vesicles obtained by this step were ﬂash-frozen in liquid nitrogen and
stored at -80°C.

Heavy SR (HSR) vesicles, enriched in the Ca2+ channel protein, were isolated by
suspending ~1-2 g crude SR in 2 ml of Buﬁ‘er III (10% sucrose, 0.4 M KCl, 5 mM Tris-
maleate, pH 7.0) and loading the suspension on discontinuous sucrose gradients (22%,
36%, 45%). The gradients were centrifuged for 5 h at 100,000 x gum,- in a Dupont-Sorvall
AH 629 rotor. The HSR was collected at interface between the 45% and 36% sucrose
layers. HSR fractions were diluted at least 1:1 with 10% sucrose and centriﬁsged at
150,000 x gum, in the Beckman Ti-70 rotor for 40 min. The pellet was suspended and
homogenized in 10% sucrose, ﬂash-frozen and stored at -80°C.

During the HSR protein puriﬁcation, the following protease inhibitors were added
at each step: PMSF at 0.2 mM, and aprotinin, benzamidine, pepstatin and leupeptin at 1
ug/ml, to Buffer I, II, III, 10% sucrose and sucrose gradients. Beta-mecaptoethanol was
also added at a ﬁnal concentration of 30 mM to Buffers I and II.

B. Ryanodine Binding Assays

Fifty ug of HSR protein were incubated in buffer containing various
[3H]ryanodine concentrations (2-70 nM), 10 mM MOPS, a CaClz-EGTA-nitrilotriacetic
acid buffer to give a speciﬁc [Ca2+]rm of 10 uM (pH 7.2), plus combinations of NaCl (0.1
M, 0.5 M, 1 M), sucrose (0.1 M, 0.5 M, 1 M) and AMP-PCP (0, 1 mM) in a 3x3x2
matrix. Triplicate samples of each condition were incubated for 6 h at 37°C, and ﬁltered

by Whatman GF/B ﬁlters. Filters were washed three times with 5 m1 ice-cold buffer (10

37

mM MOPS, 0.1 M NaCl, 50 uM CaClz, pH 7.0), and the amount of [3H]ryanodine
retained on the ﬁlters was determined by scintillation counting. Non-speciﬁc binding of
ryanodine was determined by adding unlabeled ryanodine at 1000 times the
[3H]ryanodine in the incubation buffer. Speciﬁc ryanodine binding was determined by
subtracting the non-speciﬁc binding from the total binding.

The ryanodine binding conditions selected from the 3x3 x2 matrix were also
compared with the results using the conditions of Wang et al. (1999). In the latter case,
the buffer used for ryanodine binding consisted of 0.25 M KCl, 25 mM PIPES (pH 7.0), a
CaClz-EGTA-nitrilotriacetic acid buffer to give a speciﬁc [Ca2+]:fee of 10 uM.

The K. (ryanodine binding aﬁinity) and Bmax (maximum binding capacity) values
for ryanodine binding by the HSR were calculated by Scatchard analysis using Sigma
Plot.

3. Protein Assay

HSR protein concentration was determined by the method of Lowry (1951).

Bovine serum albumin was used as a standard.
4. Statistical Analysis
The Durbin-Watson test was used to compare the consistency of both Wang’s

(Wang et al., 1999) and the current condition.

38

III. Results and Discussion

The ryanodine binding assay is a useful tool to analyze the function of the Ca2+-
channel protein in the SR, because ryanodine only binds to the open state of the Ca2+-
channel protein (RyR) with high affinity. The afﬁnity for ryanodine and open state
probability of RyR are modulated by various effectors. This property was exploited by
Mickelson et al. (1988) to deﬁne differences in channel protein activity of normal and
stress-susceptible pigs. The buﬁ‘er condition used for analysis of ryanodine binding of pig
SR, however, gives signiﬁcant variation within individual experiments when adapted for
turkey SR, suggesting that optimization of the assay condition was required for turkey
SR. The aim of this study was to deﬁne conditions that will facilitate the comparison of
the individual RyR functional differences between the random-bred and commercial
turkey populations.

The ryanodine binding assay was conducted by using two Nicholas commercial
turkey breast muscle samples (Turkey 1 and Turkey 2). Three salt concentrations (0.1 M,
0.5 M, 1 M), three sucrose concentrations (0.1 M, 0.5 M, 1 M), and two AMP-PCP
concentrations (0, 1 mM), were chosen to Optimize the ryanodine binding buffer
condition. AMP-PCP is the nonhydrolyzable analog of ATP and was used instead of ATP
to avoid possible interference from phosphorylation. The equilibrium constants obtained
by Scatchard analysis of [3H]ryanodine binding under diﬁ‘erent conditions are
summarized in Table 2.1. The eighteen combinations of salt, sucrose and AMP-PCP for
each sample caused the dissociation constant (Kd) to vary tremendously, while no

dramatic difference was found in the total binding capacity (Bmax) values within each

39

 

ox:- N.w_ 5N 0: ow ~.: m.»- oN~ o; o.—

 

 

 

2 3.: E 0.2 E 3 3 ms 3 no
3 3.2 oi S: 8 3.2 3.3 E 3 3o
2 38 S- b: 2 to S 3 no 3
ca 3.2 o.: 38 we 2: E 3 no no

3.: :3 3.2 m2 NS 3. : we 2 no 3
N3 3: nm 62 0.: E: 3 3 3o 3
no 2: we we 2 Z 3. be 3 We
SN ”.8 a; N: 3: 2 SN 3 3o 3

35 33333 35 32333 35 33333 :25 33333 96 a):
J Em i 3m J. Em J 3m 383:3 =32.

mum-e)? Es _ mum-32¢. 8 mom-$32 Ea _ mom-“3,2 8

N mod—...; g hog—...:-

 

moEEam 28:: 3.3.5 ans—...:- eBH Ea...—
maezuE-m 53:36“ omens—aeucam .935 he 95...:— 2:32:33— .8 8:33.80 Eatﬁzacm ﬁn 033,—.

40

sample. For Turkey 1, the lowest Kd value was 2.3 nM, while the highest was 23.7 nM,
and the average Bmax value was 8.9 i 1.9 mel/mg. For Turkey 2, the lowest Kd value
was 2.7 nM, while the highest was 81.9 nM, and the average Bmax value was 18.9 i 2.7
mel/mg.

The binding afﬁnity Of [3H]ryanodine to RyR is markedly dependent on the
presence of compounds in the binding buffer, which are known to alter Ca2+ release from
the SR. As a general rule, conditions that Open the channel, such as the presence of
micromolar Ca2+, millimolar adenine nucleotide, or high ionic strength, are found to
increase the afﬁnity of [3H]ryanodine to the high afﬁnity ryanodine binding site
(Meissner et al., 1997). Each Of the factors included in this study has been reported to
increase the Open state probability and afﬁnity of the channel alone.

It was Observed that either in the presence or absence of 1 mM AMP-PCP, when
the salt concentration was increased, the K4 decreased in both samples. For Turkey 1, in
the presence of 0.1 M NaCl and 0.1 M sucrose, the K; was 23.7 nM in the absence Of 1
mM AMP-PCP and 14.0 nM in the presence Of AMP-PCP. The Kd’s derived from 1 M
NaCl plus 1 M sucrose were only 3.5 nM and 5.0 nM in the absence or presence of AMP-
PCP, respectively. The same trend was Observed from the Turkey 2 skeletal muscle
sample. Therefore, high concentrations Of NaCl can increase the binding afﬁnity Of
[3H]ryanodine to the receptors. Chu et al. (1990) reported that the apparent afﬁnity of
RyR for ryanodine is increased upon increasing NaCl concentrations from 25 mM tO 2
M, via an increase in the rate of association of ryanodine. High salt is known to remove
loosely associated proteins and molecules and may, therefore, be enhancing the apparent

association of ryanodine by removing an endogenous occupant of the site. However,

41

because the effects of ionic strength (both high and low) on the properties Of the protein
appear to be totally reversible, they concluded that it is most likely that the conformation
Of the binding site is dependent on ionic strength. Meissner et al. (1997) Observed that
replacement Of Cl' with 2-[N-Morpholino]ethanesulfonic acid (Mes') in 0.25 M choline”
media lowered [3H]ryanodine binding (BM = 10.8 vs 12.4 mel/mg, K, = 1.8 vs 11.6
nM) . They suggested that an increase in [C1'] is responsible for elevating the
[3H]ryanodine binding levels by increasing the apparent Ca2+ afﬁnity Of the Ca2+
activation sites and decreasing the apparent Ca2+ binding affinity of the channel
inactivation site (Meissner et al., 1997).

In contrast to the effect of varied salt concentration on the [3H]ryanodine binding,
there is no clear trend Of the effect of sucrose on the [3H]ryanodine binding among all the
combinations Of conditions. However, for turkey 1, under the 0.1 M [NaCl] condition, the
Kd was 23.7 nM and 14.0 nM in the absence and presence of 1 mM AMP-PCP,
respectively, at a sucrose concentration Of 0.1 M. When [sucrose] was raised to 0.5 M,
the K, values were 19.5 nM and 12.2 nM in the absence and presence Of 1 mM AMP-
PCP, respectively. Then, as the [sucrose] increased to l M, the Kd was decreased tO 9.91
nM and 6.30 nM in the absence and presence of AMP-PCP, respectively. The data
suggested that the [3H]ryanodine binding is increased by sucrose through increase of the
afﬁnity under 0.1 M salt concentration. The same trend was also Observed in the Turkey
2 sample.

Certain concentrations Of salt and sucrose can increase the [3H]ryanodine binding
afﬁnity. Salt can change the ionic strength while sucrose changes the osmolarity of the

[3H]ryanodine binding buffer. A stimulation of [3H]ryanodine binding (Ogawa and

42

Harafuji, 1990) and slowing the single channel gating (Tu et al., 1994 ) by sucrose in the
presence Of salt suggests that the osmolarity and viscosity Of the assay media may play a
role in determining channel activity (Meissner, et al., 1997). There are two controversial
conclusions about whether it is osmolarity or ionic strength that stimulates the
[3H]ryanodine binding. Ogawa and Haraﬁiji (1990) examined the effect of the
composition of the reaction medium on ryanodine binding activity Of bullfrog skeletal
muscle SR. NaCl, KCI and sucrose were tested individually and in combinations at the
same osmolarity, and similar ryanodine binding results were Obtained. The [3H]ryanodine
binding reached a plateau at an osmolarity level Of 3.0 osm/liter. This suggests that
osmolarity, but not ionic strength, is a crucial factor for ryanodine binding activity. Lai et
al. (1989) showed that the tetramer, but not the monomer of the ryanodine binding
protein binds ryanodine. The high osmolarity may be favorable for oligomerization of the
ryanodine binding protein by reducing the mole fraction Of water molecules (Ogawa and
Harafuji, 1990). In contrast, Meissner et al. (1997) reported that in experiments on Ca2+
dependence Of [3H]ryanodine binding to the SR vesicles from rabbit skeletal muscle, the
nonionic solution (0.5 M sucrose) with an osmolarity comparable to that of the salts, give
very low levels of [3H]ryanodine binding (< 0.05 pmol/mg protein). The results
suggested that sucrose can activate the RyR only to a limited extent compared to the
effects of salt. Both Of these two groups used sucrose and salts at comparable osmolarity,
but Obtained Opposite results. The difference could be the result Of the different species
they used (mammalian vs amphibian), and/or the different composition Of the

[3H]ryanodine binding buffer.

43

In this study, no clear trend was Observed to determine whether salt or sucrose has
more effect on [3H]ryanodine binding than the other. The combination effect may be
playing a greater role rather than either alone.

AMP-PCP was included in some of these experiments to Optimize the
[3H]ryanodine binding because it is generally recognized that AMP-PCP can enhance
ryanodine binding and thus provide a more quantitative measurement Of high afﬁnity
ryanodine binding (Pessah et al., 1987; Michalak et al., 1988; Zarka and Shoshan-
Barmatz, 1993). Among the total thirty-six dissociation constants (Kd) through different
test conditions for both samples, 1 mM AMP-PCP decreased the Kd in most cases. Pessah
et al. (1987) indicated that 1 mM AMP-PCP with the presence Of 1 mM Mg” nearly
triples the [3H]ryanodine binding site afﬁnity, while increasing the B...“ 25-fold, AMP-
PCP possibly elicits strong positive cooperativity at the [3H]ryanodine binding site. In
this study, 10 M free Ca”, 1 mM AMP-PCP were included in the ryanodine binding
buffer solution, and this appears to efﬁciently activate the SR Ca2+release channel and
produces channel activation (Smith et al., 1986b; 1988). Under these conditions the Open-
channel form is predominant, and exhibits a full conductance characteristic for the native
channel (Serysheva, et al., 1999).

Salt, sucrose and AMP-PCP are all capable of enhancing the open state
probability Of the Ca2+ channel and thus, enhancing the [3H]ryanodine binding. Different
combinations at various concentrations have additive or even synergistic effects. Under
the condition Of 1 M NaCl, 1 M sucrose and 1 mM AMP-PCP, high [3H]ryanodine
binding afﬁnities were obtained (Kd = 5.03 nM and 3.55 nM, Turkey 1 and Turkey 2,

respectively), thus reﬂecting the increased open state of the Ca2+-release channel. The

44

BImam values Obtained at these conditions reﬂect upper limits Of maximal binding capacity.
However, in order to distinguish differences in RyR activities between random-bred and
commercial turkey populations, a lower afﬁnity range (higher Kd) is preferred. Under the
condition in which the [3H]ryanodine has high binding afﬁnity for the RylL little
difference in afﬁnity can be distinguished. In comparison Of MHS and normal pig SR, for
example, Mickelson et a1. (1990) reported that the affinity Of the MHS RyR for
[3H]ryanodine in the presence Of 6 uM Ca2+ plus 10 mM AMP-PCP (K, = 6.0 nM) is
almost identical to that of the normal SR (K; = 8.5 nM). However, in the presence Of 6
uM Ca2+ alone, the Kd values are 92 nM vs 265 nM from MHS and normal pigs,
respectively. The Kd’s derived from the condition of 0.1 M NaCl, 0.5 M sucrose and no
AMP-PCP were 19.54 nM and 27.86 nM, while the Bum were 7.52 mel/mg and 16.34
mel/mg from Turkey 1 and Turkey 2, respectively. The afﬁnity constants were the
second highest among 18 values in each sample, while the total binding capacities were
within the average range. Therefore, this condition can provide the best measurement Of
the receptor content (Bu-ax) while giving a high Kd value, and was chosen as the Optimal
condition for the turkey skeletal muscle ryanodine binding assay.

The results from the current ryanodine binding conditions were compared with
those from the conditions of Wang et al. (1999). Twelve assays were conducted using the
ryanodine-binding conditions Of Wang et al. (1999) and twenty-ﬁve assays under current
conditions were conducted simultaneously. Two assays were randomly chosen from
Wang's condition, and another two assays were chosen from the current condition to
match those from Wang's. Durbin-Watson statistical analysis showed that the consistency

Obtained from current condition is greater than that from Wang's (p<0.05). Thus, the

45

condition selected for future experiments represents an improvement in reproducibility Of
the ryanodine binding assay for turkey skeletal muscle. This assay condition was chosen
tO use for the following study to compare the functional differences in ryanodine receptor

activity between random-bred and commercial turkey populations.

46

IV. Conclusion

The Optimal buffer conditions for studying the [3H]ryanodine binding varies with
muscle type, species, pattern Of muscle stimulation, ryanodine concentration, and other
non-physiological factors. The ryanodine binding assay is a useful way to investigate the
SR Cay-release channel function. By testing different combinations of salt, sucrose and
AMP-PCP concentrations, a buffer condition consisting Of 0.1 M NaCl, 0.5 M sucrose,
with 10 M free Ca2+ and 10 mM MOPS (pH 7.2) was determined to be the best
conditions for the ryanodine binding assay of the turkey skeletal SR. These assay
conditions will provide the necessary stability and reproducibility to: 1) test a single
bird’s skeletal muscle RyR function, 2) distinguish RyR functional differences would be
suggestive Of differences in primary structure between the random-bred and commercial
turkeys, 3) measure RyR content accurately, 4) eventually correlate the individual

ryanodine binding data with meat quality characteristics.

47

Chapter 3

Ca2+-channel protein activity and regulation associated with turkey
PSE meat quality problems
I. Introduction

The modern turkey industry has grown rapidly during the last 30 years to meet
consumers’ demand for lean and inexpensive turkey meat products. To meet this demand,
breeders have intensively selected for heavy muscling and rapid growth of turkeys. Over
the past three decades, the market age of turkeys has shortened from 26-28 weeks to 14-
16 weeks while the market weight has increased from about 20 1b to about 35 1b.
However, the improvement in size and growth efﬁciency has come with a cost: increased
incidence of meat quality defects including PSE (pale, soft, exudative) meat. This product
is characterized by soft texture, lighter color, decreased water holding capacity and poor
cooking yield.

The development of PSE turkey meat during the postmortem conversion of
muscle to meat is remarkably similar to that of PSE pork. Decades of research have
shown that incidence of PSE pork is inﬂuenced by both environmental factors and by
genetic factors. Intensive genetic selection in the pork industry led to inadvertent
appearance of an inherited trait called Porcine Stress Syndrome (PSS). Pigs with PSS are
more likely than non-stress-susceptible pigs to yield PSE pork. This meat quality defect is
observed in about 10-20% of the total US pork production (Vansickle, 1989). Porcine
stress syndrome causes substantial economic losses to farmers, owing to death of animals
from stresses of transportation, heat, crowding, etc., before reaching market (Louis,

1993). Stress-susceptible pigs which go through the slaughter process exhibit increased

48

glycolytic rates during the conversion of muscle to meat, leading to higher carcass
temperatures in combination with acidic pH that favors protein denaturation. This results
in decreased protein solubility and poor water holding capacity.

It has been known since 1980’s that abnormal calcium homeostasis is a key factor
in the development of PSE pork (Gronert 1980; Nelson, 1983; Cheah et al., 1984). The
major breakthrough in the ﬁeld was the colocalization of the malignant hyperthermia
gene with the SR Ca2+ channel (RyR) to the same region of chromosome 19 in human
(MacLennan et al., 1990; McCarthy et al., 1990). Fujii et al., (1991) reported that a single
point mutation in the porcine gene for the ryanodine receptor (RyR) was correlated with
PSS in five major breeds of lean, heavily muscled swine.

The activity of RyR is modulated by several ligands including Ca2+, Mg” and
adenine nucleotides. It is also regulated by direct coupling with a protein called the
dihydropyridine receptor (DHPR). DHPR in skeletal muscle appears to have a dual
ﬁanction as both a calcium channel and voltage sensor mediating excitation-contraction
coupling. As the voltage sensor, which is the main function of skeletal DHPR, it
transduces the sarcolemma/T-tubule depolarization into a signal for opening of the SR
Ca2+ release channel (RyR) (Rios and Brum, 1987). DHPR’s role as a Ca2+ channel
appears to be minor. Mickelson et al. (1994) measured RyR and DHPR content in heavy
SR, crude SR, and total muscle homogenates. They observed that the crude membrane
fraction and total muscle homogenates from MH pigs exhibited only 61-81% of the
DHPR and RyR content of that from normal pigs. However, the ratio of DHPR to RyR in
the muscle homogenates was not different between MH and normal pigs. They concluded

that although the stoichiometry of the DHPR to RyR is not altered, the presence of the

49

MH RyR allele during muscle development results in a decreased relative content of
these two proteins. This is probably due to a lower junctional membrane content and may
be an important ultrastructural consequence of the altered sarcoplasmic Ca2+ regulation in
MHS muscle (Mickelson et al., 1994).

Based on the numerous similarities between development of PSE pork and PSE
turkey, we hypothesize that a defect in one or both of the turkey Ca2+-channel isoforms
and/or the regulation of Ca2+-release channel isoforms contributes to the increased
incidence of PSE turkey meat in modern commercial turkeys. To test this hypothesis, we
have adapted the ryanodine binding strategy used by Mickelson et al. ( 1988), coupled
with improvements in the assay developed in the previous chapter, to determine whether
biochemical differences exist between RyRs from a control population of random-bred,
genetically unimproved turkeys and modern commercial population. We have also
measured DHPR content and determined the stoichiometry of DHPR: RyR binding in the
two lines to determine whether difference in stoichiometry of DI-IPRzRyR might be

related to the incidence of PSE turkey meat.

50

11. Materials and Methods
A. Materials
1. Skeletal Muscle

Two populations of turkeys were used in this study. A group of random-bred,
genetically unimproved turkeys (McCartney, 1964) was used as a control population.
These turkeys were provided by Dr. Karl Nestor of the Ohio Agricultural Research and
Development Center (Wooster, Ohio). Commercial turkey breast muscle samples from
Cargill/Plantation Hatchery were provided by Dr. Alan Sams (Texas A&M University).
Two lots of these commercial samples were obtained approximately one and half years
apart, group I (1998) and group II (2000). Breast muscles were cut into one-inch cubes
and immediately frozen in liquid nitrogen, stored at -80°C.
2. Chemicals

Ryanodine was purchased from Wako Chemical USA, Inc. (Richmond, VA),
nifedipine was purchased from Sigma (St. Louis, MO). Radioactive ryanodine
([3H]ryanodine) and radioactive PN200-110 ([3H]P200-110) were purchased from New
England Nuclear Life Science (Boston, MA). Protease inhibitors, phenylmethylsulfonyl
ﬂuoride (PMSF), aprotinin, benzamidine, pepstatin, leupeptin, were purchased from
Sigma.
B. Methods
1. Preparation of Crude Sarcoplasmic Reticulum Vesicles (CSR)

Skeletal muscle SR was isolated ﬁ'om turkey breast muscle samples by
procedures of Mickelson et al. (1986) and Wang et al. (1999) with minor modiﬁcation.

The 200 3 frozen muscle cubes were thawed at 4°C for 20 min, incubated with 5 volumes

51

of Buffer I (0.1 M NaCl, 5 mM Tris-maleate, pH 7.0), for 5 min., and homogenized in a
Waring blender for 2 min with intervals of 15 sec. on and 15 see. off. After 30 min
centriﬁagation at 3300 x gm“, the supernatant was collected by ﬁltering through a 16-
layer cheesecloth. The pellet was discarded, and the supernatant was centrifuged for 30
min at 16,000 x gm“. This pellet was collected, resuspended with 20 ml Buffer II (0.6 M
KCl, 5 mM Tris-maleate, pH 7.0), and gently homogenized in a Dounce homogenizer.
The homogenized suspension was centrifuged for 40 min. at 150,000 x gm. using a
Beckman Ti-70 rotor. The pellet was collected and suspended in 10% sucrose solution,
and gently homogenized in a Dounce homogenizer. The crude SR vesicles Obtained by
this step were ﬂash-frozen in liquid nitrogen and stored at -80°C.

During the crude SR vesicles puriﬁcation, the following protease inhibitors were
added to each buffer, PMSF at 0.2 mM, and aprotinin, benzamidine, pepstatin and
leupeptin at l ug/ml. Beta-mecaptoethanol was also added to a ﬁnal concentration of 30
mM to Buffers I and II.

2. Preparation of Heavy Sarcoplasmic Reticulum Vesicles (HSR)

Heavy SR (HSR) vesicles, enriched in the Ca2+ channel protein, were isolated by
sucrose gradient centrifugation. Crude SR(~l-2 g) was suspended in 2 ml of Buffer III
(10% sucrose, 0.4 M KCl, 5 mM Tris-maleate, pH 7.0) and loaded on discontinuous
sucrose gradients (22%, 36%, 45%). The gradients were centriﬁrged for 5 h at 100,000 x
gm“ in a Dupont-Sorvall AH 629 rotor. The HSR was collected at interface between the
45% and 36% sucrose layers. HSR fractions were diluted at least 1:1 with 10% sucrose
and centriﬁrged at 150,000 x g...“ in the Beckman Ti-70 rotor for 40 min. The pellet was

suspended and homogenized in 10% sucrose, ﬂash-frozen and stored at -80°C.

52

During the HSR puriﬁcation, the following protease inhibitors were added at each
step, PMSF at 0.2 mM, and aprotinin, benzamidine, pepstatin and leupeptin at 1 ugml, to
Buffer III, 10% sucrose and sucrose gradients.

3. Ryanodine Binding Assays

Fifty pg of HSR protein were incubated in buffer containing 10 mM MOPS (pH
7.2), a CaClz-EGTA-nitrilotriacetic acid buffer to give a speciﬁc [Ca2+]ﬁee of 10 M, 0.1
M NaCl, 0.5 M sucrose, [3H]ryanodine (2-70 nM as ﬁnal concentration) was added to
each tube. Triplicate samples of each [3H]ryanodine concentration were incubated for 6 h
at 37°C, and ﬁltered by Whatman GF/B ﬁlters. Filters were washed three times with 5 ml
ice-cold buffer (10 mM MOPS, 0.1 M NaCl, 50 pM CaClz, pH 7.0), and the amount of
[3H]ryanodine retained on the ﬁlter was determined by scintillation counting. Non-
speciﬁc binding of ryanodine was determined by adding unlabeled ryanodine at 1000
times the [3H]ryanodine in the incubation buffer. Speciﬁc ryanodine binding was
determined by subtracting the non-speciﬁc binding fi'om the total binding.

The Kd (Wanodine binding afﬁnity) and Bmax (maximum binding capacity) values
for ryanodine binding by the HSR or CSR were calculated by Scatchard analysis using
Sigma Plot.

4. PN200-110 Binding Assays

The PN200-110 binding assays are based on those of Mickelson et al. (1994) with
minor modiﬁcations. Crude SR membranes (30 pg) were incubated in media containing
50 mM Tris-HCI (pH 7.4), 0.1 M NaCl, 0.5 M sucrose, and various concentrations (0.1 -
4 nM) of [3H]PN200-110. After 45 min incubation of duplicate samples at room

temperature (25°C), samples were ﬁltered by Whatman GF/B ﬁlters. Filters were washed

53

ﬁve times with 5 ml ice-cold buffer (200 mM Choline-Cl, 20 mM Tris-HCl), and the
amount of [3H]PN200-110 retained on the ﬁlter was determined by scintillation counting.
Nonspeciﬁc binding was determined by adding 1 11M unlabeled nifedipine in the
incubation buffer. Speciﬁc binding was determined by subtracting the non-speciﬁc
binding from the total binding. All the procedures were conducted under low lighting
condition to minimize photodegradation ofPN200-110.

Bmx (maximum binding capacity) value for [3H]PN200-110 binding by the crude
SR was calculated by Scatchard analysis using Sigma Plot.
5. Protein Assay

CSR and HSR protein concentration was determined by the method Of Lowry
(1951). Bovine serum albumin was used as a standard.
6. Statistical Analysis

Student’s t-test was used to compare the Kd, Bmax values for the crude and heavy

SR, and ratio of DHPR/RyR, between random-bred and commercial turkeys.

54

III. Results and Discussion
A. Ryanodine-dependence of [3H]ryanodine binding to Can-release channel protein
in heavy SR from random-bred and commercial turkey populations

The [3H]ryanodine binding experimental conditions optimized in the previous
study for the turkey skeletal muscle SR vesicles was chosen to analyze SR from random-
bred and commercial turkeys for ﬁanctional differences in RyR’s. Since ryanodine only
binds to the Open state of the channel proteins with high afﬁnity, the afﬁnity of RyR for
ryanodine is a useful probe of the functional difference in channel protein activity. If a
mutation exists in the RyR isoform(s), this may be manifested as a difference in
[3H]ryanodine binding afﬁnity as compared with a control sample.

[3H]Ryanodine binding activities of commercial and random-bred turkey skeletal
muscle heavy SR vesicles were measured over a range of ryanodine concentrations (2-70
nM). A total of eight random-bred turkey samples were compared with seventeen
commercial samples Obtained in 1998 (group I) and four commercial samples Obtained in
2000 (group 11). Representative binding data from one individual turkey from group I and
random-bred turkeys are shown in Figure 3.1. The equilibrium binding constants (K, and
BM) of [3H]ryanodine binding of heavy SR and crude SR for each individual random-
bred and commercial turkey sample are shown in Table 3.1 and Table 3.2, respectively.
The average values for each group are summarized in Table 3.3.

Commercial turkeys from group I exhibited a higher (p < 0.01) affinity for

ryanodine (Kd = 8.4 ir. 1.7 nM, n = 17) compared to that from genetically unimproved

turkeys (K, = 16.0 i 2.2 nM, n = 8). The mean Ca2+ channel protein content (Bmx) ﬁ'om

55

Figure 3.1 Ryanodine dependence of ryanodine binding to random-bred“)
and commercial (0) representative turkey skeletal muscle heavy sarcoplasmic
reticulum vesicles (HSR) at 10 uM Ca2+. A) Speciﬁc ryanodine binding by
random-bred and commercial turkey HSR. B) Scatchard plot of ryanodine
binding by random-bred and commercial turkey HSR.

56

 

12

ﬂ3~

Bound Ryanodine (pmol/mg HSR)
Oi

 

 

 

 

 

 

 

 

 

O T I I if I I
0 10 20 30 40 50 60 70
Free Ryanodine (nM)
10
013-
: "
B
E 0.6 ~
0
E
3 x
a
g 0.4 -
:r
= "A.
c .
d:
01!— x
A.
0.0 r I I 1 r l 7 1
0 2 4 6 8 10 12 14

Bound Ryanodine (pmol/mg HSR)

57

Table 3.1 Equilibrium Constants of Individual [3H]Ryanodine Binding of
Heavy SR from Random-bred and Commercial Turkeys (group I and II)

 

Random-bred Group Commercial Group (I) Commercial Group (II)

 

 

 

Turkey 1]) Bug! Kd Turkey ID BE K4 Turkey ID in; K‘,
A 15.7 16.1 209 5.7 5.5 4a 9.2 20.0
B 17.1 14.5 303 5.1 8.0 53 3.0 16.9
C 8.0 13.5 527 3.6 8.3 63 1.1 20.2
D 13.7 16.4 663 5.0 7.9 8a 4.4 19.3
E 12.1 15.6 11 2.1 10.0
F 10.7 15.0 36 7.1 7.8
G 8.4 15.6 117 5.4 9.1
H 14.7 20.9 435 7.6 7.1

451 5.1 7.4
459 9.1 6.7
468 12.5 9.2
567 4.2 6.0
283 5.3 8.9
75 8.3 9.2
323 5.4 12.8
290 2.8 9.3
664 6.4 9.7

n 8 8 17 17 4 4
AVG 12.5 16.0 5.9 8.4 4.4 19.1
SD 3.4 2.2 2.5 1.7 3.4 1.5

 

58

Table 3.2 Equilibrium Constants of Individual [3HlRyanodine Binding of

Crude SR from Random-bred and Commercial Turkeys (group II)

 

AVG
SD

Random-bred Group

 

 

 

Turkey 1]) BE‘ K
A 3.1 24.4
D 2.1 20.1
E 1.2 17.3
F 1.7 24.2
H 1.1 22.3
5 5
1.9 21.7
0.8 3.0

Commercial Group (II)
Turkey ID BIL“ Kd
1a 1.4 22.7
lb 2.4 26.6
2b 1.2 22.5
3b 2.8 27.5
4b 2.0 23.0
5 5
1.9 24.5
0.7 2.4

 

59

Table 3.3 Equilibrium Constant Means of [3H]Ryanodine Binding of Crude
and Heavy SR from Random-bred and Commercial Turkeys (group I, II)

 

 

 

 

 

 

K4011“) Bmax (Pmeg)

Random-bred

Heavy SR (n=8) 16.0 e 2.2‘ 12.5 i 3.4e

Crude SR (n=5) 21.7 i 3.0b 1.9 :t 0.8f
Commercial (goup I)

Heavy SR (n=17) 8.4 d: 1.7c 5.9 i 2.5g
Commercial (group II)

Heavy SR (n=4) 19.1 i 1.5d 4.4 e 3.4g

Crude SR (n=5) 24.4 i 2.4b 1.9 a: 0.7f
a,b,c,d

Different letters within a column indicate signiﬁcant differences (p < 0.05)
“’8 Different letters within a column indicate signiﬁcant differences (p < 0.05)

60

genetically unimproved turkeys (Bum = 12.5 i 3.4 pmol/mg) is higher (p < 0.01) than that
from group I commercial turkeys (Bmax = 5.9 i 2.9 pmol/mg).

The K; value obtained from the group 11 commercial turkey heavy SR vesicles
was 19.1 i 1.5 nM, which was signiﬁcantly higher than that from both random-bred
turkeys (16.0 i 2.2 nM) (0.01 < p < 0.05) and group I commercial turkeys (8.4 i 1.7 nM)
(p < 0.01). The total binding capacity (Bmax) from group 11 commercial turkey heavy SR
was not signiﬁcantly different compared to group I (4.4 i 3.4 vs 5.9 i 2.5 pmol/mg). The
Bmax from both commercial groups were signiﬁcantly lower than that ﬁ'om random-bred
turkeys (12.5 i 3.4 pmol/mg) (p < 0.01).

The data of the random-bred turkeys and commercial group I samples compare
favorably with the population averages obtained by Wang et al. (1999). They showed that
the heavy SR fractions from commercial turkeys exhibited a higher (P < 0.05) mean
afﬁnity for ryanodine when compared to that from unimproved turkeys (K; = 12.2 vs
20.5 nM, respectively). A fourfold higher Ca2+-channel protein content (BM. = 1.10 vs
4.01 pmol/mg) in heavy SR fractions from random-bred turkeys compared to that from
commercial turkeys was also observed. Differences in the Bmax are probably the result of
changes in buffer conditions which improve quantitation of Bmax as well as improvement
in the preparation of HSR.

These results also extend those of Wang et al. (1999) by showing that individual
binding characteristics can be determined for each individual turkey. Moreover, we have
shown for the ﬁrst time that there is variation in ryanodine binding within a commercial

line which may be the result of genetic differences in ryanodine receptors.

61

High ryanodine affinity is correlated with increased open state probability of
channels. This suggests that there could be more Ca2+ released from the sarcoplasmic
reticulum to the sarcoplasm during postmortem conversion of muscle to meat in
commercial turkeys with high ryanodine binding activity. This could cause accelerated
glycolysis and rapid pH drop, resulting in protein denaturation, decreased water holding
capacity and protein solubility, as observed in meat from stress-susceptible pigs.

Studies on the pork PSE problem showed that isolated SR vesicles from skeletal
muscle of MHS pigs release more Ca2+ than vesicles from normal pigs under various
physiological and pharmacological conditions (Nelson, 1983; Kim et al., 1984;
Mickelson et al., 1986). Vesicles ﬁ'om stress-susceptible pigs bind ryanodine with a 3-
fold greater affinity (K. = 92 vs 265 nM) than vesicles from normal pigs indicating a
greater open-state probability of MHS Rle’s (Mickelson et al., 1988). Moreover, in
single channel experiments using planar lipid bilayers, the mean percent open times P0
for MHS channels (30.0 i 3.5%) was signiﬁcantly greater (p < 0.0001) than the mean Po
ﬁom normal channels (4.4 i 0.6%), so the functional alteration of Rle is consistent
with increased Ca2+ release in stress-susceptible pigs that yield PSE pork (Shomer et al.,
1995). Therefore, our results showing differences in ryanodine binding in turkeys
selected for rapid growth are consistent with those obtained from pigs.

There is only one ryanodine receptor isoform expressed in mammalian Skeletal
muscle (Rle), whereas there are two in the avian skeletal muscle, on and B. Both
isoforms are expressed in equal abundance in avian skeletal muscle (Lai, et al., 1992;
Olivares, et al. 1991). The [3H]ryanodine binding afﬁnity differences observed in this

study suggests that there are functional differences in one or both isoforms of Ca”-

62

release channel protein in these turkeys. The differences in channel content in heavy SR
from the two turkey lines further suggest functional differences in Ca2+ regulation in the
muscle cell.

The difference in BM value between the commercial and random-bred control
group HSR could have resulted from the artifactual differences in distributions of the
RyR in the HSR membrane during isolation of SR. To address this question,
[3H]ryanodine binding experiments on the total SR vesicle fraction from both groups
were conducted and compared.

The [3H]ryanodine binding to the crude SR vesicles yielded a K. of 21.7 i 3.0 nM
and a Bmax of 1.9 i 0.8 pmol/mg from random-bred turkeys (Table 3.3). The results were
not different from that of group II commercial turkeys (K; = 24.5 i 2.4 nM and a Bmx =
1.9 i 0.7 pmol/mg). These results contrast with those observed by Wang et al. (1999), in
which the Kd and Bmax obtained from commercial turkey CSRs were only half of that
obtained from random-bred turkeys (p < 0.05).

Thus, both the HSR and CSR [3H]ryanodine binding data suggest that in the
commercial lines of turkeys, there may be different subpopulations. The group I and the
commercial group that Wang et al. (1999) used could be from one subpopulation that
carries altered-functional RyR, while group II turkeys, which exhibits similar RyR
function as that from random-bred control population, could be ﬁ'om another
subpopulation.

SR vesicles isolated from heterozygous pigs exhibit Ca2+ release channel
properties that are intermediate between those of SR isolated from homozygous MH and

homozygous normal pigs (Hawkes et al., 1992; Mickelson et al., 1989). Mickelson et al.

63

(1994) showed that the total [3H]ryanodine binding capacity of muscle homogenates from
heterozygous pigs was not Signiﬁcantly different from binding to homogenates of either
type of homozygote, although it was numerically intermediate. Shomer et al. (1995) also
indicated that the activities of the heterozygous Ca2+ release channels are intermediate
even at the single channel level. They concluded that the skeletal muscle SR Ca2+ release
channels of heterozygous pigs are composed of heterotetramers with unique properties.
Likewise, if the group H commercial birds were from a subpopulation with unique RyR
properties different from either group 1 commercial birds or random-bred birds, then no
difference for Bmam or K, between random-bred and commercial group H turkeys might
be detected.
B. DHPR and RyR content in random-bred and commercial turkeys

In mammalian skeletal muscle, a mechanical coupling between Rle and the
DHPR is proposed to trigger Cay-release from the SR (Meissner and Lu, 1995). Avian
muscle, however, which consists of two RyR isoforms, is believed to have the DHPR
coupled only to the a-RyR isoforms. No studies have been reported on the stoichiometry
of DHPR2RyR in avian muscle. Moreover, it is possible that difference in the coupling
ratio of DRI-IP to RyR could account for differences in regulation of Ca2+ homeostasis in
the commercial turkeys. Therefore, the stoichiometric relationship of these two receptors
were determined by using [3H]ryanodine binding and [3H]PN200-110 binding assays.

Crude SR fractions from both commercial and random-bred turkey groups were
prepared and analyzed for [3H1ryanedine binding and [3H]PN200-110 binding activities.

Results are shown in Table 3.4. Group II commercial turkeys exhibited almost identical

64

Table 3.4 Ratio of Dihydropyridine and Ryanodine Receptors Between
Random-bred and Commercial Turkey Skeletal Muscle Crude SR

 

Vesicles
PN200-110 Ryanodine Ratio
BM BM DHPR/RyR
(pmol/mg) (pmollmg)
Random-bred 1.98 2+: 0.69 1.85 :l: 0.81 1.18 :t 0.49
Commercial 1.66 d: 0.39 1.94 :i: 0.67 0.91 d: 0.30

 

There was no signiﬁcant difference
n=5

65

total binding capacity (Bmax = 1.7 i 0.4 vs 1.9 i 0.7 pmol/mg ) for [3I-I]PN200-110 and
for [3H]ryanodine, as the random-bred turkeys (Bmax = 2.0 i 0.7 vs. 1.9 i 0.8 pmol/mg).
The ratio of [3H]PN200-110 binding to [3H]ryanodine binding was ~1 and there was no
Signiﬁcant difference between the two groups.

Franzini-Armstrong and Kish (1995) indicated that since a tetrad is composed of
four DHPRS and there are two RyRs for every tetrad, the alternate RyR association
pattern would predict a 2:1 ratio of DHPR to RyR. However, the measured ratios from
different skeletal muscles and different species range from 0.5-2.0 (Anderson et al., 1994;
Bers and Stiffel, 1993; Block et al., 1988; Margreth et al., 1993; Mickelson et al., 1994).
Mickelson et al. (1994) observed that the ratio of DHPRzRyR was ~1 for muscle
homogenates of all three pig genotypes (MHS, heterozygote and normal). The DHPR
content and RyR content of MHS pig muscle homogenates were reduced to 80% and
81%, respectively, of that of normal muscle. Therefore, in MHS muscle, even though
there was a Slightly reduced content of both the triadic junctional proteins (DHPR and
RyR), the stoichiometric relationship between these proteins is not altered in MH vs
normal muscle (Mickelson et al., 1994). They also suggested that the reduced DHPR and
RyR content in MH muscle could reﬂect lower junctional membrane protein content
which may have functional consequence for Ca2+ homeostasis. It should be noted that our
results, which show no difference in DHPR or RyR levels were based on samples only
from group 11, these samples showed no difference in ryanodine binding activity from the
random-bred turkeys and thus, may be expected to Show no difference in expression

levels of these proteins.

66

Anderson et al. (1994) compared the DHPR/RyR ratio in frog and rabbit skeletal
muscle. The value of 0.61 for the frog as compared with rabbit ratio of 0.98 indicates,
according to the authors, that one-half as many RyRs in the frog are coupled to DHPRS.
They explain this difference by the presence of two RyR isoforms in amphibian muscle,
which are expressed in approximately equal amount (Lai, et al., 1992; Olivares, et
31,1991), and of which possibly only one is linked to the DHPR. In our study, which is
the ﬁrst to examine avian muscle, a ratio of 1.0 was obtained which is comparable to that
of Margreth et al. (1993) using frog sartorius muscle. This would seem to predict an
excess of ryanodine receptors. However, it is possible that there are non-junctional RyRs

which contribute to the overall binding (Sutko and Airey, 1996).

67

IV. Conclusion

Using the ryanodine binding conditions optimized for turkey skeletal muscle, two
distinct subpopulations of a commercial turkey line were observed. Both subpopulations
differed in [3H]ryanodine binding activity from that of the random-bred control turkey
population. The HSR from one commercial subpopulation exhibited a higher ryanodine
binding affinity (K; = 8.4 vs 16.0 nM) and lower Ca2+-release channel content (BM = 5.9
vs 12.5 pmol/mg) than random-bred control population. This suggests that the RyR
function in this subpopulation of commercial birds is altered, and these differences in
RyR activity could predispose these turkeys to a higher incidence of PSE meat.

The other subpopulation of commercial turkeys yielded similar ryanodine binding
affinity of crude SR vesicles and of heavy SR as that of the random-bred control (24.5 vs
21.7 nM for CSR, p > 0.05, 19.1 vs 16.0 nM for HSR, 0.01 < p <0.05, respectively). The
contents of the RyR in crude SR preparations from these two populations were also
similar (p > 0.05). The results suggest that this subpopulation might be less likely to
exhibit PSE meat than turkeys from group 1.

Direct interaction between DHPR and RyR is essential for skeletal muscle E-C
coupling. RyR regulation by the DHPR is also important for the Ca2+ release and
subsequent muscle contraction and glycolysis. The coupling ratio of DHPR:RyR in this
study was 1.0 and no DHPRzRyR ratio between commercial turkeys and random-bred
control turkeys (0.91 vs 1.18) was observed. The reasons could be: 1) ﬁinction of RyR
from the commercial turkey subpopulation used for this study is similar to that from

random-bred control turkeys and therefore, less likely to be predisposed to yielding PSE

68

meat. 2) DHPR/RyR ratio might remain constant even if a mutation is present in one of

the RyR isoforms as was observed in pig muscles.

69

CONCLUSIONS

In skeletal muscle, excitation-contraction coupling is essential for the Ca2+ release
and muscle contraction. Protein-protein interaction between two receptors,
dihydropyridine receptor (DHPR) and ryanodine receptor (RyR), plays an important role
in this process. But the speciﬁc mechanisms involved in the regulation of Ca2+ release
and the relationship between abnormal Ca2+ regulation and meat quality problems are still
not fully understood.

The PSE (pale, soﬁ, exudative) meat problem, which was ﬁrst observed in pork
industry, has now become prevalent in the turkey industry. A Ca2+-release channel (RyR)
gene mutation detected by Fujii et al. (1991) is correlated directly with porcine stress
syndrome (PSS), which is one of the causes of the PSE pork. Based on the similarities
between turkey and pork PSE problems, we hypothesized that a mutation in one or both
of the turkey Cay-channel isoforms and/or differences in the regulation of Cay-release
channel isoforms contribute to the incidence of PSE turkey meat in modern commercial
turkeys.

In the ﬁrst part of this work, the [3H]ryanodine binding assay was optimized for
turkey skeletal muscle. By testing different combinations of salt, sucrose and AMP-PCP
concentrations, a buffer condition of 0.1 M NaCl, 0.5 M sucrose, with 10 M free Ca2+
and 10 mM MOPS was determined to be the best condition for the ryanodine binding
assay for the turkey skeletal muscle SR.

In the second part of this work, two groups of commercial turkeys were compared
with a random-bred control group. The Cay-release channel biochemical functions from

one subgroup of commercial turkeys was different from the random-bred control turkeys.

7O

The SR vesicles from seventeen commercial turkeys showed a two-fold increase in
ryanodine affinity while the total Ca2+ channel content were decreased by half compared
to those from eight random-bred control turkeys. The altered affinity of the Ca2+ channel
protein for ryanodine suggests that there is a channel ﬁmctional defect in this subgroup of
commercial turkeys. The channel content difference may suggest that there are some
factors that alter the gene expression or protein Stability. Another group of commercial
turkeys was found that yields similar Ca2+ channel properties to those of random-bred
controls. Four HSR and ﬁve CSR vesicles from this subpopulation showed similar
ryanodine binding affinity compared to control eight turkeys. These results suggest that
there are at least two subpopulations of commercial turkeys, one subpopulation that may
possess altered Ca2+ channel function, which is predisposed to yielding PSE meat, while
another subpopulation possesses the relatively normal Ca2+ channel ﬁinction, and thus is
less likely to yield PSE meat. The stoichiometry of DHPR:RyR was not different between
ﬁve commercial turkeys and eight control turkeys. This could mean that either the
subpopulation of commercial turkeys studied doesn’t have obvious functional defect of
Ca2+ channel, or there might not be a stoichiometric DHPR/RyR ratio difference between

random-bred and commercial turkey lines.

71

FUTURE RESEARCH

This study showed that the SR Cap-release channel in a subpopulation of
commercial turkeys exhibited a higher ryanodine binding affinity and lower Ca2+-release
channel content. Another subpopulation appeared to be similar to the random-bred
controls. This suggested that there is a Cay-release channel functional defect in the
former commercial turkey subpopulation. However, since there are two isoforms of Ca2+-
release channel in turkey skeletal muscle, the method used in this study cannot identify
directly which isoform(s) is Speciﬁcally responsible for this defect. In order to answer
this question, the two isoforms need to be puriﬁed from both random-bred and
commercial turkeys, and biochemical characteristics from both isoforms need to be
compared (eg. ryanodine binding data) between these two populations. If ryanodine
binding activity is different ﬁ'om puriﬁed Ca2+-release channel isoform(s) between
random-bred and commercial turkey population, then this would offer more direct
evidence that: 1) there is a biochemical defect in the commercial turkey subpopulation, 2)
which isoform has the mutation, 3) which isoform(s) has the direct interaction with
dihydropyridine receptor. If a biochemical defect is identiﬁed in speciﬁc isoform(s), then
cDNA sequencing of the isoforms could be done to identify the speciﬁc mutation.

This study focused on the biochemical changes in the commercial turkey SR
based on ryanodine binding properties. However, there are no meat quality results
comparing early postmortem pH, water holding capacity, color etc, for both random-bred
turkey and commercial turkey populations slaughtered under identical circumstances. The
hypothesis of this study was that the mutation of a Cay-release channel is more

frequently found in commercial turkey population. A higher incidence of PSE is believed

72

to exist in the commercial turkeys than in random-bred, genetically unimproved turkey
population. To prove this hypothesis and also to investigate the PSE meat incidence in
the commercial subpopulations, it is necessary to collect both ryanodine binding data and
turkey meat quality data ﬁ'om individual turkeys from both random-bred and commercial
turkey populations. The correlation of meat quality data with corresponding skeletal
muscle ryanodine binding activity data could provide more evidence for the relationship
between altered Cay-release channel function and PSE meat incidence in commercial

turkey populations.

73

REFERENCES

REFERENCES

Anderson, K., Cohn, AH. and Meissner, G. 1994. High-afﬁnity [3H]PN200-110 and
[3H]ryanodine binding to rabbit and frog Skeletal muscle. Am. J. Physiol. 266:
C462-C466.

Airey, J.A., Beck, C.F., Murakami, K., Tanksley, S.J., Deerinck, T.J., Ellisman, MH.,
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. 65:
14187-14194.

Airey, J.A., Grinsell, M.M., Jones, L.R., Sutko, J .L., Witcher, D. 1993a. Three ryanodine
receptor isoforms exist in avian striated muscle. Biochemistry 32: 5739-5745.

Airey, J.A., Baring, M.D., Beck, C.F., Chelliah, Y., Deerinck, R.J., Ellisman, M.H.,
Houenou, L.J., McKemy, D.D., Sutko, J .L., and Talvenheimo, J. 1993b. Failure to
make normal or ryanodine receptor is an early event associated with the crooked
neck dwarf (cn) mutation in chicken. Dev. Dyn. 197: 169-188.

Airey, J.A., Deerinck, T.J., Ellisman, MH., Houenou, L.J., Ivanenko, A., Kenyon, J.L.,
McKemy, DD, and Sutko, J .L. 1993c. Crooked neck dwarf (cn) mutant chicken
skeletal muscle cells in low-density primary cultures fail to express normal or
ryanodine receptor and exhibit a partial mutant phenotype. Dev. Dyn. 197: 189-
202.

Ashley, RH. 1989. Activation and conductance properties of ryanodine-sensitivity
calcium channels from brain microsomal membranes incorporated into lipid lipid
bilayers. J. Membr. Biol. 111: 179-189.

Bers, D.M., and Stiffel, V.M. 1988. Ratio of ryanodine to dihydropyridine receptors in
cardiac and skeletal muscle and implications for E-C coupling. Am. J. Physiol.
264: C1587-C1593.

Bianchi, GP. 1968. Pharmacological actions on excitation-contraction coupling in
striated muscle. Fed. Proc. 27: 126-131.

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.

Britt B.A., Kalow, W. 1970. Malignant hyperthermia: a statistical review. Can. Anaesth.
Soc. J. 17: 293-315.

74

Brownell, AK. 1988. Malignant hyperthermia: relationship to other diseases. Br. J.
Anaesthes. 60: 303-308.

Bull, R., Marengo J.J., Suarez-Isla, 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.

Bull, R., and Marengo U. 1993. Sarcoplasmic reticulum release channels ﬁom frog
skeletal muscle display two types of calcium dependence. FEBS Lett. 331: 223-
227.

Cadwell, J.J., and Caswell, AH. 1982. Identiﬁcation of a constituent of the junctional
feet linking terminal cistemae to transverse tubules in skeletal muscle. J. Cell
Biol. 93: 543-550.

Campbell, K.P., Knudson, C.M., Imagawa, T., Leung, A.T., Sutko, J.L., Kahl, S.D.,
Raab, CR, and Madson, L. 1987. Identiﬁcation and characterization of the high
affinity [3H]ryanodine receptor of junctional sarc0plasmic reticulum Ca2+ release
channel. J. Biol. Chem. 262: 6460-6463.

Cannell, M.B., Cheng, H., and Lederer. W.J. 1995. The control of calcium release in
heart muscle. Science 268: 1045-1049.

Cassens, R.G., Marple, D.N. and Eikelenboom, G. 1975. Animal physiology and meat
quality. Adv. Food Res. 12: 71-155.

Caswell, AH, and Brandt, N. 1989. Does muscle activation occur by direct mechanical
coupling of transverse tubules to sarcoplasmic reticulum? TIBS. 14: 161-165.

Catterall, WA. 1996. Molecular properties of sodium and calcium channels. J. Bioenerg.
Biomembr. 28: 219-230.

Cheah, K.S., Cheah, A.M., Crosland, A.E., Casey, J.C., Webb, A]. 1984. Relationship
between Ca2+ release, sarcoplasmic Ca2+, glycolysis and meat quality in
halothane-sensitive and halothane insensitive pigs. Meat Sci. 10:117-130.

Cheng, H. Lederer, M.R., Xiao, R.P., Gomez, A.M., Zhou, Y.Y., Ziman, B., Spurgeon,
H., Lakatta, E.G., Lederer, W.J. 1996. Excitation-contraction coupling in heart:
new insights from Ca2+ sparks. Cell Calcium 20: 129-140.

Chu, A., Diaz-Munoz, M., Hawkes, M.J., Brush, K., Hamilton S.L. 1990. Ryanodine as a
probe for the functional state of the skeletal muscle sarcoplasmic reticulum
calcium release channel. Mol. Pharmacol. 37: 735-741.

Chu, A., Fill, M., Stefanis, E., and Entman, ML. 1993. Cytoplasmic Ca2+ does not inhibit
the cardiac muscle sarcoplasmic reticulum ryanodine receptor Ca2+ channel,

75

although Cay-induced Ca2+ inactivation of Ca2+ release is observed in native
vesicles. J. Membr. Biol. 135: 49-59.

Coronado, R., Mom'ssette, J., Sukhareva, M., and Vaugham, D.M. 1994. Structure and
function of ryanodine receptors. Am. J. Physiol. 266: C1485-C1504.

Denborough, M.A., Deett, X., Anderson, RM. 1973. Central-core disease and malignant
hyperthermia. BMJ. 1: 272-273.

De Ward, M. and Campbell, KR 1995 Subunit regulation of the neuronal alpha 1A
Ca2+ channel expressed in Xenopus oocytes. J. Physiol. (London) 485: 619-634.

De Waard, M., Witcher, D.R., Pragnell, M., Liu, H., and Campbell, KR 1995. Properties
of the alpha l-beta anchoring site in voltage-dependent Ca2+ channels. J. Biol.
Chem. 270: 12056-12064.

Ebashi, S. 1991. Excitation-contraction coupling and the mechanism of muscle
contraction. Annu. Rev. Physiol. 53: 1-16.

Ellis, F .R. and Heffron, J.J.A. 1985. In Recent Advances in Anaesthesia and Analgesia
(Atkison, RS, and Adams, A.P., eds) No. 15, pp. 173-207, Churchill-

Livingstone, London

Endo, M. 1977. Calcium release from the sarcoplasmic reticulum. Physiol. Rev. 37: 71-
108.

Endo, M., Yagi, S., Ishizuka, T., Horiuti, K., Koga, Y., Amaha, K. 1983. Changes in the
Ca2+ release mechanism in sarcoplasmic reticulum from a patient with malignant
hyperthermia. Biomed. Res. 4: 83-92.

Eng. G.D., Epstein, B.S., Engel, W.K., McKay, D.W., and McKay, R. 1978. Malignant
hyperthermia and central core disease in a child with congenital dislocating hips.
Arch Neurol. (Chicago) 35: (4) 189-197

Fill, M., Coronado, R., Mickelson, J.R., Vilven, J., Ma, J.J., Jacobson, BA, and Louis,
CF. 1990. Abnormal ryanodine receptor channels in malignant hyperthermia.
Biophys. J. 57: 471-475.

Fabiato, A. 1983. Calcium-induced release of calcium from cardiac sarcoplasmic
reticulum. Am. J. Physiol. 245: C1-C14.

Fleischer, S., and Inui, M. 1989. Biochemistry and biophysics of excitation-contraction
coupling. Annu. Rev. Biophys. Biophys. Chem. 18: 333-364.

Foegeding, EA. 1992. Factors affecting texture and yield of cooked turkey. Zootechnica
Int]. 2: 24-27.

76

Fosset, M., Jaimovich, E., Delpont, E., and Lazdunski M., 1983. [3H]Nitrendipine
receptors in skeletal muscle: properties and preferential location in transverse
tubules. J. Biol. Chem. 258: 6086-6092.

Frank, J.P., Harati, Y., Butler, I.J., Nelson, TE, and Scott, CL 1980. Central core
disease and malignant hyperthermia syndrome. Ann. Neurol. 7: 11-17.

Franzini-Armstrong, C.1980. Structure of sarcoplasmic reticulum. Fed. Proc. 39: 2403-
2409.

Franzini-Armstrong, C., and Jorgensen, A0. 1994. Structure and development of E-C
coupling units in skeletal muscle. Annu. Rev. Physiol. 56: 509-534.

Franzini-Armstrong, C., and Kish, J.W. 1995. Alternate disposition of tetrads in
peripheral couplings of skeletal muscle. J. Muscle Res. Cell Motil. 16: 319-324.

Franzini-Armstrong, C., and Protasi, F. 1997. The ryanodine receptor of striated muscles,
a complex capable of multiple interactions. Physiol. Rev. 77: 699-729.

Franzini-Armstrong, C. 1999. The sarcoplasmic reticulum and the control of muscle
contraction. FASEB J. 13 (Suppl): $266-$270.

Froning, G.W., Babji, A.S., Mather RB. 1978. The effect of pre-slaughter temperature,
stress, struggle, and anaesthetization on color and textural characteristics of turkey
muscle. Poultry Sci. 57: 630-633.

Fujii, J., Ostu, K., Zorato, F., Leon, S.D., Khama, V.K., Weiler, J.E., O’Brien, P.J., and
MacLennan, DH. 1991. Identiﬁcation of mutation in porcine ryanodine receptor
associated with malignant hyperthermia. Science. 253: 448-451.

Greaser, ML. 1986. Conversion of muscle to meat. In: P. Bechtel (Ed) Muscle as Food.
Pp 37-102. Academic Press, New York.

Gregg, R.G., Messing, A., and Strube, C. 1996. Absence of the beta subunit (cchbl) of
the skeletal muscle dihydropyridine receptor alters expression of the alpha 1
subunit and eliminates excitation-contraction coupling. Proc. Natl. Acad. Sci.
USA. 93: 13961-13966.

Gronert, GA. 1980. Malignant hyperthermia. Anesthesiology. 53: 395-423.

Gronert, GA 1986. In Myology (Engel, AG, and Banker, B.Q., eds) pp. 1763-1784,
McGraw-Hill Publications, Minneapolis, MN

Hall, G.M., Lucke, J.N., and Liser, D. 1980. Malignant hyperthermia: Pearls out of
swine. Brit. J. Anaesth. 52: 165-171.

77

Harrison, G.G. 1979. Porcine malignant hyperthermia. Intl. Anaesth. Clinics. 17: 25-61.

Hawkes, M.J., Nelson, T.E., Hamilton, S.L. 1992. [3H]ryanodine as a probe of changes in
the functional state of the Cay-release channel in malignant hyperthermia. J. Biol.
Chem. 267: 6702-6709.

Hymel, L., Inui, M., Fleischer, S., and Schindler, H. 1988. Puriﬁed ryanodine receptor of
skeletal muscle sarcoplasmic reticulum forms Cay-activated oligomeric Ca2+
channels in planar bilayers. Proc. Natl. Acad. Sci. USA 85: 441-445.

Ikemoto, N.A., Antoniu, B. and Meszaros, LG. 1985. Rapid ﬂow chemical quench
studies of calcium release from isolated sarcoplasmic reticulum. J. Biol. Chem.
260: 14096-14100.

Ivanenko, A., McKemy, D.D., Kenyon, J.L., Airey, J.A., and Sutko, J.L. 1995.
Embryonic chicken skeletal muscle cells fail to develop normal excitation-
contraction coupling in the absence of the alpha ryanodine receptor. Implications
for a two-ryanodine receptor system. J. Biol. Chem. 270: 4220-4223.

Jaggar, J.H., Porter, V.A., Lederer, W.J., and Nelson, MT, 2000. Calcium sparks in
smooth muscle. Am. J. Physiol. Cell. Physiol. 278: C235-C256.

Jeyakumar, L.H., Copello, J.A., O'Malley, A.M., Wu, G.M., Grassucci, R., Wagenknecht,
T., and Fleischer, S. 1998. Puriﬁcation and Characterization of Ryanodine
Receptor 3 from Mammalian Tissue J. Biol. Chem. 273: 16011-16020.

Kim, D.H., Sreter, F.A., Ohnishi, S.T., Ryan, J.F., Roberts, J., Allen, P.D., Meszaros,
L.G., Antoiu, B., and Ikemoto, N. 1984. Kinetic studies of Ca2+ release from
sarcoplasmic reticulum of normal and malignant hyperthermia susceptible pig
muscles. Biochim. Biophys. Acta 775: 320-327.

Kirino, Y., Osakabe, M., Shimizu, H. 1983. Ca2+-induced Ca2+ release from fragmented
sarcoplasmic reticulum: Cay-dependent passive Ca2+ efflux. J. Biochem. 94:
l 1 1 1-1 1 l8.

Klaus, M.M., Scordilis, S.P., Rapalus, J.M., Briggs, RT, and Powell, IA. 1983.
Evidence for dysfunction in the regulation of cytosolic Ca“ in excitation-
contraction uncoupled dysgenic muscle. Dev. Biol. 99: 152-165.

Klein, M.G., Cheng, H., Santana, L.F., Jiang, Y.H., Lederer, W.J., and Schneider, M.F.
1996. Two mechanisms of quantized calcium release in skeletal muscle. Nature
(London). 379: 455-458.

Lacerda, A.E., Kim, H.S., Ruth, P., Perez-Reyes, E., Flockerzi, V., Hofmann, F.
Birnbaumer, L., and Brown, AM. 1991. Normalization of current kinetics by

78

interaction between the alpha] and beta subunits of the skeletal dihydropyridine-
sensitive Ca2+ channel. Nature (London) 352: 527-530.

Laemmli, UK. 1970. Cleavage of the structural protein during the assembly of the head
of bacteriophage T4. Nature. 227: 680-685.

Lai, F.A., Erickson, H.P., Rousseau, E., Liu, Q.Y., and Meissner, G. 1988. Puriﬁcation
and reconstitution of the calcium release channel ﬁ'om skeletal muscle. Nature
(London) 331: 315-319.

Lai, F.A., Misra. M., Xu, L., Smith, HA, and Meissner, G. 1989. The ryanodine
receptor-Ca2+ release channel complex of skeletal muscle sarcoplasmic reticulum.
Evidence for a cooperatively coupled, negatively charged homotetramer. J. Biol.
Chem. 264: 16776-16785.

Lai, F.A., Liu, 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: C365-C372.

Leeson, TS, and Leeson, CR. 1970. Histology, 2nd Ed. Saunders, Philadelphia,
Pennsylvania.

Leong, P., and MacLennan, DH. 1998. Complex interactions between skeletal muscle
ryanodine receptor and dihydropyridine receptor proteins. Biochem. Cell Biol. 76:
681-694.

Liu, Q.Y., Lai, A., Rousseau, E., Jones, R.V., and Meissner, G., 1989. Multiple
conductance states of the puriﬁed calcium release channel complex from skeletal
sarcoplasmic reticulum. Biophys. J. 55: 415-425.

Lopez-Lopez, J.R., Shacklock, P.S., Balke, C.W., and Wier, W.G. 1995. Local calcium
transients triggered by single L-type calcium channel currents in cardiac cells.
Science 268: 1042-1045.

Louis, CF. 1993. Porcine stress syndrome: Biochemical and genetic basis of this
inherited syndrome of skeletal muscle. Reciprocal Meat Conference Proceeding.
46:89-96.

Lowry, O.H., Rosebrough, N.J., Farr, AL, and Randall, R]. 1951. Protein measurement
with the folin phenol reagent. J. Biol. Chem. 193: 65-275.

MacLennan D.H., Duff, C., Zorzato, F ., Fujii, J., Phillips, M., Komeluk, R.G., Frodis,

W., Britt, BA, and Worton, KG. 1990. Ryanodine receptor gene is a candidate
for predisposition to malignant hyperthermia. Nature. 343: 559-561.

79

Margreth, A., Damiani, E. and Tobaldin, G. 1993. Ratio of dihydropyridine to ryanodine
receptors in mammalian and frog twitch muscles in relation to the mechanical
hypothesis of excitation-contraction coupling. Biochem. Biophys. Res. Commun.
197: 1303-1311.

Martonosi, A.N. 1984. Mechanisms of Ca2+ release from sarcoplasmic reticulum of
skeletal muscle. Physiol. Rev. 64: 1204-1319.

Marty, 1., Robert, M., Villaz, M., De Jongh, K., Lai, Y., Catterall, W.A., and Ronjat, M.
1994. Biochemical evidence for a complex involving dihydropyridine receptor
and ryanodine receptor in triad junctions of skeletal muscle. Proc. Natl. Acad. Sci.
USA. 91: 2270-2274.

Meissner, G. 1975. Isolation and characterization of two types of sarcoplasmic reticulum
vesicles. Biochim. Biophys. 389: 51-68.

Meissner, G. 1984. Adenine nucleotide stimulation of Ca2+-induced Ca2+ release in
sarcoplasmic reticulum. J. Biol. Chem. 259: 2365-2371.

Meissner, G., Darling, E., and Eveleth, J. 1986a. Kinetics of rapid Ca2+ release by
sarcoplasmic reticulum. Effects of Ca”, Mg”, and adenine nucleotides.
Biochemistry 25: 236-244.

Meissner, G. 1986b. Ryanodine activation and inhibition of the Ca2+ release channel of
sarcoplasmic reticulum. J. Biol. Chem. 261: 6300-6306.

Meissner, G., and Henderson, J .S. 1987 . Rapid calcium release from cardiac sarcoplasmic
reticulum vesicles is dependent on Ca2+ and is modulated by Mg2+ adenine
nucleotide and calmodulin. J. Biol. Chem. 262: 3065-3073.

Meissner, G., and El-Hashem, A. 1992. Ryanodine as a functional probe of the skeletal
muscle sarcoplasmic reticulum Ca + release channel. Mol. Cell. Biochem. 114:
119-123.

Meissner, G. 1994. Ryanodine receptor/Ca2+ release channels and their regulation by
endogenous effectors. Annu. Rev. Physiol. 56: 485-508.

Meissner, G., and Lu, X. 1995. Dihydropyridine receptor-ryanodine receptor interactions
in skeletal muscle excitation-contraction coupling. Biosci. Rep. 15: 39-408.

Meissner, G., Rios, E, Tripathy, A., and Pasek, DA. 1997. Regulation of skeletal muscle
Ca2+ release channel (ryanodine receptor) by Ca2+ and monovalent cations and
anions. J. Biol. Chem. 272(3): 1628-1638.

Melzer, W., Rios, E., and Schneider, M.F. 1984. Time course of calcium release and
removal in skeletal muscle ﬁbers. Biophys. J. 45: 637-641.

80

Melzer, W., Rios, E., and Schneider, M.F. 1987. A general procedure for determining the
rate of calcium release ﬁ'om the sarcoplasmic reticulum in skeletal muscle ﬁbers.
Biophys. J. 51: 849-863.

Melzer, W., Herrmann-Frank, A., and Luttgau, HQ 1995. The role of Ca2+ ions in
excitation-contraction coupling of skeletal muscle ﬁbers. Biochim. Biophys. Acta.
1241: 59-116.

McCarthy, T.V., Healy, J .M.S., Heffron, J .J .A. Lehane, M., Deufel, T. Lehmannhom, F.,
Farrall, M., and Johnson, K. 1990. Localization of the malignant hyperthermia
susceptibility locus to human-chromosome 19Q12-13.2. Nature 343: 562-564.

McCartney, MG. 1964. A randombred control population of turkeys. Poultry Sci. 43:
739-744.

McGrew, S.G., Wolleben, C., Siegl, P., Inui, M., Fleischer, S. 1989. Positive
cooperativity of ryanodine binding to the calcium release channel of sarcoplasmic
reticulum from heart and skeletal muscle. Biochemistry 28: 1686-1691.

McKee, SR. and Sams, AR. 1997. The effect of seasonal heat stress on rigor
development and the incidence of pale, exudative turkey meat. Poultry Sci. 76:
1616-1620.

Michalak, M., Dupraz, P., and Shoshan-Barmatz, V. 1988. Ryanodine binding to
sarcoplasmic reticulum membranes: comparison between cardiac and skeletal
muscle. Biochim. Biophys. Acta 939: 587-594.

Mickelson, J.R., Ross, J.A., Reed, B.K., and Louis, CF. 1986. Enhanced Cay-induced
calcium release by isolated sarcoplasmic reticulum vesicles form malignant
hyperthermia susceptible pig muscle. Biochim. Biophys. Acta. 862: 318-328.

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,
BA, and Louis, CF. 1989. Effects of halothane-sensitivity gene on sarcoplasmic
reticulum function. Am. J. Physiol. 257: C787-C794.

Mickelson, J.R., Litterer, L.A., Jacobson, BA, and Louis, CF. 1990. Stimulation and
inhibition of [3H]ryanodine binding to sarcoplasmic reticulum ﬁom malignant
hyperthermia susceptible pigs. Arch. Biochem. Biophys. 278: 251-257.

Mickelson, J.R., Ervasti, J.M., Campbell KB, and Louis, CF. 1994. Skeletal muscle

junctional membrane protein content in pigs with different ryanodine receptor
genotypes. Am. J. Physiol. 267 (Cell Physiol. 36): C282-C292.

81

Mikami, A., Imoto, K., Tanabe, T., Niidome, T., Mori, Y., Takeshima, H., Narumiya, S.,
and Numa, S. 1989. Primary structure and functional expression of the cardiac
dihydropyridine-sensitive calcium channel. Nature (London) 340: 230-233.

Mills, J. 2000. The effect of selection for fast growth rate and high meat yield on skeletal
muscle in commercial turkeys. 23rd Technical Turkey Conference, UK.

Mitchell, G. and Heffron, J.J.A. 1982. Porcine stress syndromes. Adv. Food Res. 28: 167-
230.

Monnier, N., Procaccio, V., Stieglitz, P., and Lunardi, J. 1997. Malignant-hyperthermia
susceptibility is associated with a mutation of the alpha l-subunit of the human
dihydropyridine-sensitive L-type voltage-dependent calcium-channel receptor in
skeletal muscle. Am. J. Hum. Genet. 60: 1316-1325.

Moutin M.J., and Dupont, Y. 1988. Rapid ﬁltration of Ca2+-induced Ca2+ release from
skeletal sarcoplasmic reticulum. J. Biol. Chem. 263: 4228-4235.

Murayama, T., and Ogawa, Y. 1992. Puriﬁcation and characterization of two ryanodine-
binding protein isoforms from sarcoplasmic reticulum of bullfrog Skeletal muscle.
J. Biochem. 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.

Nakai, J., Dirksen, R.T., Nguyen, H.T., Pessah, I.N., Beam, K.G., and Allen, PD. 1996.
Enhanced dihydropyridine receptor channel activity in the presence of ryanodine
receptor. Nature (London) 380: 72-75.

Nelson, TE. 1983. Abnormality in calcium release from skeletal sarcoplasmic reticulum
of pigs susceptible to malignant hyperthermia. J. Clin. Invest. 72: 862-870.

O’Brien, PJ. 1987. Etiopathogenetic defect of malignant hyperthermia: hypersensitive
calcium-release channel of skeletal muscle sarcoplasmic reticulum. Vet. Res.
Commun. 11: 527-559.

O’Brien, P.J., Meissner, G., and Block, BA. 1993. The fastest contracting skeletal
muscle of non-mammalian vertebrates express only one isoform of the ryanodine
receptor. Biophys. J. 65: 2418-2427.

O’Brien, P.J., Valdivia, H.H., and Block, BA. 1995. Physiological difference between

the or and B ryanodine receptors of ﬁsh Skeletal muscle. Biophysical J. 68: 471-
482.

82

Ogawa, Y., and Haraﬁiji, H. Effect of temperature on [3H]ryanodine binding to
sarcoplasmic reticulum from bullfrog skeletal muscle. 1990. J. Biochem. 107:
887-893.

Ogawa, Y. 1994. Role of ryanodine receptor. Criti. Rev. Biochem. Molec. Biol. 29: 229-
274.

Ogawa, Y., Murayama, T., and Kurebayashi, N. 1999. Comparison of properties of Ca2+
release channels between rabbit and frog skeletal muscles. Mol. Cell. Biochem.
190: 191-201.

Ohnishi, ST. 1987. Effects of halothane, caffeine, dantrolene and tetracaine on the
calcium permeability of skeletal sarcoplasmic reticulum of malignant
hyperthermic pigs. Biochim. Biophys. Acta. 897: 261-268.

Olcese, R., Qin N., Schneider, T., Neely, A., Wei, X., Stefani, E., and Birnbaumer, L.
1994. The amino terminus of a calcium channel beta subunit sets rates of channel
interaction independently of the subunit effect on activation. Neuron, 13: 1433-
1438.

Olivares, E.B., Tanksley, S.J., Airey, J.A., Beck, C.F., Ouyang, Y., Deerinck, T.J.,
Ellisman, MH., and Sutko, J .L. 1991. Nonmammalian vertebrate skeletal muscles
express two triad junctional foot protein isoforms. Biophys. J ., 59: 1153-1163.

ording, H. 1985. Incidence of malignant hyperthermia in Denmark. Anesthes Analges.
64: 700-704.

ording, H. 1996. Investigation of malignant hyperthermia susceptibility in Denmark. Dan
Med Bull. 43: 111-125.

Orlova, E.V., Serysheva, I.I., Heel, M.V., Hamilton, S.L., and Chiu, W. 1996. Two
structural conﬁgurations of the skeletal muscle calcium release channel. Nature
Structural Biol. 3: 547-551.

Oyamada, H., Murayama, T., Takagi, T., Iino, M., Iwabe, N., Miyata, T., Ogawa, Y., and
Endo, M. 1994. Primary structure and distribution of ryanodine-binding protein
isoforms of the bullfrog skeletal muscle. J. Biol. Chem. 269: 17206-17214.

Palade, P. 1987. Drug-induced Ca2+ release ﬁom isolated sarcoplasmic reticulum; 11.
Release involving a Cay-induced Ca2+ release channel. J. Biol. Chem. 262: 6135-
6141.

Pessah, I.N., Waterhouse, AL, and Casida, J.E. 1985. The calcium ryanodine receptor

complex of skeletal and cardiac muscle. Biochem. Biophys. Res. Commun. 128:
449-456.

83

Pessah, I.N., Stambuk, RA, and Casida, J.E. 1987. Cay-activated ryanodine binding:
mechanisms of sensitivity and intensity modulation by Mg2+, caffeine, and
adenine nucleotides. Mol. Pharmacol. 31: 232-238.

Pessah, IN, and Zimany, I. 1991. Characterization of multiple [3H]ryanodine binding
sites on the calcium release of sarcoplasmic reticulum from skeletal and cardiac
muscle: Evidence for a sequential mechanism in ryanodine action. Mol.
Pharmacol. 39: 679-689.

Pietrzak, M., Greaser, ML and Sosnicki, AA. 1997. Effect of rapid rigor mortis
processes on protein functionality in pectoralis major muscle of domestic turkeys.
J. Anim. Sci. 75: 2106-2116.

Powell, J.A. and Fambrough. OM. 1973. Electrical properties of normal and dysgenic
mouse skeletal muscle in culture. J. Cell Physiol. 82: 21-38.

Pragnell, M., De Waard, M., Mori, Y., Tanabe, T., Snutch, TB, and Campbell, KR
1994. Calcium channel beta-subunit binds to a conserved motif in the I-11
cytoplasmic linker of the alpha I-subunit. Nature (London). 368: 67-70.

Protasi, F., Sun, X.H., and Franzini-Armstrong, C. 1996. Formation and maturation of
calcium release apparatus in developing and adult avian myocardium. Dev. Biol.
173: 265-278.

Protasi, F ., Franzini-Armstrong, C., and F lucher, BE. 1997. Coordinated incorporation of
skeletal muscle dihydropyridine receptors and ryanodine receptors in peripheral
couplings of BCng cells. J. Cell Biol. 137: 859-870.

Protasi, F., Franzini-Armstrong, C., and Allen, PD. 1998. Restoration of tetrads by
transfection of dyspedic cells with RyR-l. Biophys. J. 74: A141.

Rios, E., and Brum, G. 1987. Involvement of dihydropyridine receptors in excitation-
contraction coupling in skeletal muscle. Nature (London). 325: 717-720.

Rios, E., and Pizarro, G. 1991. Voltage sensor of excitation-contraction coupling in
skeletal muscle. Physiol. Rev. 71: 849-908.

Rogers, E.F., Koniuszy, F.R., Shave], J., Fr., and Folker, K. 1948. Plant insecticides. I.
Ryanodine, a new alkaloid from Ryania speciosa Vahl. J. Am. Chem. Soc. 70:
3086-3088.

Rousseau E, Ladine, J., Liu, Q.Y., Meissner, G. 1988. Activation of the Cay-release

channel of skeletal muscle sarcoplasmic reticulum by caffeine and related
compounds. Arch Biochem. Biophys. 267: 75-86.

84

 

Saito, A., Inui, M., Radermacher, M., Frank, J., and Fleischer, S. 1988. Ultrastructure of
the calcium release channel of sarcoplasmic reticulum. J. Cell Biol. 107: 211-219.

Serysheva, I.I., Schatz, M., van Heel, M., Chiu, W., and Hamilton, S.L. 1999. Structure
of the skeletal muscle calcium release channel activated with Ca2+ and AMP-PCP.

Biophys. J. 77: 1936-1944.

Schneider, M.F., and Simon, 3.]. 1988. Inactivation of calcium release ﬁom the
sarcoplasmic reticulum in frog skeletal muscle. J. Physiol. (London) 405: 727-
745.

Schwartz, L.M., McCleskey, E.W. and Almers, W. 1985. Dihydropyridine receptors in
muscle are voltage-dependent but most are not functional calcium channels,

Nature (London) 314: 747-751.

Shirokova, N., and Rios, E. 1997. Small event Ca2+ release: a probable precursor of Ca2+
sparks in frog skeletal muscle. J. Physiol. (London) 502: 3-11.

Shirokova, N., and Rios, E. 1998. Local calcium release in mammalian skeletal muscle.
Biophys. J. 74: A269.

Shomer, N.H., Mickelson, JR. and Louis, CF. 1995. Ca2+ release channels of pigs
heterozygous for malignant hyperthermia. Muscle and Nerve 18: 1167-1176.

Shoshan-Barmatz, V., and Ashley, RH. 1998. The structure, function, and cellular
regulation of ryanodine-sensitive Ca2+ release channels. Intl. Rev. Cytology 183:
185-270.

Smith, J.S., Coronado, R., and Meissner, G. 1985. Sarcoplasmic reticulum contains
adenine nucleotide activated 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 sarcoplasmic reticulum. Biophys. J.
50: 921-928.

Smith, J.S., Coronado, R., Meissner, G. 1986b. Single channel measurements of the
calcium release channel from skeletal muscle sarcoplasmic reticulum. Activation
by Ca2+ and ATP and modulation by Mg2+. J. Gen. Physiol. 88: 573-588.

Smith J.S., Imagawa, T., Ma, 1., Fill, M., Campbell, KR 1988. Puriﬁed ryanodine
receptor from rabbit skeletal muscle is the calcium-release channel of
sarc0plasmic reticulum. J. Gen. Physiol. 92: 1-26.

Soeller, C., and Cannell, MB. 1997. Numerical simulation of local calcium movements
during L-type calcium channel gating in the cardiac diad. Biophys. J. 73: 97-111.

85

Sorenson, M.M., Coelho, H.S.L., and Reuben, JP. 1986. Caffeine inhibition of calcium
accumulation by the sarcoplasmic reticulum in mammalian skinned ﬁbers. J.
Membr. Biol. 90: 219-230.

Sosnicki, A. A. and Wilson, B.W. 1991. Pathology of turkey skeletal muscle: Implications
for the poultry industry. Food Struct. 10: 317-326.

Sosnicki, A. A. and Wilson, B.W. 1992. Relationship of focal myopathy of turkey skeletal
muscle to meat quality. Proc. 19th World’s Poult. Congr. 3: 43-47.

Sosnicki, A.A. 1993a. Focal myonecrosis effects in turkey muscle tissue. Proc. 46‘“
Annu. Recip. Meat Conf. 46: 97-102.

Sosnicki, A.A. 1993b. Is the modern turkey prone to pale, soft, exudative (PSE) breast
muscle conditions? Meat Focus Int. 2: 75-78.

Sosnicki, AA. 1995. The domestic turkey: A model of the impact selection production
practices on meat quality. In: A. Ouali, D.I. Demeyer, and F.J.M. Smulders (Ed.)
Expression of tissue to proteinases and regulation of protein degradation as
related to meat quality. pp. 363-380. Audet Tijdschriften bv, Nijmegen, The
Netherlands.

Stephenson, E.W. 1981. Activation of fast skeletal muscle: contributions of studies on
skinned ﬁbers. Am. J. Physiol. 240: C1-C19.

Strasburg, G.M., and Byrem, TM. 2000. Red meats. In: Food Chemistry: Principles and
applications, edited by G. Christen and Smith, J .S.. ADNC Press, California.

Suda, N. 1995. Involvement of dihydropyridine receptor in terminating Ca2+ release in rat
skeletal myotubes. J. Physiol. (London), 486: 105-112.

Sun, X-H., Protasi, F., Takahashi, M., Takeshima, H., Gerﬁison, D.G., and Franzini-
Armstrong, C. 1995. Molecular architecture of membranes involved in excitation-
contraction coupling of cardiac muscle. J. Cell. Biol. 129: 659-671.

Sutko, J.L., and Airey, J.A. 1996. Ryanodine receptor Ca2+ release channels: does
diversity in form equal diversity in function? Physiol. Rev. 76: 1027-1071.

Takagi, A., Suohara, N., Ishizuka, T., Nonaka, I., and Sugita, H. 1983. Malignant
hyperthermia and related neuromuscular diseases: caffeine contracture of the
skinned muscle ﬁbers. Muscle Nerve 6: 510-514.

Takekura, H., Bennet. L, Tanabe, T., Beam, K.G., and Franzini-Armstrong, C. 1994.

Restoration of junctional tetrads in dysgenic myotubes by dihydropyridine
receptor cDNA. BiOphys. J. 67: 793-804.

86

Takeshima, H., Nishimura, S., Matsumoto, T., Ishida, H., Kangawa, K., Minamino, N.,
Matsuo, H., Ueda, M., Hanaoka, M., Hirose, T., and Numa, S. 1989. Nature 339:
439-445.

Takeshima, H. 1993. Primary structure and expression from cDNAs of the ryanodine
receptor. Ann. NY. Acad. Sci. 707: 165-177.

Takeshima, H., lino, M., Takekura, H., Nishi, M., Kuno, J., Minowa, O., Takano, H.
Noda, T. 1994. Excitation-contraction uncoupling and muscular degeneration in
mice lacking functional skeletal muscle ryanodine-receptor gene. Nature 369:
556-559.

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 (London). 346: 567-569.

Tu, Q., Velez, P., Brodwick, M., and Fill, M. 1994. Slowing of single cardiac muscle
ryanodine receptor channel gating by viscous solutions. Biophys. J. 66: A418.

Valdivia, H.H., Fuentes, O., and Block, BB. 1994. Imperatoxin A, a selective activator
of skeletal ryanodine receptors (RYR), distinguishes between or and B RYR
isoforms in non-mammalian muscle. Biophys. J. 66: A418.

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. Natl. Acad. Sci.
USA. 82: 6352-6356.

Volpe, P., Salviati, G., Di Virgilio, F., and Pozzan, T. 1985. Inositol 1,4,5-trisphospate
induces calcium release from sarcopasmic reticulum of skeletal muscle. Nature
316: 347-349.

Wagenknecht, T., Grassucci, R., Frank, J., Saito, A., Unui, M., and Fleischer, S. 1989.
Three-dimensional architecture of the calcium channel/foot structure of
sarcoplasmic reticulum. Nature 338: 167-170. -

Wagenknecht, T., and Radermacher, M. 1997. Ryanodine receptors: structure and
macromolecular interactions. Curr. Opin. Struct. Biol. 7: 258-265.

Wang, L., Byrem, T.M., Zarosley, J., Booren, AM. and Strasburg, GM. 1999. Skeletal
muscle calcium channel ryanodine binding activity in genetically unimproved and
commercial turkey populations. Poultry Sci. 78: 792-797.

Wei, X., Perez-Reyes, E., Lacerda, A.E., Schuster, G., Brown, AM, and Birnbaumer, L.
1991. Heterologous regulation of the cardiac Caz? channel alpha 1 subunit by

87

skeletal muscle beta and gamma subunits. Implications for the structure of cardiac
L-type Ca2+ channels. J. Biol. Chem. 266: 21943-21947.

Wei, X., Neely, A., Lacerda, A.E., Olcese, R., Stefani, E., Perez-Reyes, E., and
Birnbaumer, L. 1994. Modiﬁcation of Ca2+ channel activity by deletions as the
carboxyl terminus of the cardiac alpha 1 subunit. J. Biol. Chem. 269: 1635-1640.

Yano, M., El-Hayek, R., and Ikemoto, N. 1995. Conformational changes in the junctional
foot protein/Ca2+ release channel mediate depolarization-induced Ca + release
ﬁ'om sarcoplasmic reticulum. J. Biol. Chem. 270: 3017-3021.

Yamazawa, T., Takeshima, H., Sakurai, T., Endo, M., and Iino, M. 1996. Subtype
Speciﬁcity of the ryanodine receptor for Ca2+ signal ampliﬁcation in Excitation-
Contraction coupling. EMBO J. 15: 6172-6177.

Zarka, A., and Shoshan-Barmatz, V. 1993. Characterization ad photoafflnity labeling of
the ATP binding site of the ryanodine receptor from skeletal muscle. Eur. J.
Biochem. 214: 147-155.

Zhang, J .F., Ellinor, P.T., Aldrich, R.W., and Tsien, R.W. 1994. Molecular determinants
of voltage-dependent inactivation in calcium channels. Nature (London). 372: 97-
100.

Zhang, W., Kulers, D.L., and Rempel, W.E. 1992. Halothane gene and swine
performance. J. Amin. Sci. 70: 1307-1313.

Zarka, A., and Shoshan-Barmatz, V. 1993. Characterization and photoafﬁnity-labeling of
the ATP binding-site of the ryanodine receptor from skeletal muscle. Eur. J
Biochem. 213: 147-154.

Zot, AS, and Potter, JD. 1987. Structural aspects of troponin-tropomyosin regulation of
skeletal muscle contraction. Annu. Rev. Biophys. Biochem. 16: 535-559.

88

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

""111111111111"11111