From the Department of Medicine, Cardiovascular
Institute, Mount Sinai School of Medicine, New York, New York 10029 and
the ¶ Department of Molecular Pharmacology and Biological
Chemistry, Northwestern University Medical School,
Chicago, Illinois 60611
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ABSTRACT |
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Intracellular Ca2+
release in muscle is governed by functional communication between the
voltage-dependent L-type Ca2+ channel and the
intracellular Ca2+ release channel by processes that are
incompletely understood. We previously showed that sorcin binds to
cardiac Ca2+ release channel/ryanodine receptors and
decreases channel open probability in planar lipid bilayers. In
addition, we showed that sorcin antibody immunoprecipitates ryanodine
receptors from metabolically labeled cardiac myocytes along with a
second protein having a molecular weight similar to that of the
1 subunit of cardiac L-type Ca2+ channels.
We now demonstrate that sorcin biochemically associates with cardiac
and skeletal muscle L-type Ca2+ channels specifically
within the cytoplasmically oriented C-terminal region of the
1 subunits, providing evidence that the second protein
recovered by sorcin antibody from cardiac myocytes was the 240-kDa
L-type Ca2+ channel
1 subunit. Anti-sorcin
antibody immunoprecipitated full-length
1 subunits from
cardiac myocytes, C2C12 myotubes, and transfected non-muscle cells
expressing
1 subunits. In contrast, the anti-sorcin antibody did not immunoprecipitate C-terminal truncated forms of
1 subunits that were detected in myotubes. Recombinant
sorcin bound to cardiac and skeletal HIS6-tagged
1 C termini immobilized on Ni2+ resin.
Additionally, anti-sorcin antibody immunoprecipitated C-terminal
fragments of the cardiac
1 subunit exogenously expressed in mammalian cells. The results identified a putative sorcin binding domain within the C terminus of the
1 subunit. These
observations, along with the demonstration that sorcin accumulated
substantially during physiological maturation of the
excitation-contraction coupling apparatus in developing postnatal rat
heart and differentiating C2C12 muscle cells, suggest that sorcin
may mediate interchannel communication during
excitation-contraction coupling in heart and skeletal muscle.
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INTRODUCTION |
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The release of Ca2+ from muscle sarcoplasmic reticulum (SR)1 is the principal link between electrical excitation of the sarcolemma and mechanical activation of the myofilaments, a process known as excitation-contraction (E-C) coupling. Ca2+ release from stores in the cardiac SR occurs via Ca2+ release channels that are referred to as ryanodine receptors (RyRs). In the heart, Ca2+ release from the SR is largely triggered by Ca2+ influx at the plasma membrane via voltage-dependent L-type Ca2+ channels that are also dihydropyridine receptors (DHPRs) (1-7). In skeletal muscle, DHPRs serve as voltage sensors to detect depolarization of the sarcolemmal transverse tubules (T-tubules) and provide the physical impetus for opening of the SR Ca2+ release channels (1, 8-11). In both tissues, the geometry and close spatial relationship between sarcolemmal L-type channels and SR RyRs is critical in determining the time course of Ca2+ release and E-C coupling; however, the molecular mechanisms that mediate interchannel communication are incompletely understood (1, 12-14). The possibility that additional proteins or factors might be interposed to facilitate cross-talk has often been suggested (1, 8, 12).
Sorcin, a 22-kDa Ca2+-binding protein first identified in multidrug-resistant cells, is widely distributed among mammalian tissues, including heart and skeletal muscle (15-19). At the subcellular level, sorcin localizes to T-tubule junctions of cardiac SR (19) and co-localizes with brain RyR in rat brain caudate-putamen nucleus (20, 21). We previously demonstrated that introduction of sorcin into nonmuscle cells confers the property of caffeine-activated intracellular Ca2+ release, suggesting a role for sorcin in modulating RyR function (19). This hypothesis was further strengthened by the demonstration that sorcin completely inhibits ryanodine binding to cardiac RyRs and substantially decreases the open probability of the Ca2+ release channels reconstituted in lipid bilayers (22). Sorcin is, therefore, one of a group of modulators of RyR gating that includes calmodulin and FK506-binding protein, ligands that bind to a RyR domain that connects directly to a cytoplasmic extension of the transmembrane assembly of the receptor (23-27).
Anti-sorcin antibody immunoprecipitates two proteins from metabolically
labeled cardiac myocytes (19), one of which we previously identified as
the 565-kDa cardiac RyR (19), and the other (~220 kDa) displayed an
electrophoretic mobility similar to that of the major, pore-forming
1 subunit of the cardiac L-type Ca2+ channel
(
1C) (28, 29). Here we directly test the possibility that the unidentified protein is
1C by analyzing the
interaction of sorcin and the
1 subunits of cardiac and
skeletal muscle Ca2+ channels. The ability of sorcin to
interact with sarcolemmal L-type channels, in concert with its
modulatory effect on RyR gating, positions sorcin as a candidate
regulator of interchannel cross-talk.
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MATERIALS AND METHODS |
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Cell Lines and Cultured Cardiac Myocytes-- COS-1 cells were obtained from ATCC (Rockville, MD) and grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Culture conditions for human embryonic kidney (HEK) 293 and Sf9 insect cells have been described (29-31). Mouse C2C12 myoblasts (32-34) were maintained in Dulbecco's modified Eagle's medium containing 15% fetal bovine serum and were transferred to Dulbecco's modified Eagle's medium containing 2% horse serum to initiate differentiation, a process that serves as a model of normal myogenesis. Formation of multinucleated myotubes was observed within 48 h after medium transfer. Preparation of rat cardiac myocytes was carried out according to published procedures (19, 35).
Membrane Preparations--
Sf9 and HEK 293 cell membranes
were prepared as described previously (29, 30). Fractionation of rat
heart tissue for soluble and crude membrane components was carried out
by differential centrifugation according to our published procedures
(29). In brief, rat heart tissue was homogenized in buffer containing
0.25 M sucrose, 0.25 M KCl, 10 mM
imidazole (pH 7.4), 5 mM MgCl2, 10 mM EDTA, and protease inhibitors. After centrifugation at
5000 × g for 10 min, supernatants were removed and
frozen, and pellets were quickly washed with lysis buffer containing
0.6 M KCl to extract myosin, which obscures
1c on gels. Washed pellets were solubilized and analyzed
for
1c content, and supernatants were analyzed for
sorcin by Western blot as described below. In some experiments, heart
tissue was lysed by sonication in 50 mM Tris (pH 7.4)
containing 1% Nonidet P-40, 150 mM NaCl, 20 mM
NaF, 10 µg/ml aprotinin, and 2 mM phenylmethylsulfonyl
fluoride (buffer A).
Antibodies and Expression Vectors--
Preparation and
characterization of sorcin antibodies (19); of SKN, SKC, Card I, and
Card C antibodies; and of 1C and
1S expression vectors has been described previously (30, 36). Briefly, the
SKN and SKC antibodies recognize N-terminal and C-terminal domains on
the
1S subunit (36), while the Card I and Card C antibodies recognize internal and C-terminal domains of the
1C subunit (30). The RyR antibody was a generous gift of
Dr. Gerhard Meissner (University of North Carolina) (37). The
anti-myosin heavy chain (MHC) antibody F59 has been described (34).
Anti-polyhistidine was purchased from Sigma, and horseradish
peroxidase-conjugated secondary antibodies were obtained from Santa
Cruz Biotechnology, Inc. (Santa Cruz, CA) and used according to the
manufacturer's directions.
Western Blot Analysis and Immunoprecipitation--
Soluble
fractions of cultured cells and tissues, as well as solubilized
membrane fractions (described above) were prepared for analysis in
buffer A. Samples containing 60 µg of protein (38) were separated by
gel electrophoresis on 6% (for analysis of 1 subunits,
RyR, and MHC) or 11% (for sorcin and HIS6-tagged proteins)
polyacrylamide gels under denaturing conditions (39). Proteins were
transferred to nitrocellulose, and the membranes were processed for
Western blotting with detection by chemiluminescence (Amersham
Pharmacia Biotech) as described previously (19). Additional details are
given in the figure legends. For immunoprecipitation assays, aliquots
of protein samples in buffer A containing 100 or 200 µg of protein in
a total of 0.25 ml of buffer A were incubated with 1 µg of antibody
raised against peptides from either the N or C terminus of sorcin (19)
for 1 h at 4 °C. Antigen-antibody complexes were precipitated
with protein G (Sigma), washed with buffer A, and solubilized in
Laemmli (39) buffer at room temperature for 15 min. Immunoprecipitated
proteins were analyzed by Western blot as described above and in the
figure legends. Molecular weight markers were purchased from Amersham
Pharmacia Biotech or Bio-Rad.
In Vitro Binding Assays--
Fusion proteins of the L-type
Ca2+ channel 1C and
1S
C-terminal domains were expressed in bacteria with vectors constructed by ligation of a genomic BglII-BamHI fragment of
1C (GenBankTM accession number X15539) or
1S (GenBankTM accession number X05921) into
the BamHI site of pQE (QIAGEN, Valencia, CA). The proteins
were expressed with an N-terminal HIS6 tag fused to amino
acids 1622-2171 (40) of the
1C C terminus or to amino
acids 1497-1873 (40) of the
1S C terminus. For immobilization of fusion peptides on Probond Ni2+ resin
(Invitrogen Corp., San Diego, CA), bacterial lysates containing the
fusion proteins were prepared according to the manufacturer's directions by sonication and freeze-thaw in phosphate-buffered saline
(PBS) at pH 7.8 (binding buffer). Cleared supernatants were applied to
washed resin beds by batch absorption, and treated resin samples were
washed three times with binding buffer, twice with PBS at pH 6.0, and
three times with PBS at pH 7.4. Aliquots of 10 µg of recombinant
sorcin (41) in PBS (pH 7.4) were then combined with the
protein-containing resins for 30 min at 4 °C. Resins were washed
three times with PBS (pH 7.0), and bound proteins were eluted in 0.5 M imidazole. HIS6-tagged p27, a
cyclin-dependent kinase inhibitor (42), was used as a
negative control. Eluted proteins were analyzed by Western blot after
electrophoresis on 11% acrylamide. Nitrocellulose membranes containing
transferred proteins were sequentially probed with mouse monoclonal
anti-polyhistidine antibody and then with rabbit polyclonal antibody to
a peptide from the C terminus of sorcin (19).
Preparation and Transfection of HIS6-tagged
C-terminal Fragments of 1C--
A fragment (designated
fragment A) of the C-terminal cytoplasmic domain of
1C
(amino acids 1622-2171) (41) was removed from the
1C
expression vector (30) and cloned in frame into the pHISA expression
vector (Invitrogen), which added 30 amino acids, including the
His6 epitope, at the N terminus. Fragments B (amino acids
1622-1872), C (amino acids 1749-1978), D (amino acids 1622-1772),
and E (amino acids 1622-1748) from the
1C C terminus
were prepared by polymerase chain reaction and cloned into pHISA.
Polymerase chain reaction primers included restriction sites to
facilitate subsequent cloning. The following primer pairs were used:
fragment B, sense GGAAACCTGGAACAAGCCAAT (also used for fragments D and
E) and antisense GCTCTAGATCCTCACGTCGTAGTTGTC; fragment C, sense
GCGGATCCTCCCCCAGACCTTCACTACG and antisense GCTCTAGAGCGAGGTGGGGGAATGGCT; fragment D, antisense GCTCTAGACTAGGAGTCCACCAGCTTCTCGTG; and fragment E,
antisense GCTCTAGATCCTCACGTCGTAGTTGTC. Plasmids encoding the fragments were transiently co-transfected with the full-length sorcin
vector pFRCMVSOR (19) in COS-1 cells or alone in HEK cells with the use
of lipofectamine (Life Technologies, Inc.). Association of sorcin and
fragments was studied by immunoprecipitation with sorcin antibody and
Western blot analysis.
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RESULTS |
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Sorcin Antibody Immunoprecipitates 1C--
We
previously showed that sorcin antibody recovered two proteins from rat
cardiac myocytes metabolically labeled with
[35S]methionine, a 565-kDa protein shown to be the RyR
and an unidentified ~220-kDa protein (19). Here we demonstrate that a
single protein recognized by Card I, an
1C-specific
antibody shown to recognize the 240-kDa
1C (29, 30), was
immunoprecipitated from rat heart tissue by sorcin antibody (Fig.
1A, lane
1). Card C, an antibody directed against the C terminus of
1C (30), also recognized this protein (see Fig. 4).
Results of immunoprecipitations carried out in the absence of
Ca2+ (not shown) were indistinguishable from those shown in
Fig. 1A. Rat heart contained the 22-kDa sorcin, which
co-migrated with recombinant sorcin (41) (Fig. 1B,
lane 1), and an 18-kDa species (Fig.
1B, lane 1). Both sorcin forms were
detected in most rat (and mouse) heart samples; tissues from some
animals contained only the 22-kDa form (19).
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Sorcin Antibody Immunoprecipitates Full-length
1S--
We next addressed the question of whether the
1C/sorcin association was
1
isoform-specific. SKN, an
1S-specific antibody generated
against the N terminus of
1S and shown to recognize full-length (214-kDa) and C-terminal truncated (170-190-kDa) forms of
1S (31, 36, 44), recognized three proteins in C2C12 myotubes (Fig. 1C, lane 2). None of
these proteins were detected in undifferentiated C2C12 myoblasts (Fig.
1C, lane 1); up-regulation of the
1S subunit during skeletal muscle development has been established (45, 46). The anti-sorcin antibody immunoprecipitated only
the most slowly migrating form that was detected in myotubes, while
lower Mr forms that were reactive with SKN were
not recovered (Fig. 1C, lane 4). The
SKC antibody, directed against the
1S C terminus,
recognizes the full-length 214-kDa
1S subunit but does
not detect the C-terminal truncated forms of
1S that are commonly observed in skeletal muscle (44, 47). In the present experiments, SKC recognized only the full-length subunit in C2C12 myotubes (Fig. 1C, lane 6), a band of
similar size recovered by sorcin antibody (Fig. 1C,
lane 4). The Mr of
full-length
1S has been established by Ferguson plot
analysis to be 214 kDa, although under most conditions it migrates
anomalously as an ~190-kDa protein (44). From these data, we
concluded that the lower Mr bands in C2C12
myotubes that were immunoreactive with SKN, but not with SKC, were
C-terminal truncations of
1S and that the sorcin
antibody only immunoprecipitated full-length
1S. These
results suggested that an intact C terminus in
1S was
necessary for interaction with sorcin. As in the
1C
studies, we examined whether sorcin antibody would recover
1S heterologously expressed in Sf9 and HEK 293 cells. SKN (and SKC, not shown) recognized a single protein immunoprecipitated by sorcin antibody from
1S-expressing
Sf9 (Fig. 1C, lane 9) and HEK
293 (Fig. 1C, lane 11) cells. The
bands co-migrated with proteins recognized by SKN and SKC by direct Western blot (data not shown). No specific proteins identified by SKN
were recovered by sorcin antibody immunoprecipitation of uninfected
Sf9 (Fig. 1C, lane 7),
Sf9 infected with
1C (Fig. 1C,
lane 8), or untransfected HEK cells (Fig.
1C, lane 10). The results are
consistent with the interpretation that sorcin associates with the
1S and
1C subunits and that an intact C
terminus in each protein is necessary for this interaction.
Association of Sorcin with 1 C Terminus
Domains--
In order to test the hypothesis that sorcin interacts
with C-terminal domains of
1C and
1S,
HIS6-tagged C terminus fragments of
1C and
1S were immobilized on Ni2+ resin and
incubated with recombinant sorcin protein. As shown in Fig.
2, recombinant sorcin (22 kDa)
specifically bound to immobilized
1S (Fig. 2,
lane 4) and
1C (Fig. 2,
lane 6) C termini (57 and 70 kDa, respectively).
In contrast, no specific association of sorcin with the irrelevant
HIS6-tagged p27 protein was observed (Fig. 2,
lane 2).
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Increase in Sorcin Expression during Muscle Development--
To
further probe potential relationships between sorcin and L-type
Ca2+ channel 1 subunits, we examined the
developmental expression of sorcin during physiological maturation of
E-C coupling in developing muscle. It is well established that the
expression of sarcolemmal and SR channel proteins is regulated in
concordance with a program of muscle maturation (1, 45, 46, 48-52).
Sorcin abundance in postnatal rat heart substantially increased from
day 1 to day 12 after birth (Fig.
4A). Both the 22- and 18-kDa
species, depicted in Fig. 1B, increased in parallel. The
1C subunit was readily detectable as early as postnatal
day 1 and also increased in abundance during this time period (Fig.
4A), in agreement with previous reports (45).
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DISCUSSION |
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The voltage-sensing L-type Ca2+ channels in T-tubules play a major role in E-C coupling in both heart and skeletal muscle (1, 28). The close apposition of the L-type channels and RyRs in specific spatial relationships is required to accurately bridge the gap between SR and the sarcolemma for interchannel cross-talk (12-14). Indeed, a change in the relationship between SR Ca2+-release channels and sarcolemmal Ca2+ channels may be a defect in cardiac hypertrophy that contributes to contractile failure (53). Functional coupling between L-type Ca2+ channels and RyRs in neurons has also been demonstrated (54), and sensors for synaptic signaling just beneath sites of Ca2+ entry in the hippocampus have been proposed (55). In normal cardiac myocytes, the opening of voltage-activated L-type channels results in the development of a high local intracellular [Ca2+] in the microenvironment of the junctional space surrounding the sarcolemmal and SR membranes (56-59). How the local L-type channel-generated signals are transmitted to and activate RyRs is incompletely understood as is the potential retrograde signal (60) by which RyRs may affect L-type channel function. Proteins or factors interposed between the L-type Ca2+ channels and RyRs that might facilitate the relay of signals between them have been postulated (1, 8, 12). The results of the present study indicate that the Ca2+-binding protein, sorcin, associates with voltage-dependent Ca2+ channels. These new data, together with previous studies, which established that sorcin binds to and modulates RyR, suggest a role for sorcin in interchannel communication and E-C coupling.
Sorcin binds Ca2+ (Kd ~1 µM) and undergoes both a Ca2+-dependent decrease in intrinsic fluorescence and a Ca2+-mediated intracellular translocation from soluble to membrane components (41, 61). The protein inhibits binding of ryanodine to cardiac RyR and is a modulatory ligand for RyR gating (22). In contrast to sorcin's effect on cardiac RyR, sorcin increases binding of ryanodine to skeletal muscle RyRs, perhaps reflecting structural differences between the cardiac and skeletal muscle RyR isoforms (22).
Our finding that sorcin associates with the cytoplasmically oriented
C-terminal domains of L-type Ca2+ channel 1
subunits together with our previous demonstration that the addition of
sorcin to the cytoplasmic side of cardiac RyRs in planar bilayers
decreases the single channel open probability (22) places sorcin within
the sarcolemmal/SR junctional space, a strategic location for
facilitation of interchannel communication. The
1C
domain from amino acids 1622-1748 appeared to be sufficient for sorcin
binding in vitro; however, the sorcin/
1
interaction may be more complex in vivo. Shortened forms of
1 subunits, reportedly cleaved at C-terminal residues
distal to the delineated sorcin binding domain (43, 44), were not
immunoprecipitated by sorcin antibody. This suggests that an intact
C-terminal is required for sorcin interaction with the subunits
in vivo and that the distal C-terminal regions may
participate in stabilization of the sorcin/
1
interaction.
Sorcin modulates RyR gating in planar bilayers, but whether it affects
L-type Ca2+ channel function is not known. We have no
evidence that sorcin antibody recovered functional channels. The
cytoplasmically oriented C terminus of 1C has been shown
to be involved in Ca2+-sensitive channel inactivation, and
a 142-amino acid segment from amino acids 1572-1717 in the
1C C terminus has been suggested to be required for
inactivation (62). The putative sorcin-binding domain delineated in
this report is within the margins of the potential inactivation domain
and suggests that sorcin's involvement in this aspect of L-type
Ca2+ channel regulation should be investigated.
We found that sorcin expression increases in abundance in both developing heart and differentiating muscle cells. The contractile machinery in developing rodent heart is activated largely by Ca2+ entering the cell through L-type channels (1, 49). As the T-tubule/SR system gradually develops, contraction becomes more dependent on SR Ca2+ release activated by Ca2+ entry through sarcolemmal channels (49). The increase in sorcin expression in postnatal rat heart coincides with the developing Ca2+-induced Ca2+ release mechanism, consistent with a putative role for sorcin in L-type channel/RyR interchannel communication. The increase in sorcin expression during C2C12 myotube formation, commensurate with L-type Ca2+ channel and contractile apparatus development, is in accord with a role for sorcin in interchannel communication in skeletal muscle as well. A program of coordinate expression of myocyte proteins involved in contraction and Ca2+ regulation has been suggested (51). Treatment of heart cells with verapamil initiates divergent expression of proteins that may be involved in this program along with contractile arrest (51). Whether sorcin, whose expression was substantially reduced in verapamil-arrested myocytes, is a component of that Ca2+ regulation program remains to be determined.
Sorcin may undergo Ca2+-mediated dynamic changes in
structure and subcellular localization, allowing it to either
simultaneously or sequentially interact with L-type Ca2+
channels and RyR in response to changing Ca2+ levels.
[Ca2+] changes in the sarcolemmal/SR microenvironment may
result in conformational changes and translocation of sorcin from
cytoplasmic to membrane locations (41, 61) in a manner analogous to the Ca2+-mediated conformational modification of recoverin
(63). Ca2+-mediated dimerization of sorcin, shown to occur
in vitro (60), may also occur in vivo. In
addition, the recent demonstration of sorcin binding to annexin VII
(64), thought to play a role in E-C coupling in skeletal muscle (65),
and our demonstration that calmodulin exerts an additive effect on
sorcin's inhibition of ryanodine binding to cardiac RyR (22) suggests
that sorcin may function in conjunction with other channel accessory
proteins. Whether these proteins might include L-type Ca2+
channel subunits other than 1 is speculative; direct
interaction between sorcin and other channel subunits was not in
evidence.
Our studies demonstrating that sorcin interacts with both sarcolemmal L-type Ca2+ channels and SR Ca2+ release channels suggest a role for sorcin in interchannel communication. Sorcin may act as a sensor of the T-tubule junctional environment to ultimately participate in regulation of intracellular Ca2+ mobilization and E-C coupling.
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ACKNOWLEDGEMENT |
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We thank Dr. Richard Pestell of Albert Einstein College of Medicine for bacterial cultures expressing HIS6-tagged p27.
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FOOTNOTES |
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* This work was supported in part by a grant from the American Heart Association (to G. I. F.), by National Institutes of Health (NIH) Grant HL23306 (to M. M. H.), and by NIMH (NIH) National Research Service Award Grant 1 F30-MH10770 (to A. J. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Medicine, Cardiovascular Institute, Box 1030, Mount Sinai School of Medicine, One Gustave L. Levy Pl., New York, New York 10029-6574. Tel.: 212-241-5143; Fax: 212-860-7032; E-mail: marian_meyers{at}smtplink.mssm.edu.
Established Investigator of the American Heart
Association.
1 The abbreviations used are: SR, sarcoplasmic reticulum; DHPR, dihydropyridine receptor; E-C coupling, excitation-contraction coupling; HEK, human embryonic kidney; MHC, myosin heavy chain; RyR, ryanodine receptor; TBST, Tris-buffered saline with Tween 20; T-tubule, transverse tubule; PBS, phosphate-buffered saline.
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REFERENCES |
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