Skeletal Muscle Calcium Channel Ryanodine Binding Activity in Genetically Unimproved and Commercial Turkey Populations LI-JU WANG, TODD M. BYREM, JEFFERY ZAROSLEY, ALDEN M. BOOREN, and GALE M. STRASBURG1 Department of Food Science and Human Nutrition, Michigan State University, East Lansing, Michigan 48824 difference (P < 0.05) in mean Ca2+-channel protein content or Bmax (1.10 pmol/mg vs 4.01 pmol/mg) was observed between commercial and unimproved turkey SR fractions. The apparent difference in channel protein content between the two populations may be partially accounted for by the high abundance of a 75-kDa protein, as yet unidentified, observed in most commercial turkey samples on SDS polyacrylamide gels. The differences in ryanodine binding activity between these two populations of turkeys suggest that altered SR calcium channel protein activity, or altered channel regulation, may be associated with the increased incidence of PSE meat from turkeys selected for growth characteristics.
ABSTRACT The biochemical basis for the incidence of pale, soft, exudative (PSE) turkey meat was investigated by conducting ryanodine binding experiments on sarcoplasmic reticulum (SR) vesicles prepared from genetically unimproved and commercial turkeys. Ryanodine binding to the Ca2+ channel protein in SR vesicles from both populations of turkeys was activated at a threshold concentration of approximately 0.2 mM Ca2+, reached a plateau over the range of 3 to 30 mM free Ca2+, and was only slightly inhibited at 1 mM Ca2+. The SR fractions, enriched in the Ca2+-channel protein, from commercial turkeys exhibited a higher (P < 0.05) mean affinity for ryanodine when compared to that from unimproved turkeys (Kd = 12.2 vs 20.5 nM, respectively). A fourfold
(Key words: turkey, muscle, calcium, channel, pale, soft, and exudative meat) 1999 Poultry Science 78:792–797
The modern turkey industry has grown rapidly over the past 20 yr to meet consumer demand for lean, inexpensive, and convenient meat products. To meet this demand, the industry has intensely bred birds for efficient growth and heavy muscling. Over the past few years, the turkey processing industry has been experiencing severe meat quality problems that closely resemble the development of pale, soft, and exudative (PSE) pork, the incidence of which results, in part, from an inherited skeletal muscle disorder in swine. Meat from affected turkeys shows a very rapid pH decline, resulting in a product that is very exudative, and has softer texture, poorer bind, and higher cooking losses than meat from normal birds (Sosnicki, 1993; Pietrzak et al., 1997). The striking similarity of factors leading to development of PSE meat in turkey to that of swine suggests that there may be a genetic basis for this syndrome in turkeys (Pietrzak et al., 1997). Porcine stress syndrome (PSS) is an inherited skeletal muscle disorder associated with the genetic selection of
lean, heavily muscled, and fast-growing pigs (Zhang et al., 1992) and affects 10 to 20% of the swine population in the U.S. (Vansickle, 1989). This syndrome results in substantial economic losses to farmers, owing to the death of animals from stresses of transportation, heat, and crowding before reaching the market (Louis, 1993), and to the development of PSE pork directly attributable to PSS (Pommier and Houde, 1993). Biochemical studies demonstrated an abnormal Ca2+ release in skinned muscle fibers and purified skeletal muscle sarcoplasmic reticulum (SR) vesicles from PSS-susceptible swine (Endo et al., 1983; Mickelson et al., 1986, 1988). Calcium release in skeletal muscle is governed by the SR Ca2+ channel, frequently referred to as the ryanodine receptor because the plant alkaloid ryanodine binds to the channel with high affinity. Mickelson et al. (1988) found that purified SR vesicles from stress-susceptible pigs bind ryanodine with a threefold greater affinity (Kd = 92 vs 265 nM) than vesicles from normal pigs, which suggested that there were differences in structure and function of the Ca2+ channel. The ultimate cause of PSS in pigs has been identified as an Arg615 to Cys615
Received for publication August 1, 1998. Accepted for publication December 8, 1998. 1To whom correspondence should
[email protected]
Abbreviation Key: ATPase = adenosine triphosphatase; Bmax = binding capacity; CSR = crude total sarcoplasmic reticulum vesicles; H = heavy; Kd = dissociation constant; PSE = pale, soft, and exudative; PSS = porcine stress syndrome; SR = sarcoplasmic reticulum;
INTRODUCTION
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substitution in the skeletal muscle SR Ca2+-channel protein (Fujii et al., 1991). This mutation gives rise to altered Ca2+ homeostasis, hypermetabolism, and malignant hyperthermia in stressed, PSS-susceptible pigs. Muscle from PSS-susceptible pigs may also experience hypermetabolism in the conversion of muscle to meat, thus leading to the PSE condition (Mickelson and Louis, 1996). It is hypothesized that a growing subpopulation of commercial turkeys has an increased frequency of an altered SR Ca2+-channel protein, resulting in the abnormal Ca2+-channel protein activity associated with the development of PSE meat. Ryanodine binding assays were conducted on SR vesicles prepared from commercial turkeys and from a randombred control group to determine whether Ca2+-channel protein activity is altered in a subpopulation of commercial turkeys.
MATERIALS AND METHODS The animal experiments were approved by the AllUniversity Committee on Animal Care and Use at Michigan State University. Seven turkeys from both a randombred genetically unimproved group of turkeys (McCartney, 1964)2 and a genetically select group of commercial turkeys3 were killed by a 5-mL intravenous injection of sodium pentobarbital (12.5 mg/mL). The breast muscles were removed, cut into 2.5-cm cubes, and immediately frozen in liquid nitrogen. Samples were stored at –80 C until used for the purification of SR vesicles. Skeletal muscle SR vesicles were purified from breast muscle by the procedure of Mickelson et al. (1986). Briefly, the frozen muscle cubes were homogenized in a blender4 for 60 s in 5 vol of 0.1 M NaCl,5 5 mM Trismaleate5 (pH 6.8). The procedure was modified to include three proteinase inhibitors6 (0.1 mM PMSF, 1 mg/mL aprotinin, and 1mg/mL leupeptin) in the homogenization buffer and in each subsequent step of the preparation. After centrifugation7 of the homogenate for 30 min at 3,500 × g, the pellet was discarded, and the resultant supernatant was centrifuged for 30 min at 10,000 × g. This pellet was resuspended in 0.6 M KCl,5 5 mM Tris-maleate (pH 6.8), and centrifuged in a Ti-70 rotor8 for 40 min at 180,000 × g. For preparation of crude total SR vesicles (CSR), the pellet was resuspended in 10% sucrose4 and centrifuged at 180,000 × g for 40 min.
2Ohio Agricultural Experiment Station, Wooster, OH 44691. 3BilMar Foods, Zeeland, MI 49464. 4Waring Products Division, New Hartford, CT 06057. 5Sigma Chemical Co., St. Louis, MO 63178-9916. 6Boehringer Mannheim Biochemicals, Indianapolis, IN 46250. 7Ivan Sorvall Inc., Norwalk, CT 06852. 8Beckman Instruments, Inc., Palo Alto, CA 94304. 9Dupont-New England Nuclear, Boston, MA 02118. 10Wako Pure Chemical Industries, LTD., Richmond, VA 23237. 11Whatman GF/B; Fisher Scientific, Pittsburgh, PA 15205. 12Biosoft, Cambridge, U.K. CB2 1JP.
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The final pellet was resuspended in 10% sucrose, quickfrozen and stored at –80 C. Protein concentration in SR vesicles were determined by the method of Lowry et al. (1951) using bovine serum albumin5 as a standard. The yield of CSR was approximately 150 mg from 100 g of randombred or commercial turkey breast muscle. For purification of the high density or “heavy” SR vesicles enriched in the Ca2+-channel protein (HSR), the pellet from the 0.6 M KCl extraction was resuspended in 10% sucrose, 0.4 M KCl, 20 mM CaCl2, 5 mM Trismaleate (pH 6.8), and placed on discontinuous sucrose gradients (22, 36, and 45%) containing 0.4 M KCl, 20 mM CaCl2, 5 mM Tris-maleate (pH 6.8). The tubes were centrifuged for 5 h at 112,000 × g in an AH-629 rotor,7 and the material located at the interface between each sucrose layer was harvested. Harvested fractions were diluted at least 1:2 with 10% sucrose and centrifuged at 180,000 × g in a Ti-70 rotor for 40 min. The lower density fraction collected between the 22 and 36% sucrose gradient layers was light SR; the SR fraction obtained between 36 and 45% sucrose gradient layers was HSR. The pellets were resuspended in 10% sucrose, quickfrozen, and stored at –80 C. The yields were typically 17 mg HSR from 100 g of unimproved turkey muscle and 30 mg HSR from 100 g of commercial turkey muscle. The ryanodine binding assays were based on that of Mickelson et al. (1988). Sarcoplasmic reticulum vesicles (0.2 mg of HSR protein/mL or 1 mg of CSR protein/ mL) were incubated for 90 min at 37 C in media containing 0.25 M KCl, 25 mM PIPES5 (pH 7.0), 4 or 10 nM [3H] ryanodine,9 and a CaCl2-EGTA5-nitrilotriacetic5 acid buffer to give specific free calcium concentrations (Ca2+free) ranging from 0.01 to 1,000 mM (Perrin and Sayce, 1967). This information was used to derive optimal [Ca2+free] for subsequent ryanodine binding studies designed to obtain the capacity (Bmax) and affinity (Kd) of ryanodine binding to SR vesicles. The ryanodine concentration was varied in these assays by the addition of unlabeled ryanodine.10 After incubation, all samples were filtered through glass fiber filters11 and washed three times with 5 mL of ice-cold buffer (0.25 M KCl, 25 mM PIPES, pH 7.0). Radioactivity remaining on the filters was quantified by liquid scintillation counting (LS 100C8) and specific ryanodine binding was determined by subtracting nonspecific binding obtained in the presence of 100 mM unlabeled ryanodine. The Kd and Bmax values for ryanodine binding by the SR vesicles were calculated from the fit of bound vs free ryanodine using the Enzfitter computer program12 when the ryanodine concentrations were varied from 1 to 200 nM. The electrophoresis procedure (SDS-PAGE) employed was that described by Laemmli (1970) with the modification that 4 to 10% linear acrylamide5 gradient gels were used for the separation of SR proteins. Slab gels were stained with 0.05% Coomassie blue5 in 50% methanol,5 10% acetic acid,5 and destained with 50% methanol and 10% acetic acid followed by 10% methanol and 10% acetic acid.
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The SDS-PAGE gels were also stained with the cationic carbocyanine dye Stains-all5 (1-ethyl-2-[3-(1ethyl-naphtho [1,2d] thiazolin-2-ylidene)-2methylpropenyl] naptho [1,2d] thiazolium bromide) as described by Campbell et al. (1983). Slab gels were fixed overnight with 25% isopropyl alcohol5 and washed exhaustively in 25% isopropyl alcohol to remove SDS. The gel was then stained in the dark for at least 48 h with 0.0025% Stains-all, 25% isopropyl alcohol, 7.5% formamide,5 and 30 mM Tris-base5 (pH 8.8). This staining procedure results in blue staining of Ca 2+binding proteins (such as calsequestrin), whereas other proteins are stained pink. Comparisons of mean Kd and Bmax values for the unimproved and commercial turkey, for CSR and HSR, and for CSR at two different Ca2+ concentrations were made with Student’s t test.
RESULTS AND DISCUSSION Figure 1 shows the SDS-PAGE of HSR preparations purified from skeletal muscle of seven genetically unimproved turkeys (Lanes 1 to 7) and seven commercial turkeys (Lanes 8 to 14). The arrows shown near the top of the gel indicate the two isoforms (a and b) of the Ca2+-channel protein from both populations of turkeys, consistent with SDS-PAGE analysis of SR vesicles of other avian, amphibian, piscine, and reptilian species (Sutko and Airey, 1996). In contrast, mammalian species possess one skeletal muscle channel protein isoform (Ogawa, 1994). Five of the seven preparations of commercial turkey SR vesicles showed particularly high abundance of a 75-kDa protein (lower arrow). Preliminary attempts at
FIGURE 1. SDS-PAGE of heavy sarcoplasmic reticulum (HSR) from individual unimproved and commercial skeletal muscles. Lanes 1 to 7: HSR from seven genetically unimproved turkeys; Lanes 8 to 14: HSR from seven commercial turkeys; Lane 15: molecular weight markers (kilodaltons). The upper two arrows indicate the Ca2+-channel protein subunits that occur as two isoforms in turkey. The lower arrow indicates the 75-kDa protein band.
FIGURE 2. The Ca2+ dependence of ryanodine binding to unimproved and commercial turkey skeletal muscle heavy sarcoplasmic reticulum vesicles. Points represent the means ± SE for seven individual unimproved (o) and commerical (ÿ) heavy SR preparations.
identifying this protein have been unsuccessful (discussed below), which is interesting considering that in HSR vesicles from five of seven commercial turkeys, this protein is in greater abundance than the Ca 2+-adenosine triphosphatase (Ca2+-ATPase) at 100 kDa (Figure 1). Furthermore, the difference in abundance in the 75-kDa protein within the commercial turkey population suggests the presence of subpopulations within the commercial group of turkeys with respect to abundance of this protein. As the commercial group of turkeys has been selected for rapid body growth, it is possible that this protein plays a role in turkey muscling. Ryanodine binding assays were conducted to determine whether there are differences in SR Ca2+-channel activity between genetically unimproved and commercial turkeys. If a defect in function is present in the Ca2+channel of SR from commercial turkeys, the results would probably be manifested as an altered affinity for ryanodine, as an altered Ca2+ dependence for ryanodine binding, or both. The Ca2+-concentration dependence of ryanodine binding to unimproved and commercial turkey HSR vesicles from seven individual preparations is indicated in Figure 2. For both unimproved and commercial turkey HSR, ryanodine binding was activated at a threshold concentration of approximately 0.2 mM free Ca2+, and reached a plateau of binding over the range of 3 to 30 mM free Ca2+. When the free Ca2+ concentration reached 1 mM, the average ryanodine binding decreased in both populations of turkeys. Maximum ryanodine binding between 3 and 30 mM Ca2+ concentrations was greater (P < 0.05) for HSR from unimproved turkeys than for HSR from commercial turkeys (0.59 pmol/mg HSR protein 0.24 pmol/mg of HSR protein). This difference could be a result of the large abundance of the 75-kDa protein in HSR from commercial turkeys, which would inevitably dilute binding capacity. At high Ca2+ concentrations (∼1 mM),
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ryanodine binding to HSR was inhibited by 20 to 25% in both populations of turkeys compared to maximal binding at 3 to 30 mM Ca2+. These results contrast with those observed in pigs (Mickelson et al., 1988) and rabbits (O’Brien et al., 1995), which show a bell-shaped Ca2+-dependence curve and nearly complete inhibition of ryanodine binding and, thus, calcium release activity at millimolar Ca2+ concentrations. Because ryanodine binding is an indicator of Ca2+-channel protein activity, our results suggest that millimolar cytoplasmic levels of Ca2+ have only a slight effect on reducing the open state probability of the Ca2+channel protein. O’Brien et al. (1995) reported that the b isoform of the Ca2+-channel protein from fish is less sensitive to inactivation by millimolar concentrations of Ca2+ than the a isoform. Thus, the difference in Ca2+ dependence of ryanodine binding between porcine and turkey HSR probably results from the fact that the Ca2+channel protein of turkey skeletal muscle consists of two functionally distinct isoforms, whereas only one isoform that functionally resembles the a Ca2-channel protein isoform predominates in porcine skeletal muscle. The affinity of the Ca2+-channel protein from unimproved and commercial turkey skeletal muscle was measured in the presence of 10 mM Ca2+ over a range of ryanodine concentrations from 0 to 300 nM. Figure 3 shows the ryanodine binding curves of unimproved and commercial turkey HSR; the binding constants are presented in Table 1. The HSR from commercial turkeys bound ryanodine with greater (P < 0.05) affinity (Kd = 12.2 ± 5.9) than HSR from unimproved turkeys (20.5 ± 7.6 nM). The Ca2+-channel protein content in HSR from commercial turkeys (Bmax = 1.10 ± 0.06) was lower (P < 0.05) than the Ca2+-channel protein content in HSR from unimproved turkeys (4.01 ± 0.17 pmol/mg). The differences in affinity of channel proteins between unimproved and commercial populations of turkeys suggest functional differences in one or both channel isoforms. The large difference in channel protein content reflected by the large difference in ryanodine Bmax may be accounted for, in part, by the increased abundance of the 75-kDa protein in commercial turkey SR. In addition, these differences could reflect a differential distribution of the Ca2+-channel protein between the heavy and light fractions of the SR membranes from the two populations of turkeys. To address this question, ryanodine binding by CSR from commercial turkeys was compared with unimproved turkey preparations. Ryanodine binding to commercial turkey CSR vesicles yielded a Kd of 8.9 ± 5.1 nM and a Bmax of 0.29 ± 0.12 pmol/mg compared to a Kd of 19 ± 4.3 nM and a Bmax of 0.52 ± 0.13 pmol/mg obtained for CSR vesicles from unimproved turkeys (Figure 4 and Table 1). As expected, there was little change in Kd of ryanodine for the channel protein when comparing results from the HSR fraction with CSR. Differences in Bmax between these preparations were smaller, but are still significantly different. Studies on PSS suggest that alterations
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in Ca2+-channel protein content as well as differences in Kd may play a role in the defect. Mickelson et al. (1994) indicated that whereas the Ca2+ channel content of HSR was not significantly different between PSS and normal pigs, CSR preparations from PSS pigs had only 75% of the ryanodine Bmax as preparations of CSR isolated from normal pigs. Likewise, the present results showed that CSR from commercial turkeys displayed only 56% of ryanodine binding of CSR from unimproved turkeys. Because HSR preparations are highly enriched in junctional membranes, our results suggest that there may be differences in the localization of Ca2+-channel proteins between the two populations. The results support the hypothesis that there is a functional distinction between Ca2+-channel proteins of unimproved and commercial turkey SR. Because of the presence of the additional ryanodine binding peak at 300 mM Ca2+ (Figure 2), ryanodine
FIGURE 3. Ryanodine dependence of ryanodine binding to unimproved (o) and commercial (ÿ) turkey skeletal muscle heavy sarcoplasmic reticulum vesicles (HSR) at 10 mM Ca2+. A) Specific ryanodine binding by unimproved and commercial HSR. B) Scatchard plot of ryanodine binding by unimproved and commercial turkey heavy SR. Points represent the means ± SE for seven HSR preparations.
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TABLE 1. Equilibrium constants of ryanodine binding of crude (n = 4) and heavy (n = 7) sarcoplasmic reticulum (SR) fractions from breast muscle of unimproved and commercial turkeys at 10 or 300 mM Ca2+ Sample Unimproved 10 mM Ca2+ Heavy SR Crude SR 300 mM Ca2+ Crude SR Commercial 10 mM Ca2+ Heavy SR Crude SR 300 mM Ca2+ Crude SR
Kd1
Bmax1
(nM)
(pmol/mg protein)
20.5 ± 7.6a 19.0 ± 4.3a
4.01 ± 0.17a 0.52 ± 0.13b
25.1 ± 2.6b
0.58 ± 0.22b
12.2 ± 5.9c 8.9 ± 5.1c
1.10 ± 0.06c 0.29 ± 0.12d
15.7 ± 3.7d
0.34 ± 0.06d
a-dMeans
within a column with no common superscript differ significantly (P < 0.05). 1K = binding affinity; B d max = maximal binding capacity.
binding assays were conducted at this Ca2+ concentration to determine whether functional differences in channel activity might emerge at Ca2+ concentrations corresponding to those of contracting muscle. At 300 mM Ca2+, the ryanodine binding by commercial turkey CSR vesicles yielded a Kd of 15.7 ± 3.7 nM and Bmax of 0.34 ± 0.06 pmol/mg compared to a Kd of 25.1 ± 2.6 nM and Bmax of 0.58 ± 0.22 pmol/mg for unimproved turkey CSR (Figure 5 and Table 1). No additional differences between the two turkey populations were evident at this high Ca2+ concentration. The presence of the 75-kDa protein in such great abundance in most commercial turkey SR preparations could be an important indicator associated with growth or meat quality problems. The identity of this protein on
FIGURE 5. Ryanodine dependence of ryanodine binding to unimproved (») and commercial (…) turkey skeletal muscle crude SR vesicles at 300 mM Ca2+. Points represent the means ± SE of four CSR preparations.
the basis of molecular weight alone is unknown. Chadwick et al. (1988) showed that a 71-kDa protein crosslinked with the Ca2+-channel protein of terminal cisternae in rabbit skeletal muscle. However, they did not identify this protein, which was present in low abundance. Calsequestrin, a Ca2+-binding protein normally present in high abundance in SR preparations exhibits varying mobility on SDS-PAGE (normally Mr = 44,000 to 63,500) depending on the type of gel used and the tissue source. Because of its abundance and variable mobility on gels, the cationic carbocyanin dye Stains-all was used to determine whether the 75-kDa protein could be calsequestrin, which, because of its very acidic amino acid composition, stains dark blue (Campbell et al., 1983). The pink staining of the 75-kDa protein rather than blue staining (data not shown) clearly showed that the 75-kDa protein in commercial turkey heavy SR preparations is not calsequestrin, nor is it an acidic Ca2+binding protein. Experiments are ongoing to determine the sequence and identity of this protein. The present studies, which compare a randomly backcrossed, genetically unimproved line of turkeys with a group of commercially raised turkeys selected for rapid growth and muscling, suggest that there are significant differences in proteins comprising the SR that could have resulted from genetic selection. Studies are continuing in our laboratory to define the relationship between altered channel protein activity and the susceptibility of muscle from turkeys to produce PSE meat.
ACKNOWLEDGMENTS
FIGURE 4. Ryanodine dependence of ryanodine binding to unimproved (◊) and commercial (♦) turkey skeletal muscle crude sarcoplasmic reticulum (CSR) vesicles at 10 mM Ca2+. Points represent the means ± SE for four CSR preparations.
This study was supported by the USDA Animal Health and Disease Program, the USDA National Research Initiative Competitive Grants Program (Award Number 97-35503-4372), and the Michigan Agriculture Experiment Station.
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REFERENCES Campbell, K. P., D. H. MacLennan, and A. O. Jorgensen, 1983. Staining of the Ca2+-binding proteins, calsequestrin, calmodulin, troponin C, and S-100, with the cationic carbocyanine dye “Stains-all”. J. Biol. Chem. 258: 11267–11273. Chadwick, C. C., M. Inui, and S. Fleischer, 1988. Identification and purification of a transverse tubule coupling protein which binds to the ryanodine receptor of terminal cisternae at the triad junction in skeletal muscle. J. Biol. Chem. 263:10872–10877. Endo, M., S. Yagi, T. Ishizuka, K. Horiuti, Y. Koga, and K. Amaha, 1983. Changes in the Ca2+-induced Ca2+ release mechanism in sarcoplasmic reticulum from a patient with malignant hyperthermia. Biomed. Res. 4:83–92. Fujii, J. , K. Ostu, F. Zorzato, S. D. Leon, V. K. Khanna, J. E. Weiler, P. J. O’Brien, and D. H. MacLennan, 1991. Identification of a mutation in porcine ryanodine receptor associated with malignant hyperthermia. Science 253: 448–451. Laemmli, U. K., 1970. Cleavage of the structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. Louis, C. F., 1993. Porcine stress syndrome: biochemical and genetic basis of this inherited syndrome of skeletal muscle. Recipr. Meat Conf. Proc. 46:89–96. Lowry, O. H., N. J. Rosenbrough, A. L. Farr, and R. J. Randall, 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275. McCartney, M. G., 1964. A randombred control population of turkeys. Poultry Sci. 43:739–744. Mickelson, J. R., and C. F. Louis, 1996. Malignant hyperthermia: excitation-contraction coupling, Ca2+ release channel, and cell Ca2+ regulation defects. Physiol. Rev. 76:537–591. Mickelson, J. R., J. M. Ervasti, L. A. Litterer, K. P. Campbell, and C. F. Louis, 1994. Skeletal muscle junctional mem-
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