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Functional Interactions between Cytoplasmic Domains of the Skeletal Muscle Ca2+ Release Channel

1997; Elsevier BV; Volume: 272; Issue: 40 Linguagem: Inglês

10.1074/jbc.272.40.25051

ISSN

1083-351X

Autores

Yili Wu, Bahman Aghdasi, Shu Jun Dou, Jia Zheng Zhang, Si Qi Liu, Susan L. Hamilton,

Tópico(s)

Heat shock proteins research

Resumo

The skeletal muscle Ca2+release channel (RYR1), which plays a critical role in excitation-contraction coupling, is a homotetramer with a subunit molecular mass of 565 kDa. Oxidation of the channel increases its activity and produces intersubunit cross-links within the RYR1 tetramer (Aghdasi, B., Zhang, J., Wu, Y., Reid, M. B., and Hamilton, S. L. (1997) J. Biol. Chem.272, 3739–3748). Alkylation of hyperreactive sulfhydryls on RYR1 withN-ethylmaleimide (NEM) inhibits channel function and blocks the intersubunit cross-linking. We used calpain and tryptic cleavage, two-dimensional SDS-polyacrylamide gel electrophoresis, N-terminal sequencing, sequence-specific antibody Western blotting, and [14C]NEM labeling to identify the domains involved in these effects. Our data are consistent with a model in which 1) diamide, an oxidizing agent, simultaneously produces an intermolecular cross-link between adjacent subunits within the RYR1 tetramer and an intramolecular cross-link within a single subunit; 2) all of the cysteines involved in both cross-links are in either the region between amino acids ∼2100 and 2843 or the region between amino acids 2844 and 4685; 3) oxidation exposes a new calpain cleavage site in the central domain of the RYR1 (in the region around amino acid 2100); 4) sulfhydryls that react most rapidly with NEM are located in the N-terminal domain (between amino acids 426 and 1396); 5) alkylation of the N-terminal cysteines completely inhibits the formation of both inter- and intrasubunit cross-links. In summary, we present evidence for interactions between the N-terminal region and the putatively cytoplasmic central domains of RYR1 that appear to influence subunit-subunit interactions and channel activity. The skeletal muscle Ca2+release channel (RYR1), which plays a critical role in excitation-contraction coupling, is a homotetramer with a subunit molecular mass of 565 kDa. Oxidation of the channel increases its activity and produces intersubunit cross-links within the RYR1 tetramer (Aghdasi, B., Zhang, J., Wu, Y., Reid, M. B., and Hamilton, S. L. (1997) J. Biol. Chem.272, 3739–3748). Alkylation of hyperreactive sulfhydryls on RYR1 withN-ethylmaleimide (NEM) inhibits channel function and blocks the intersubunit cross-linking. We used calpain and tryptic cleavage, two-dimensional SDS-polyacrylamide gel electrophoresis, N-terminal sequencing, sequence-specific antibody Western blotting, and [14C]NEM labeling to identify the domains involved in these effects. Our data are consistent with a model in which 1) diamide, an oxidizing agent, simultaneously produces an intermolecular cross-link between adjacent subunits within the RYR1 tetramer and an intramolecular cross-link within a single subunit; 2) all of the cysteines involved in both cross-links are in either the region between amino acids ∼2100 and 2843 or the region between amino acids 2844 and 4685; 3) oxidation exposes a new calpain cleavage site in the central domain of the RYR1 (in the region around amino acid 2100); 4) sulfhydryls that react most rapidly with NEM are located in the N-terminal domain (between amino acids 426 and 1396); 5) alkylation of the N-terminal cysteines completely inhibits the formation of both inter- and intrasubunit cross-links. In summary, we present evidence for interactions between the N-terminal region and the putatively cytoplasmic central domains of RYR1 that appear to influence subunit-subunit interactions and channel activity. In skeletal muscle, the Ca2+ release channel (RYR1) of the sarcoplasmic reticulum (SR) 1The abbreviations used are: SR, sarcoplasmic reticulum; NEM, N-ethylmaleimide; DTT, dithiothreitol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; DHPR, dihydropyridine receptor.1The abbreviations used are: SR, sarcoplasmic reticulum; NEM, N-ethylmaleimide; DTT, dithiothreitol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; DHPR, dihydropyridine receptor. responds to T tubule depolarization by releasing lumenal Ca2+ into the myoplasmic space (1Martonosi A.N. Physiol. Rev. 1984; 64: 1240-1320Crossref PubMed Google Scholar), triggering the sequence of events that leads to muscle contraction. Single amino acid changes in RYR1 (2Quane K.A. Healy J.M.S. Keating K.E. Manning B.M. Couch F.J. Palmucci L.M. Doriguzzi C. Fagerlund T.H. Berg K. Ording H. Bendixen D. Mortier W. Linz U. Muller C.R. McCarthy T.V. Nat. Genet. 1993; 5: 51-55Crossref PubMed Scopus (307) Google Scholar, 3Quane K.A. Keating K.E. Manning B.M. Healy J.M. Monsieurs K. Hefron J.J.A. Lehane M. Heytens L. Krivosic-Hober R. Adnet P. Ellis F.R. Monnier N. Lunardi J. McCarthy T.V. Hum. Mol. Genet. 1994; 3: 471-476Crossref PubMed Scopus (105) Google Scholar, 4Phillips M. Khanna V.K. DeLeon S. Frodis W. Britt B.A. MacLennan D.H. Hum. Mol. Genet. 1994; 3: 2181-2186Crossref PubMed Scopus (51) Google Scholar, 5Zhang Y. Chen H.S. Khanna V.K. DeLeon S. Phillips M.S. Schapper K. Britt B.A. Brownell A.K.W. MacLennan D.H. Nat. Genet. 1993; 5: 46-50Crossref PubMed Scopus (290) Google Scholar, 6Fujii J. Otsu K. Zorzato F. DeLeon S. Khanna V.K. Weiler J.E. O'Brien P.J. MacLennan D.H. Science. 1991; 253: 448-451Crossref PubMed Scopus (1249) Google Scholar, 7Gillard E.F. Otsu K. Fujii J. Khanna V.K. DeLeon S. Derdemezi J. Britt B.A. Duff C.L. Worton R.G. MacLennan D.H. Genomics. 1991; 11: 751-755Crossref PubMed Scopus (277) Google Scholar, 8Gillard E.F. Otsu K. Fujii J. Duff C.L. DeLeon S. Khanna V.K. Britt B.A. Worton R.G. MacLennan D.H. Genomics. 1992; 13: 1247-1254Crossref PubMed Scopus (130) Google Scholar) are thought to produce the human diseases malignant hyperthermia and central core disease. Based on a hydropathy analysis of the primary amino acid sequence of RYR1, the monomeric subunit is predicted to have a short cytoplasmic C terminus and between 4 and 10 membrane-spanning regions in the C-terminal one-fifth of the molecule (9Takeshima H. Nishimura S. Matsumoto T. Ishida H. Kangawa K. Minamino N. Matsuo H. Ueda M. Hanaoka M. Hirose T. Numa S. Nature. 1989; 339: 439-440Crossref PubMed Scopus (866) Google Scholar, 10Zorzato F. Fujii J. Otsu K. Phillips M. Green N.M. Lai F.A. Meissner G. MacLennan D.H. J. Biol. Chem. 1990; 265: 2244-2256Abstract Full Text PDF PubMed Google Scholar). The transmembrane regions of the monomers may combine to form the pore of the homotetrameric Ca2+ release channel. The large N-terminal part of the molecule is thought to extend into the cytoplasm as a "foot" structure (11Franzini-Armstrong C. J. Cell Biol. 1970; 47: 488-499Crossref PubMed Scopus (297) Google Scholar), and this region of the protein plays an important role in regulation of the channel activity of RYR1. Most of the mutations in RYR1 that produce malignant hyperthermia and central core disease have been found in this region and cluster in two cytoplasmic locations (2Quane K.A. Healy J.M.S. Keating K.E. Manning B.M. Couch F.J. Palmucci L.M. Doriguzzi C. Fagerlund T.H. Berg K. Ording H. Bendixen D. Mortier W. Linz U. Muller C.R. McCarthy T.V. Nat. Genet. 1993; 5: 51-55Crossref PubMed Scopus (307) Google Scholar, 3Quane K.A. Keating K.E. Manning B.M. Healy J.M. Monsieurs K. Hefron J.J.A. Lehane M. Heytens L. Krivosic-Hober R. Adnet P. Ellis F.R. Monnier N. Lunardi J. McCarthy T.V. Hum. Mol. Genet. 1994; 3: 471-476Crossref PubMed Scopus (105) Google Scholar, 4Phillips M. Khanna V.K. DeLeon S. Frodis W. Britt B.A. MacLennan D.H. Hum. Mol. Genet. 1994; 3: 2181-2186Crossref PubMed Scopus (51) Google Scholar, 5Zhang Y. Chen H.S. Khanna V.K. DeLeon S. Phillips M.S. Schapper K. Britt B.A. Brownell A.K.W. MacLennan D.H. Nat. Genet. 1993; 5: 46-50Crossref PubMed Scopus (290) Google Scholar, 6Fujii J. Otsu K. Zorzato F. DeLeon S. Khanna V.K. Weiler J.E. O'Brien P.J. MacLennan D.H. Science. 1991; 253: 448-451Crossref PubMed Scopus (1249) Google Scholar, 7Gillard E.F. Otsu K. Fujii J. Khanna V.K. DeLeon S. Derdemezi J. Britt B.A. Duff C.L. Worton R.G. MacLennan D.H. Genomics. 1991; 11: 751-755Crossref PubMed Scopus (277) Google Scholar, 8Gillard E.F. Otsu K. Fujii J. Duff C.L. DeLeon S. Khanna V.K. Britt B.A. Worton R.G. MacLennan D.H. Genomics. 1992; 13: 1247-1254Crossref PubMed Scopus (130) Google Scholar). Also, most modulators of the channel are thought to interact with cytoplasmic domains of RYR1 (12Wagenknecht T. Berkowitz J. Grassucci R. Timerman A.P. Fleischer S. Biophys. J. 1995; 66: A416Google Scholar, 13Wagenknecht T. Grassucci R. Berkowitz J. Wiederrecht G.J. Xin H.-B. Fleischer S. Biophys. J. 1996; 70: 1709-1715Abstract Full Text PDF PubMed Scopus (63) Google Scholar, 14Chen S.R.W. Zhang L. MacLennan D.H. Proc. Natl. Acad. Sci., U. S. A. 1994; 91: 11953-11957Crossref PubMed Scopus (53) Google Scholar, 15Chen S.R.W. MacLennan D.H. J. Biol. Chem. 1994; 269: 22698-22704Abstract Full Text PDF PubMed Google Scholar, 16Wang J.P. Needleman D.H. Seryshev A.B. Aghdasi B. Slavik K.J. Liu S.-Q. Pedersen S.E. Hamilton S.L. J. Biol. Chem. 1996; 271: 8387-8393Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Reactive oxygen intermediates are produced in resting muscle and their production increases with muscle activity (17Reid M.B. Shoji T. Moody M.R. Entmam M.L. J. Appl. Physiol. 1992; 73: 1805-1809Crossref PubMed Scopus (269) Google Scholar, 18Reid M.B. Haack K.E. Franchek K.M. Valberg P.A. Kobzik L. West M.S. J. Appl. Physiol. 1992; 73: 1797-1804Crossref PubMed Scopus (394) Google Scholar, 19Reid M.B. Khawli F.A. Moody M.R. J. Appl. Physiol. 1993; 75: 1081-1087Crossref PubMed Scopus (245) Google Scholar, 20Kobzik L. Reid M.B. Bredt D.S. Stamler J.S. Nature. 1994; 372: 546-548Crossref PubMed Scopus (842) Google Scholar, 21Balon T.W. Nadler J.L. J. Appl. Physiol. 1994; 77: 2519-2521Crossref PubMed Scopus (356) Google Scholar, 22Diaz P.T. She Z.W. Davis W.B. Clanton T.L. J. Appl. Physiol. 1993; 75: 540-545Crossref PubMed Scopus (78) Google Scholar). The reactive oxygen intermediates appear to enhance contractile function in muscle, and one mechanism for their effects of contractility could be a modulation of the activity of the Ca2+ release channel. Consistent with this, oxidation reduction reactions have been reported to modulate RYR1 activity (23Favero T.G. Zable A.C. Abramson J.J. J. Biol. Chem. 1995; 270: 25557-25563Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 24Zaidi N.F. Lagenaur C.F. Abramson J.J. Pessah I. Salama G. J. Biol. Chem. 1989; 264: 21725-21736Abstract Full Text PDF PubMed Google Scholar, 25Abramson J.J. Trimm J.L. Weden L. Salama G. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 1526-1530Crossref PubMed Scopus (147) Google Scholar, 26Abramson J.J. Buck E. Salama G. Casida J.E. Pessah I.N. J. Biol. Chem. 1988; 263: 18750-18758Abstract Full Text PDF PubMed Google Scholar, 27Abramson J.J. Cronin J. Salama G. Arch. Biochem. Biophys. 1988; 263: 245-255Crossref PubMed Scopus (49) Google Scholar, 28Zorzato F. Margreth A. Volpe P. J. Biol. Chem. 1986; 261: 13252-13257Abstract Full Text PDF PubMed Google Scholar, 29Abramson J.J. Zable A.C. Favero T.G. Salama G. J. Biol. Chem. 1995; 270: 29644-29647Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Previously, we have demonstrated that the sulfhydryl oxidizing agent, diamide (30Kosower N.S. Kosower E.M. Wertheim B. Correa W.S. Biochem. Biophys. Res. Commun. 1969; 37: 593-596Crossref PubMed Scopus (408) Google Scholar), produces intersubunit cross-links between subunits of the RYR1 tetramer, enhances [3H]ryanodine binding, and activates the Ca2+release channel (31Aghdasi B. Zhang J.-Z. Wu Y. Reid M.B. Hamilton S.L. J. Biol. Chem. 1997; 272: 3739-3748Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). In addition to redox modulation, RYR1 is also sensitive to reagents that react with free sulfhydryls but do not form disulfide bonds (31Aghdasi B. Zhang J.-Z. Wu Y. Reid M.B. Hamilton S.L. J. Biol. Chem. 1997; 272: 3739-3748Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 32Bindoli S. Fleischer S. Arch. Biochem. Biophys. 1983; 221: 458-466Crossref PubMed Scopus (48) Google Scholar). Treatment of RYR1 with the alkylating reagentN-ethylmaleimide (NEM) for very short periods of time alkylates a small percentage of the total sulfhydryls on the protein and inhibits both channel activity and [3H]ryanodine binding (3Quane K.A. Keating K.E. Manning B.M. Healy J.M. Monsieurs K. Hefron J.J.A. Lehane M. Heytens L. Krivosic-Hober R. Adnet P. Ellis F.R. Monnier N. Lunardi J. McCarthy T.V. Hum. Mol. Genet. 1994; 3: 471-476Crossref PubMed Scopus (105) Google Scholar). Alkylation of these hyperreactive sulfhydryls (representing less than 10% of the total sulfhydryls on RYR1) completely blocks the diamide-induced intersubunit cross-linking (3Quane K.A. Keating K.E. Manning B.M. Healy J.M. Monsieurs K. Hefron J.J.A. Lehane M. Heytens L. Krivosic-Hober R. Adnet P. Ellis F.R. Monnier N. Lunardi J. McCarthy T.V. Hum. Mol. Genet. 1994; 3: 471-476Crossref PubMed Scopus (105) Google Scholar). The domain locations of the sulfhydryls on RYR1 involved in the NEM and oxidative effects are not known. In particular, it is not clear whether the same sulfhydryls are involved in both the alkylation and oxidation-reduction reactions or whether the alkylation of hyperreactive sulfhydryls has a long distance effect on the sulfhydryls involved in cross-linking. Formation of intersubunit disulfides activates the channel and the localization of the cysteine residues involved may help to identify a functionally important site of subunit-subunit contact. Proteolysis has been used as a tool for probing the functional domains of RYR1 (16Wang J.P. Needleman D.H. Seryshev A.B. Aghdasi B. Slavik K.J. Liu S.-Q. Pedersen S.E. Hamilton S.L. J. Biol. Chem. 1996; 271: 8387-8393Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 33Callaway C. Seryshev A. Wang J.-P. Slavik K.-J. Needleman D.H. Cantu III, C. Wu Y Jayaraman T. Marks A.R. Hamilton S.L. J. Biol. Chem. 1994; 269: 15876-15884Abstract Full Text PDF PubMed Google Scholar, 34Meissner G. Rousseau E. Lai F.A. J. Biol. Chem. 1989; 264: 1715-1722Abstract Full Text PDF PubMed Google Scholar). For example, both high and low affinity ryanodine binding sites are found between the tryptic cleavage site Arg-4475 and the carboxyl terminus. If ryanodine is bound prior to digestion, it remains bound. If, however, ryanodine does not occupy the site, the ability of the channel to bind [3H]ryanodine is rapidly lost upon tryptic digestion (33Callaway C. Seryshev A. Wang J.-P. Slavik K.-J. Needleman D.H. Cantu III, C. Wu Y Jayaraman T. Marks A.R. Hamilton S.L. J. Biol. Chem. 1994; 269: 15876-15884Abstract Full Text PDF PubMed Google Scholar, 34Meissner G. Rousseau E. Lai F.A. J. Biol. Chem. 1989; 264: 1715-1722Abstract Full Text PDF PubMed Google Scholar). The tryptic complex composed only of RYR1 fragments from amino acid 4476 to the carboxyl terminus retains the ability to form channels in planar lipid bilayers (16Wang J.P. Needleman D.H. Seryshev A.B. Aghdasi B. Slavik K.J. Liu S.-Q. Pedersen S.E. Hamilton S.L. J. Biol. Chem. 1996; 271: 8387-8393Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). In contrast to trypsin, digestion of SR membranes with calpain II does not cause loss of ability of RYR1 to bind [3H]ryanodine (35Rardon D.P. Cefali D.C. Mitchell R.D. Seiler S.M. Hathaway D.R. Jones L.R. Circ. Res. 1990; 67: 84-96Crossref PubMed Scopus (54) Google Scholar, 36Gilchrist J.S.C. Wang K.K.W. Katz S. Belcastro A.N. J. Biol. Chem. 1992; 267: 20857-20865Abstract Full Text PDF PubMed Google Scholar). RYR1 is the major SR substrate for endogenous calpains (37Seiler S. Wegener A.D. Whang D.D. Hathaway D.R. Jones L.R. J. Biol. Chem. 1984; 259: 8550-8557Abstract Full Text PDF PubMed Google Scholar), and calpain II digestion enhances the activity of the Ca2+release channel (35Rardon D.P. Cefali D.C. Mitchell R.D. Seiler S.M. Hathaway D.R. Jones L.R. Circ. Res. 1990; 67: 84-96Crossref PubMed Scopus (54) Google Scholar). Calpain digestion does not appear to alter the sedimentation properties of RYR1, and the cleaved fragments appear to remain associated with the remainder of the complex (38Grunwald R. Meissner G. J. Biol. Chem. 1995; 270: 11338-11347Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 39Brandt N.R. Caswell A.H. Brandt T. Brew K. Mellgren R.L. J. Membr. Biol. 1992; 127: 35-47Crossref PubMed Scopus (42) Google Scholar). The purpose of the study described here was to identify domains on RYR1 that contribute to the regulation of channel activity by oxidation and by alkylation. Our strategy to localize functional domains was to identify the calpain-derived fragments of the RYR1 cross-linked by diamide and/or alkylated by [14C]NEM. We chose the proteolytic enzyme calpain for these studies because endogenous calpain is known to produce large fragments of RYR1 (35Rardon D.P. Cefali D.C. Mitchell R.D. Seiler S.M. Hathaway D.R. Jones L.R. Circ. Res. 1990; 67: 84-96Crossref PubMed Scopus (54) Google Scholar, 36Gilchrist J.S.C. Wang K.K.W. Katz S. Belcastro A.N. J. Biol. Chem. 1992; 267: 20857-20865Abstract Full Text PDF PubMed Google Scholar, 37Seiler S. Wegener A.D. Whang D.D. Hathaway D.R. Jones L.R. J. Biol. Chem. 1984; 259: 8550-8557Abstract Full Text PDF PubMed Google Scholar, 38Grunwald R. Meissner G. J. Biol. Chem. 1995; 270: 11338-11347Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 39Brandt N.R. Caswell A.H. Brandt T. Brew K. Mellgren R.L. J. Membr. Biol. 1992; 127: 35-47Crossref PubMed Scopus (42) Google Scholar), and large fragments allow us to discriminate between inter- and intramolecular cross-links. We used three types of cross-linking experiments: 1) diamide cross-linking of purified RYR1 previously digested by calpain while in the SR membrane to eliminate the possibility of cross-linking between neighboring tetramers or with other proteins in the SR membrane; 2) calpain digestion of RYR1 followed by cross-linking in membranes to demonstrate that the cross-linking pattern was not altered by membrane solubilization, and 3) cross-linking of RYR1 followed by calpain digestion in SR membranes to assess whether cross-linking altered the sites of the calpain digestion or if calpain digestion altered the sites of cross-linking. [3H]Ryanodine (81.5 Ci/mmol) and [14C]NEM (0.05 Ci/mmol) were purchased from NEN Life Science Products. Ryanodine and calpain II were purchased from Calbiochem (La Jolla, CA). Diamide, dithiothreitol (DTT), NEM, CHAPS, MOPS, and CAPS were obtained from Sigma. SR membranes were prepared from rabbit leg white skeletal muscle and were purified using sucrose gradient centrifugation (40Hamilton S.L. Tate C.A. McCormack J.G. Cobbold P.H. Cellular Calcium. IRL Press, Oxford1991: 313-343Google Scholar, 41Hawkes M.J. Diaz-Munoz M. Hamilton S.L. Membr. Biochem. 1989; 8: 133-145Crossref PubMed Scopus (32) Google Scholar). Antipeptide antibodies corresponding to 2727–2743 (Ab 2727), 4363–4373 (Ab 4363), and 4685–4697 (Ab 4685) were made as described previously (33Callaway C. Seryshev A. Wang J.-P. Slavik K.-J. Needleman D.H. Cantu III, C. Wu Y Jayaraman T. Marks A.R. Hamilton S.L. J. Biol. Chem. 1994; 269: 15876-15884Abstract Full Text PDF PubMed Google Scholar). Ab 0001 is a polyclonal antibody against a fusion protein containing the first 280 amino acids from the N-terminal of RYR1. The GST fusion construct was prepared by ligation of the N-terminal RYR1 cDNA into toBamHI-digested pGEX. The GST fusion protein was expressed inEscherichia coli strain XL1-blue and purified with a glutathione affinity column. The antiserum against the purified fusion protein was prepared by Pel-Freez Biologicals (Rogers, AR), and the antibody was then affinity-purified with an Affi-Gel Protein A column (Bio-Rad). Ab 5029 was graciously provided by Dr. Andrew Marks (Mount Sinai School of Medicine, New York). The polyclonal RyR antibody against the intact RYR1 was prepared in collaboration with Dr. John Dedman (University of Cincinnati, OH). Rabbit SR membranes (10 mg/ml) were labeled with [3H]ryanodine (10 nm) in binding buffer containing 300 mmNaCl, 50 mm MOPS (pH 7.4), 100 μmCaCl2, and protease inhibitors for 1 h at 37 °C. Unlabeled ryanodine (130 μm) was then added, and the incubation was continued for an additional 10 min. This latter treatment slows dissociation of [3H]ryanodine from the high affinity site (33Callaway C. Seryshev A. Wang J.-P. Slavik K.-J. Needleman D.H. Cantu III, C. Wu Y Jayaraman T. Marks A.R. Hamilton S.L. J. Biol. Chem. 1994; 269: 15876-15884Abstract Full Text PDF PubMed Google Scholar). Subsequently, the membranes were centrifuged for 5 min at 30 p.s.i. in a Beckman Airfuge to remove the protease inhibitors and unbound [3H]ryanodine. The pellet was then resuspended in digestion buffer (50 mm NaCl, 20 mm MOPS, 3 mm CaCl2, 2 mm DTT, pH 7.4) and digested with endogenous and exogenous calpain (1:50–1:25 calpain:protein ratio) at 37 °C for 1.5 h. The reaction was terminated by the addition of leupeptin (10 μm), and the samples were centrifuged for 35 min at 190,000 × g. The resulting pellets were solubilized in a solution of 2% CHAPS in binding buffer with protease inhibitors for 30 min at 4 °C. The solubilized complex was layered onto 17 ml of 5–20% sucrose gradients containing 0.4% CHAPS, 200 mm NaCl, and 20 mm MOPS (pH 7.4) and centrifuged for 17 h at 110,000 × g. Fractions of 20 drops each were collected from the bottom of the sucrose gradient. Aliquots of 50 μl were taken from each fraction to determine radioactivity. 5% polyacrylamide gel electrophoresis was performed as described by Laemmli (42Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207012) Google Scholar). The molecular mass of the high molecular weight fragments of RYR1 was estimated using Rainbow molecular markers (46–220 kDa, Amersham Corp.), the full-length RYR1 as a 565-kDa marker, and the commonly observed second large fragment as a 410-kDa marker (36Gilchrist J.S.C. Wang K.K.W. Katz S. Belcastro A.N. J. Biol. Chem. 1992; 267: 20857-20865Abstract Full Text PDF PubMed Google Scholar, 39Brandt N.R. Caswell A.H. Brandt T. Brew K. Mellgren R.L. J. Membr. Biol. 1992; 127: 35-47Crossref PubMed Scopus (42) Google Scholar). Protein samples subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) were transferred to Immobilon-P membrane (Millipore Corp., Bedford, MA) for 16–18 h at 25 V in 5% methanol, 10 mm CAPS (pH 11.0) at 4 °C. The blots were developed with primary antibodies and alkaline phosphatase-conjugated second antibodies as described previously (33Callaway C. Seryshev A. Wang J.-P. Slavik K.-J. Needleman D.H. Cantu III, C. Wu Y Jayaraman T. Marks A.R. Hamilton S.L. J. Biol. Chem. 1994; 269: 15876-15884Abstract Full Text PDF PubMed Google Scholar). The calpain- and trypsin-digested RYR1 fragments were separated on 5% SDS-polyacrylamide gels and then prepared for sequencing as described previously (33Callaway C. Seryshev A. Wang J.-P. Slavik K.-J. Needleman D.H. Cantu III, C. Wu Y Jayaraman T. Marks A.R. Hamilton S.L. J. Biol. Chem. 1994; 269: 15876-15884Abstract Full Text PDF PubMed Google Scholar). Sequencing was performed by Dr. Richard Cook at Baylor College of Medicine. RYR1 proteolyzed by endogenous calpain in the SR membrane was purified by sucrose gradient centrifugation and DEAE-Trisacryl ion exchange as described previously (41Hawkes M.J. Diaz-Munoz M. Hamilton S.L. Membr. Biochem. 1989; 8: 133-145Crossref PubMed Scopus (32) Google Scholar). The sample was stored in 300 mm NaCl, 10 mm MOPS (pH 7.4), 0.4% CHAPS, 5% sucrose, and 1 mm DTT. For the diamide titration, diamide at final concentrations of 0–2 mm in 200 mm NaCl, 20 mm MOPS (pH 7.4) was added to the purified sample before incubation at 4 °C for 30 min. Subsequently, the samples were incubated with 4 mm NEM at room temperature for 20 min, and then with sample buffer (0.06m Tris, 10% glycerol, 2% SDS, 0.001% bromphenol blue) for another 20 min prior to electrophoresis. The purified proteolytic complex was cross-linked by diamide and then treated with 4 mm NEM for 20 min before solubilization in SDS sample buffer. Polyacrylamide gels (5%) were used for the first dimension electrophoresis. Electrophoresis was continued for a certain time period after the dye front ran off the gel. The time period varied according to the required resolution of the fragments. Subsequently, the lanes were excised, and the gel strips were incubated with 25–50 mm DTT in the sample buffer without SDS and bromphenol blue for 1 h at room temperature (23 °C). The gel strip was then loaded on top of another 5% polyacrylamide gel for the second dimension electrophoresis. The gap between the gel strip and the two-dimensional resolving gel was sealed with melted agarose (1% agarose, 2% SDS, 50 mmDTT), and the gels were electrophoresed for 1–2 h at 120–170 V at 4 °C. The two-dimensional gels were subsequently stained with Coomassie Blue or silver nitrate. Rabbit SR membranes (2 mg/ml) were incubated either with calpain (calpain:protein ratio of 1:25 and 3 mmCaCl2) at 37 °C for 40 min or without calpain at room temperature for 20 min in the presence of protease inhibitors in digestion buffer (50 mm NaCl, 20 mm MOPS, 2 mm DTT, pH 7.4). Subsequently, the membranes were centrifuged for 5 min at 30 p.s.i. in a Beckman Airfuge to remove the calpain, CaCl2, and DTT. The pellet was then resuspended and cross-linked with diamide (0.1–0.5 mm) in 200 mm NaCl and 20 mm MOPS (pH 7.4) at 4 °C for 30 min. Following the cross-linking, the preproteolyzed sample was applied to two-dimensional electrophoresis. The nonproteolyzed sample was centrifuged for 5 min at 30 p.s.i. to remove the diamide and then resuspended and digested with calpain at 37 °C for 40 min in the absence of DTT (50 mm NaCl, 20 mm MOPS, 3 mm CaCl2, pH 7.4). After the calpain was removed by centrifugation, the pellet was resuspended in buffer (200 mm NaCl and 20 mm MOPS, pH 7.4) and either applied to two-dimensional electrophoresis or reduced by 25 mm DTT for one-dimensional electrophoresis and Western blot analysis. SR membranes (10 μg/assay) were incubated with [3H]ryanodine (5 nm) at room temperature (23 °C) for 16 h in binding buffer (300 mm NaCl, 50 mm MOPS (pH 7.4), 100 μg/ml bovine serum albumin, 0.1% CHAPS, and 1 mm EGTA) in the absence and presence of 1.1 mm CaCl2. Nonspecific binding was defined in the presence of 10 μm unlabeled ryanodine. The bound [3H]ryanodine was separated from free ligand by filtering through Whatman GF/F glass fiber filters. The filters were washed with 5 × 3 ml of ice-cold buffer containing 300 mm NaCl, 100 μm CaCl2, and 20 mm MOPS (pH 7.4). The radioactivity bound to the filters was quantitated by liquid scintillation counting. SR membrane was incubated with 0.3 mm [14C]NEM for 5, 15, and 45 min. The reaction was stopped by the addition of 20 mm DTT, and samples were pelleted in an Airfuge to remove the free [14C]NEM and DTT. Subsequently, the sample was resuspended in digestion buffer and digested by calpain (calpain:protein ratio of 1:25) and then electrophoresed on 5% gel. After stained with Coomassie Brilliant Blue, the fragment optical density (OD) was analyzed by densitometry (Pharmacia Biotech Inc.). Then each lane of the gel was sliced in 1-mm intervals, and the slices were dried to remove acetic acid from the staining solutions and then rehydrated in 100 μl of H2O. The radioactivity of the gel slices was quantitated by liquid scintillation counting of the slices in nonaqueous scintillant containing 10% (v/v) TS-2 tissue solubilizer (Beckman). Since the calpain cleavage sites on RYR1 were not known, it was necessary to identify the calpain-derived fragments of RYR1 prior to performing the cross-linking experiments. SR membranes were labeled with [3H]ryanodine, proteolyzed with varying amounts of calpain II (0–1:25 calpain:protein weight ratio), and solubilized. The proteolyzed complexes were then purified on sucrose gradients. In contrast to tryptic digestion (33Callaway C. Seryshev A. Wang J.-P. Slavik K.-J. Needleman D.H. Cantu III, C. Wu Y Jayaraman T. Marks A.R. Hamilton S.L. J. Biol. Chem. 1994; 269: 15876-15884Abstract Full Text PDF PubMed Google Scholar), neither the sedimentation behavior nor the ability to bind [3H]ryanodine was significantly altered by calpain digestion (data not shown). The isolated proteolytic complex contained a large number of polypeptides, and the presence of bound ryanodine did not alter the cleavage pattern. To identify these polypeptides, we analyzed the proteolytic fragments of RYR1 by SDS-PAGE and Western blotting. Some of the smaller fragments were also analyzed by N-terminal sequencing. Fig. 1 shows proteolysis of RYR1 by calpain and the identification of these fragments with a polyclonal antibody against full-length RYR1 and an antibody against the last 9 amino acids of the RYR1 (Ab 5029). The protein fragments in all lanes are matched and alphabetically listed in Table I, along with their apparent molecular mass. In the control lane of Fig. 1 A, RYR1 was already slightly proteolyzed, and several RYR1-derived fragments were detected: the full-length 565-kDa RYR1 (band a), the 410-kDa fragment (fragment b), and a doublet of 172 and 166 kDa (fragments h and i). This proteolysis detected in the membrane preparation may have occurred either in the intact muscle or during the isolation of membranes. Incubation of the membranes in the digestion buffer alone (calpain:protein ratio of 0) prior to the purification of RYR1 produced several new fragments, c (338 kDa), m (98 kDa), n (82 kDa), p (67

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