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• W2000590691 abstract "We have analyzed the effects of the endogenous redoxactive agents S-nitrosoglutathione and glutathione disulfide, and the NO donor NOR-3, on calcium release kinetics mediated by ryanodine receptor channels. Incubation of triad-enriched sarcoplasmic reticulum vesicles isolated from mammalian skeletal muscle with these three agents elicits different responses. Glutathione disulfide significantly reduces the inhibitory effect of Mg2+ without altering Ca2+ activation of release kinetics, whereas NOR-3 enhances Ca2+ activation of release kinetics without altering Mg2+ inhibition. Incubation with S-nitrosoglutathione produces both effects; it significantly enhances Ca2+ activation of release kinetics and diminishes the inhibitory effect of Mg2+ on this process. Triad incubation with [35S]nitrosoglutathione at pCa 5 promoted 35S incorporation into 2.5 cysteine residues per channel monomer; this incorporation decreased significantly at pCa 9. These findings indicate that S-nitrosoglutathione supports S-glutathionylation as well as the reported S-nitrosylation of ryanodine receptor channels (Sun, J., Xu, L., Eu, J. P., Stamler, J. S., and Meissner, G. (2003) J. Biol. Chem. 278, 8184–8189). The combined results suggest that S-glutathionylation of specific cysteine residues can modulate channel inhibition by Mg2+, whereas S-nitrosylation of different cysteines can modulate the activation of the channel by Ca2+. Possible physiological and pathological implications of the activation of skeletal Ca2+ release channels by endogenous redox species are discussed. We have analyzed the effects of the endogenous redoxactive agents S-nitrosoglutathione and glutathione disulfide, and the NO donor NOR-3, on calcium release kinetics mediated by ryanodine receptor channels. Incubation of triad-enriched sarcoplasmic reticulum vesicles isolated from mammalian skeletal muscle with these three agents elicits different responses. Glutathione disulfide significantly reduces the inhibitory effect of Mg2+ without altering Ca2+ activation of release kinetics, whereas NOR-3 enhances Ca2+ activation of release kinetics without altering Mg2+ inhibition. Incubation with S-nitrosoglutathione produces both effects; it significantly enhances Ca2+ activation of release kinetics and diminishes the inhibitory effect of Mg2+ on this process. Triad incubation with [35S]nitrosoglutathione at pCa 5 promoted 35S incorporation into 2.5 cysteine residues per channel monomer; this incorporation decreased significantly at pCa 9. These findings indicate that S-nitrosoglutathione supports S-glutathionylation as well as the reported S-nitrosylation of ryanodine receptor channels (Sun, J., Xu, L., Eu, J. P., Stamler, J. S., and Meissner, G. (2003) J. Biol. Chem. 278, 8184–8189). The combined results suggest that S-glutathionylation of specific cysteine residues can modulate channel inhibition by Mg2+, whereas S-nitrosylation of different cysteines can modulate the activation of the channel by Ca2+. Possible physiological and pathological implications of the activation of skeletal Ca2+ release channels by endogenous redox species are discussed. Ca2+-induced Ca2+ release (CICR) 1The abbreviations used are: CICR, Ca2+-induced Ca2+ release; RyR, ryanodine receptor; SR, sarcoplasmic reticulum; SH, sulfhydryl; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; GSH, glutathione; GSNO, glutathione disulfide; PTIO, 2-phenyl-4,4,5,5-tetramethylimidazolin-1-oxyl-3-oxide; MOPS, 4-morpholinepropanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; E-C, excitation-contraction; DTT, dithiothreitol.1The abbreviations used are: CICR, Ca2+-induced Ca2+ release; RyR, ryanodine receptor; SR, sarcoplasmic reticulum; SH, sulfhydryl; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; GSH, glutathione; GSNO, glutathione disulfide; PTIO, 2-phenyl-4,4,5,5-tetramethylimidazolin-1-oxyl-3-oxide; MOPS, 4-morpholinepropanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; E-C, excitation-contraction; DTT, dithiothreitol. mediated by ryanodine receptors/Ca2+ release channels (RyR channels) has a central role in very dissimilar processes. Among other processes, CICR mediates muscle contraction, neuronal plasticity, and secretion (1Svoboda K. Mainen Z.F. Neuron. 1999; 22: 427-430Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 2Rose C.R. Konnerth A. Neuron. 2001; 31: 519-522Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 3Fill M. Copello J.A. Physiol. Rev. 2002; 82: 893-922Crossref PubMed Scopus (880) Google Scholar, 4Cancela J.M. Van Coppenolle F. Galione A. Tepikin A.V. Petersen O.H. EMBO. J. 2002; 21: 909-919Crossref PubMed Scopus (158) Google Scholar). Not surprisingly, these Ca2+ release channels are extensively regulated by a variety of endogenous ions and molecules, as well as through interactions with other proteins (3Fill M. Copello J.A. Physiol. Rev. 2002; 82: 893-922Crossref PubMed Scopus (880) Google Scholar, 5Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (842) Google Scholar, 6Zucchi R. Ronca-Testoni S. Pharmacol. 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Nature. 1989; 339: 439-445Crossref PubMed Scopus (866) Google Scholar). In the native channel, ∼50 of these residues appear to be in the reduced state, and, of these, ∼10–12 are highly susceptible to oxidation/modification by exogenous sulfhydryl (SH) reagents (13Sun J. Xu L. Eu J.P. Stamler J.S. Meissner G. J. Biol. Chem. 2001; 276: 15625-15630Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Sulfhydryl modification enhances Ca2+ release from skeletal SR vesicles (14Trimm J.L. Salama G. Abramson J.J. J. Biol. Chem. 1986; 261: 16092-16098Abstract Full Text PDF PubMed Google Scholar, 15Zaidi 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, 16Salama G. Abramson J.J. Pike G.K. J. Physiol. 1992; 454: 389-420Crossref PubMed Scopus (80) Google Scholar, 17Koshita M. Miwa K. Oba T. Experientia (Basel). 1993; 49: 282-284Crossref PubMed Scopus (17) Google Scholar, 18Abramson J.J. 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J. 1998; 74: 1263-1277Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 24Suko J. Hellmann G. Biochim. Biophys. Acta. 1998; 1404: 435-450Crossref PubMed Scopus (14) Google Scholar, 25Oba T. Ishikawa T. Murayama T. Ogawa Y. Yamaguchi M. Am. J. Physiol. 2000; 279: C1366-C1374Crossref PubMed Google Scholar, 26Oba T. Murayama T. Ogawa Y. Am. J. Physiol. 2002; 282: C684-C692Crossref PubMed Scopus (36) Google Scholar). Sulfhydryl modification also increases [3H]ryanodine binding to skeletal SR membranes (18Abramson 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, 21Favero 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, 24Suko J. Hellmann G. Biochim. Biophys. Acta. 1998; 1404: 435-450Crossref PubMed Scopus (14) Google Scholar, 27Aghdasi 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). Highly reactive SH residues of the RyR1 channel protein participate in interactions between homotetrameric channel subunits (24Suko J. Hellmann G. Biochim. Biophys. Acta. 1998; 1404: 435-450Crossref PubMed Scopus (14) Google Scholar), participate in the formation of high molecular weight complexes with triadin (29Wu Y. Aghdasi B. Dou S.J. Zhang J.Z. Liu S.Q. Hamilton S.L. J. Biol. Chem. 1997; 272: 25051-25061Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 30Liu G. Pessah I.N. J. Biol. Chem. 1994; 269: 33028-33034Abstract Full Text PDF PubMed Google Scholar), and modulate calmodulin binding to the channel (31Zhang J.Z. Wu Y. Williams B.Y. Rodney G. Mandel F. Strasburg G.M. Hamilton S.L. Am. J. Physiol. 1999; 276: C46-C53Crossref PubMed Google Scholar, 32Porter M.C. Zhang J.Z. Hamilton S.L. J. Biol. Chem. 1999; 274: 36831-36834Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Endogenous redox active molecules, such as O2 and superoxide anion (33Eu J.P. Sun J. Xu L. Stamler J.S. Meissner G. Cell. 2000; 102: 499-509Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, 34Kawakami M. Okabe E. Mol. Pharmacol. 1998; 53: 497-503Crossref PubMed Scopus (121) Google Scholar), H2O2 (21Favero 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, 35Suzuki Y.J. Cleemann L. Abernethy D.R. Morad M. Free Radic. Biol. Med. 1998; 24: 318-325Crossref PubMed Scopus (51) Google Scholar), and glutathione disulfide (GSSG) (13Sun J. Xu L. Eu J.P. Stamler J.S. Meissner G. J. Biol. Chem. 2001; 276: 15625-15630Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 36Zable A.C. Favero T.G. Abramson J.J. J. Biol. Chem. 1997; 272: 7069-7077Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 37Aracena P. Hidalgo C. Proceedings of the XI Biennial Meeting of the Society for Free Radical Research International. Monduzzi Editore S.p.A.-Medimond Inc., Bologna, Italy2003: 221-224Google Scholar, 38Sun J. Xin C. Eu J.P. Stamler J.S. Meissner G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11158-11162Crossref PubMed Scopus (255) Google Scholar), enhance RyR channel activity. Likewise, changes in the glutathione/glutathione disulfide ratio (GSH/GSSG) (26Oba T. Murayama T. Ogawa Y. Am. J. Physiol. 2002; 282: C684-C692Crossref PubMed Scopus (36) Google Scholar, 39Xia R. Stangler T. Abramson J.J. J. Biol. Chem. 2000; 275: 36556-36561Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 40Feng W. Liu G. Allen P.D. Pessah I.N. J. Biol. Chem. 2000; 275 (33590): 35902Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) or incubation with NO or NO donors also affect RyR1 channel activity (13Sun J. Xu L. Eu J.P. Stamler J.S. Meissner G. J. Biol. Chem. 2001; 276: 15625-15630Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 33Eu J.P. Sun J. Xu L. Stamler J.S. Meissner G. Cell. 2000; 102: 499-509Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, 41Suko J. Drobny H. Hellmann G. Biochim. Biophys. Acta. 1999; 1451: 271-287Crossref PubMed Scopus (26) Google Scholar, 42Sun J. Xu L. Eu J.P. Stamler J.S. Meissner G. J. Biol. Chem. 2003; 278: 8184-8189Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). In particular, S-nitrosoglutathione (GSNO) and NO donors modify RyR1 channel activity through S-nitrosylation of a few critical SH residues (13Sun J. Xu L. Eu J.P. Stamler J.S. Meissner G. J. Biol. Chem. 2001; 276: 15625-15630Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 42Sun J. Xu L. Eu J.P. Stamler J.S. Meissner G. J. Biol. Chem. 2003; 278: 8184-8189Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). However, incubation of skeletal SR vesicles with 0.2–1.0 mm GSNO removes more free SH residues than those modified by S-nitrosylation (42Sun J. Xu L. Eu J.P. Stamler J.S. Meissner G. J. Biol. Chem. 2003; 278: 8184-8189Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). These findings indicate that GSNO induces additional SH modifications, which may include S-glutathionylation because in other systems GSNO acts both as S-nitrosylating and S-glutathionylating agent (43Padgett C.M. Whorton A.R. Am. J. Physiol. 1995; 269: C739-C749Crossref PubMed Google Scholar, 44Ji Y. Akerboom T.P. Sies H. Thomas J.A. Arch. Biochem. Biophys. 1999; 362: 67-78Crossref PubMed Scopus (163) Google Scholar). S-Glutathionylation, through the formation of a mixed disulfide between a protein SH residue and glutathione (45Lau K.H. Thomas J.A. J. Biol. Chem. 1983; 258: 2321-2326Abstract Full Text PDF PubMed Google Scholar), and S-nitrosylation appear to be reversible post-translational protein modifications, which may modulate intracellular signaling pathways by targeting critical molecules (46Klatt P. Lamas S. Eur. J. Biochem. 2000; 267: 4928-4944Crossref PubMed Scopus (656) Google Scholar). In fact, the activities of several signaling molecules including PP2A (47Rao R.K. Clayton L.W. Biochem. Biophys. Res. Commun. 2002; 293: 610-616Crossref PubMed Scopus (143) Google Scholar), Ras (48Mallis R.J. Buss J.E. Thomas J.A. Biochem. J. 2001; 355: 145-153Crossref PubMed Scopus (149) Google Scholar), and NFκB (49Pineda-Molina E. Klatt P. Vazquez J. Marina A. Garcia D.L. Perez-Sala D. Lamas S. Biochemistry. 2001; 40: 14134-14142Crossref PubMed Scopus (343) Google Scholar) are modified as a consequence of S-glutathionylation. This reaction is also markedly activated in response to oxidative stress, as shown by redox proteome analysis (50Fratelli M. Demol H. Puype M. Casagrande S. Eberini I. Salmona M. Bonetto V. Mengozzi M. Duffieux F. Miclet E. Bachi A. Vandekerckhove J. Gianazza E. Ghezzi P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3505-3510Crossref PubMed Scopus (490) Google Scholar, 51Lind C. Gerdes R. Hamnell Y. Schuppe-Koistinen I. von Lowenhielm H.B. Holmgren A. Cotgreave I.A. Arch. Biochem. Biophys. 2002; 406: 229-240Crossref PubMed Scopus (278) Google Scholar). We have previously reported that thimerosal enhances single RyR channel activity both by increasing channel activation by micromolar [Ca2+] and decreasing inhibition by 0.5 mm [Ca2+] (23Marengo J.J. Hidalgo C. Bull R. Biophys. J. 1998; 74: 1263-1277Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Thimerosal, in a concentration- and time-dependent manner, also enhances CICR rates from SR vesicles from mammalian skeletal muscle and decreases or abolishes the inhibition of CICR by 1 mm [Mg2+] (20Donoso P. Aracena P. Hidalgo C. Biophys. J. 2000; 79: 279-286Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Thimerosal, however, is an exogenous organomercapturial compound that modifies SH residues most likely via S-alkylation (52Elferink J.G. Gen. Pharmacol. 1999; 33: 1-6Crossref PubMed Scopus (77) Google Scholar). If endogenous GSNO had the potential to produce similar modifications of RyR channel activity as thimerosal, a significant enhancement of the CICR process would be expected in response to GSNO-induced endogenous redox modification of the channel protein. Therefore, we assessed the ability of GSNO to modify Ca2+ release kinetics and determined whether, in addition to its reported S-nitrosylating activity (42Sun J. Xu L. Eu J.P. Stamler J.S. Meissner G. J. Biol. Chem. 2003; 278: 8184-8189Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), GSNO also induced S-glutathionylation of RyR1 channels. We found that GSNO produced similar activation of CICR as thimerosal, and promoted calcium-dependent S-glutathionylation of the RyR channel protein. In addition, we tested the effects on CICR of GSSG and of the NO donor NOR-3, as pure S-glutathionylating or S-nitrosylating agents, respectively. The present results suggest that S-glutathionylation modifies specific cysteine residues and, in doing so, either directly or indirectly modulates channel inhibition by Mg2+ whereas S-nitrosylation modifies other cysteines, thereby altering activation by Ca2+. Possible physiological and pathological implications of these findings are discussed. Materials—All reagents used were of analytical grade. Protease inhibitors (leupeptin, pepstatin A, benzamidine, and phenylmethylsulfonyl fluoride) and bovine serum albumin were obtained from Sigma. Calcium Green-2 and Calcium Green-5N were obtained from Molecular Probes, Inc. (Eugene, OR). The redox reagents GSH, GSSG, DTT, and NOR-3 were purchased from Calbiochem (La Jolla, CA). When stored, fresh GSH solutions were bubbled with N2 and frozen at –20 °C. To avoid oxidation of GSH, aliquots were thawed only once and the unused portion was discarded. [35S]GSH and [3H]ryanodine were purchased from PerkinElmer Life Sciences. Primary antibodies against RyR1 (MA3–925) or triadin (MA3–931) and the secondary antibody (SA1–100) were purchased from Affinity BioReagents, Inc. (Golden, CO). Membrane Preparations—Triad-enriched SR vesicles (from now on triads) were isolated from rabbit fast skeletal muscle in the presence of a combination of protease inhibitors, as described previously (53Hidalgo C. Jorquera J. Tapia V. Donoso P. J. Biol. Chem. 1993; 268: 15111-15117Abstract Full Text PDF PubMed Google Scholar). To avoid spontaneous oxidation, all membrane fractions were rapidly frozen and kept under liquid N2. Experiments were performed within 24–48 h of membrane isolation to minimize possible oxidation during storage. Before use, the functionality of each preparation was verified. Native vesicles (0.2 mg/ml) that, following addition of 5 mm Mg-ATP, took longer than 2 min at 25 °C to actively decrease extravesicular free [Ca2+] from 25 μm to <0.2 μm (measured with Calcium Green-2) were discarded. It is possible that, because of the steps taken to avoid oxidation, release rate constants reported in this work for native triads were somewhat lower than previously reported values (20Donoso P. Aracena P. Hidalgo C. Biophys. J. 2000; 79: 279-286Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Protein concentration was determined according to Hartree (54Hartree E.F. Anal. Biochem. 1972; 48: 422-427Crossref PubMed Scopus (4541) Google Scholar) using bovine serum albumin as standard. [3H]Ryanodine Binding—[3H]Ryanodine binding to triads was determined essentially as described (55Bull R. Marengo J.J. Suárez-Isla B. Donoso P. Sutko J.L. Hidalgo C. Biophys. J. 1989; 56: 749-756Abstract Full Text PDF PubMed Scopus (62) Google Scholar). The composition of the solution was (in mm): 500 KCl, 0.5 adenosine 5′-(β,γ-imino)triphosphate, 20 MOPS-Tris, pH 7.2, pCa 5. Total binding was routinely measured in the presence of 10 nm [3H]ryanodine and nonspecific binding in the additional presence of 10 μm ryanodine. The isolated triad preparations displayed, on average, B max values of ryanodine binding of 18 pmol/mg of protein. Synthesis of GSNO—The synthesis of GSNO was performed as described (56Rossi R. Lusini L. Giannerini F. Giustarini D. Lungarella G. Di Simplicio P. Anal. Biochem. 1997; 254: 215-220Crossref PubMed Scopus (58) Google Scholar). Briefly, equimolar quantities of GSH and NaNO2 were incubated in 0.75 n HCl at room temperature. After 5 min of incubation, the solution was neutralized by addition of solid Trizma (Tris base). Concentrations of GSNO were determined from its absorbance, using an extinction coefficient of 767 m–1 cm–1 at 334 nm (44Ji Y. Akerboom T.P. Sies H. Thomas J.A. Arch. Biochem. Biophys. 1999; 362: 67-78Crossref PubMed Scopus (163) Google Scholar). Routinely, yields of GSNO were in the 90–95% range. Synthesis of [35S]GSNO was performed as above, using [35S]GSH at a specific activity of 600 mCi/mmol, after extraction with ethyl acetate of the DTT present in this reagent. Ca2 + Release Kinetics—Ca2+ release kinetics was measured in a SX.18MV fluorescence stopped flow spectrometer from Applied Photophysics Ltd. (Leatherhead, United Kingdom) as described (20Donoso P. Aracena P. Hidalgo C. Biophys. J. 2000; 79: 279-286Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 57Sanchez G. Hidalgo C. Donoso P. Biophys. J. 2003; 84: 2319-2330Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Vesicles were actively loaded with Ca2+ before eliciting Ca2+ release. For this purpose triads (1 mg/ml) were incubated for 20 min at 25 °C in a solution containing (in mm): 0.05 CaCl2, 100 KCl, 5 ATP, 5 MgCl2, 10 phosphocreatine plus 15 units/ml creatine kinase, 20 imidazole-MOPS, pH 7.2. Calcium release was initiated by mixing 1 volume of Ca2+-loaded triads with 10 volumes of releasing solution. Releasing solutions were designed to produce after mixing pCa 5 (unless indicated otherwise), 0.6–1.5 mm free [ATP], and variable free [Mg2+], from 25 μm (the lowest value attainable after active loading) to 900 μm. The increase in extravesicular [Ca2+] was determined by measuring the fluorescence of the Ca2+ indicator Calcium Green-5N (20Donoso P. Aracena P. Hidalgo C. Biophys. J. 2000; 79: 279-286Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The free [Ca2+], free [Mg2+] and free ATP concentration of releasing solutions were calculated with the WinMaxC program using the constants provided in the file cmc1002e.tcm (www.stanford.edu/~cpatton/winmaxc2.html). To study the effect of SH redox modification on calcium release, kinetics, prior to active loading triads, were incubated with redox agents: GSSG (1 mm for 20 min at 25 °C), GSNO (100 - 500 μm for 20 min at 25 °C), or NOR-3 (50 μm for 10 min at 25 °C). In all cases, control vesicles were incubated in the same conditions. To ensure maximal calcium loading after SH modification, which slowed down Ca2+ uptake rates, the time needed to actively reduce extravesicular free [Ca2+] from 50 μm to <0.2 μm was determined in each case as described in detail elsewhere (57Sanchez G. Hidalgo C. Donoso P. Biophys. J. 2003; 84: 2319-2330Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Identification of RyR1 and Triadin by Western Blot—Protein samples, denatured at 100 °C for 5 min, were separated by SDS-PAGE as detailed below. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes and probed with primary antibodies against RyR1 or triadin, diluted 1:1000 in phosphate-buffered saline. After washing, membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (1:5000 dilution in phosphate-buffered saline). Protein-antibody reactions were detected with an ECL kit (Amersham Biosciences, Uppsala, Sweden). Radioactive Labeling of SR Proteins with [35S]GSNO—Vesicles were incubated for 20 min at 25 °C with [35S]GSNO (25 μm to 1 mm). The incubation solution contained (in mm): 100 KCl, 20 imidazole-MOPS, pH 7.2, variable free [Ca2+], plus or minus 0.8 mm free [Mg2+]. The amount of CaCl2, EGTA, and MgCl2 needed for a given condition was calculated with the WinMaxC software as above. Incorporation of 35S radioactivity into RyR1 channels was studied either in sucrose gradient fractions enriched in the channel protein or directly following electrophoresis of labeled triads in non-reducing SDS-containing polyacrylamide gels. For sucrose gradient RyR1 channel purification (58Lee H.B. Xu L. Meissner G. J. Biol. Chem. 1994; 269: 13305-13312Abstract Full Text PDF PubMed Google Scholar), SR vesicles incubated with [35S]GSNO were sedimented at 45,000 × g for 30 min (4 °C). The resulting pellets were resuspended at 2 mg/ml and incubated with 0.5% Triton X-100, 1 mm CaCl2, 20 mm imidazole-MOPS, pH 7.2, for 20 min at room temperature under constant stirring, and sedimented at 45,000 × g for 30 min at 4 °C. The pellet was resuspended at 2 mg/ml in 3 mm EGTA, 20 mm MOPS-Tris, pH 9.0, incubated for 10 min at 4 °C, and sedimented at 45,000 × g for 30 min at 4 °C. This procedure allows recovery of a fraction enriched in RyR channels, essentially devoid of Ca2+-ATPase and calsequestrin. The RyR1-enriched fraction was resuspended at 1 mg/ml in solubilization buffer solution, containing (in mm): 1000 NaCl, 0.2 CaCl2. 0.1 EGTA, 20 MOPS-Tris, pH 7.2, plus 2% CHAPS and 1% l-α-phosphatidylcholine. This mixture was incubated for 60 min at 25 °C, followed by 60 min at 4 °C, layered onto a 5–20% linear sucrose gradient, and centrifuged at 120,000 × g for 17 h at 4 °C. The sucrose solutions used to generate the gradients were made in solubilization buffer plus 1% CHAPS and 0.5% l-α-phosphatidylcholine. To identify channel-containing fractions, parallel sucrose gradients were run with RyR1 channels solubilized from triads incubated with non-radioactive 4 mm GSNO and 2 nm [3H]ryanodine. Gradient fractions, collected in 1-ml aliquots, were analyzed for protein content (60Feng W. Pessah I.N. Methods Enzymol. 2002; 353: 240-253Crossref PubMed Scopus (12) Google Scholar) and for 35S or 3H radioactivity in a liquid scintillation counter. The protein composition of the fractions was analyzed by electrophoresis in 6% polyacrylamide-SDS gels under reducing conditions. Alternatively, the 35S radioactivity incorporated into the RyR1 channel protein was determined in RyR1-containing gel fractions separated by electrophoresis under non-reducing conditions. To this purpose, triads incubated with [35S]GSNO were sedimented at 178,000 × g for 5 min at room temperature in a Beckman Airfuge. The resulting pellets were resuspended in non-reducing sample buffer (6 m urea, 1% SDS, 0,02% bromphenol blue, 48 mm NaH2PO4, 170 mm Na2HPO4). Samples, denatured at 100 °C for 5 min, were separated in polyacrylamide gradient gels (3.5–8%) using the Tris-acetate buffer system (Novex NuPAGE®, Invitrogen, Carlsbad, CA). Gels, stained with Coomassie Brilliant Blue, were cut in 2-mm slices, the slices were trypsinized, and their radioactivity was determined in a liquid scintillation counter. Alternatively, gels were dried and phosphorimaging was performed using a Phosphor Screen CP (Eastman Kodak Co.) and the Molecular Imager FX system (Bio-Rad). Screens were scanned, and the images were quantified using the Quantity One software (Bio-Rad). Fast Release Kinetic Measurements—The time course of CICR, measured in the presence of 1.5 mm free ATP, from native and SH-modified triads is illustrated in Fig. 1. In native triads Ca2+ release followed a single exponential time course with an average rate constant((k) value of 11.7 s–1 at <25 μm free [Mg2+] Table I). The experimental record obtained in one preparation that displayed a k value of 11.4 s–1 is illustrated in Fig. 1, panel A. In agreement with previous results (20Donoso P. Aracena P. Hidalgo C. Biophys. J. 2000; 79: 279-286Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), increasing free [Mg2+] to 400 μm produced a strong inhibition of CICR (Fig. 1, panel B), decreasing k in this case to 1.6 s–1, with an average value of 1.4 s–1 (Table I). At free [Mg2+] < 25 μm, the same triad preparation incubated with 1 mm GSSG exhibited a k value similar to that for controls, 12 s–1 (Fig. 1, panel C); the average k value was 12.1 s–1 (Table I). However, in 400 μm free [Mg2+], GSSG-treated vesicles had a 3-fold higher k value than control, 4.6 s–1 (Fig. 1, panel D), with an average value of 5.0 s–1 (Table I). At <25 μm free [Mg2+], triads incubated with 100 μm GSNO displayed a higher k value than control vesicles, 17.2 s–1 (Fig. 1, panel E), with an average value of 15.8 s–1 (Table I). In 400 μm free [Mg2+], these same triads also displayed higher k values than controls, 8.2 s–1 (Fig. 1, panel F), with an average k value of 8.9 s–1 (Table I). Incubation with the NO donor NOR-3 produced at <25 μm free [Mg2+] a k value of 21 s–1 (Fig. 1, panel G), with an average value of 25.2 s–1, which is 2-fold higher than control (Table I). However, when release was measured in 400 μm free [Mg+2], triads incubated with NOR-3 displayed a low k value of 1.9 s–1 (Fig. 1, panel H), with an average value of 2.3 s–1 (Table I). These results indicate that, in the incubation conditions used in this work, GSNO and NOR-3, but not GSSG, stimulated CICR when measured at <25 μm free [Mg2+]. These results suggest that GSNO and NOR-3 promoted through S-nitrosylation an increase in the Ca2+ sensitivity of CICR. In addition, GSSG and GSNO, but not NOR-3, relieved the strong inhibitory effect on k exerted by Mg+2.Table IEffects of redox agents on calcium release rate constantsConditionRelease rate constant<25 μm [Mg2+]0.4 mm [Mg2+]s-1Control11.7 ± 0.8 (4)1.4 ± 0.2 (4)1 mm GSSG12.1 ± 0.8 (3)5.0 ± 0.6 (3)100 μm GSNO15.8 ± 3.8 (5)8.9 ± 3.0 (5)500 μm GSNO26.9 ± 7.3 (2)aThe error range of only two determinations is given.50 μm NOR-325.2 ± 5.0 (3)2.3 ± 0.6 (3)a The error range of only two determinations is given." @default.
• W2000590691 created "2016-06-24" @default.
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• W2000590691 date "2003-10-01" @default.
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• W2000590691 title "S-Glutathionylation Decreases Mg2+ Inhibition and S-Nitrosylation Enhances Ca2+ Activation of RyR1 Channels" @default.
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• W2000590691 doi "https://doi.org/10.1074/jbc.m306969200" @default.
• W2000590691 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12920114" @default.
• W2000590691 hasPublicationYear "2003" @default.
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