Low N-Ethylmaleimide Concentrations Activate Ryanodine Receptors by a Reversible Interaction, Not an Alkylation of Critical Thiols

Abstract

Previous studies proposed thatN-ethylmaleimide (NEM) alkylates 3 classes of thiols on skeletal muscle ryanodine receptors (RyRs) producing 3 phases of channel modification, as function of time and concentration. NEM (5 mm) decreased, increased, and then decreased the open probability (P o) of the channel by thiol alkylation, a reaction not reversed by reducing agents. We now show that low NEM concentrations (20–200 μm) elicit Ca2+ release from sarcoplasmic reticulum (SR) vesicles, but contrary to expectations, the effect was fully reversed by reducing agents or by washing SR vesicles. In bilayers, NEM (0.2 mm) increased P o of RyRs within seconds when added to the cis (not trans) side, and dithiothreitol (DTT; 1 mm) decreased P o in seconds. High (5 mm) NEM concentrations elicited SR Ca2+ release that was not reversed by DTT, as expected for an alkylation reaction. A non-sulfhydryl reagent structurally related to NEM, N-ethylsuccinimide (0.1–0.5 mm), also elicited SR Ca2+ release that was not reversed by DTT (1 mm). Other alkylating agents elicited SR Ca2+ release, which was fully (N-methylmaleimide) or partially (iodoacetic acid) reversed by DTT and inhibited by ruthenium red. Nitric oxide (NO) donors at concentrations that did not activate RyRs inhibited NEM-induced Ca2+ release, most likely by an interaction of NO with NEM rather than an inactivation of RyRs by NO. Thus, at low concentrations, NEM does not act as a selective thiol reagent and activates RyRs without alkylating critical thiols indicating that the multiple phases of ryanodine binding are unrelated to RyR activity or to NEM alkylation of RyRs. Previous studies proposed thatN-ethylmaleimide (NEM) alkylates 3 classes of thiols on skeletal muscle ryanodine receptors (RyRs) producing 3 phases of channel modification, as function of time and concentration. NEM (5 mm) decreased, increased, and then decreased the open probability (P o) of the channel by thiol alkylation, a reaction not reversed by reducing agents. We now show that low NEM concentrations (20–200 μm) elicit Ca2+ release from sarcoplasmic reticulum (SR) vesicles, but contrary to expectations, the effect was fully reversed by reducing agents or by washing SR vesicles. In bilayers, NEM (0.2 mm) increased P o of RyRs within seconds when added to the cis (not trans) side, and dithiothreitol (DTT; 1 mm) decreased P o in seconds. High (5 mm) NEM concentrations elicited SR Ca2+ release that was not reversed by DTT, as expected for an alkylation reaction. A non-sulfhydryl reagent structurally related to NEM, N-ethylsuccinimide (0.1–0.5 mm), also elicited SR Ca2+ release that was not reversed by DTT (1 mm). Other alkylating agents elicited SR Ca2+ release, which was fully (N-methylmaleimide) or partially (iodoacetic acid) reversed by DTT and inhibited by ruthenium red. Nitric oxide (NO) donors at concentrations that did not activate RyRs inhibited NEM-induced Ca2+ release, most likely by an interaction of NO with NEM rather than an inactivation of RyRs by NO. Thus, at low concentrations, NEM does not act as a selective thiol reagent and activates RyRs without alkylating critical thiols indicating that the multiple phases of ryanodine binding are unrelated to RyR activity or to NEM alkylation of RyRs. sarcoplasmic reticulum ryanodine receptor(s) N-ethylmaleimide nitric oxide S-nitrosocysteine S-nitroso-N-acetyl-d-l-penicillamine dithiothreitol reduced glutathione 1,4-piperazinediethanesulfonic acid antipyrylazo N-methylmaleimide iodoacetic acid N-ethyl succinimide MAHMA/NO, (z)-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-1-ium-1,2-diolate PAPA-NONOate, PAPA/NO, (z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate In striated muscle, the sarcoplasmic reticulum (SR)1 is the major storage compartment of intracellular Ca2+ that controls cytosolic free Ca2+ and developed force by sequestering and releasing Ca2+ during each contraction. Studies of Ca2+flux across the SR membrane and single channel recordings in planar lipid bilayers have suggested that Ca2+ release from skeletal muscle SR is mediated by a high conductance Ca2+channel (ryanodine receptor; RyR) (1Rousseau E. Smith J.S. Henderson J.S. Meissner G. Biophys. J. 1986; 50: 1009-1014Abstract Full Text PDF PubMed Scopus (173) Google Scholar, 2Meissner G. J. Biol. Chem. 1986; 261: 6300-6306Abstract Full Text PDF PubMed Google Scholar). A wide range of intracellular signaling molecules and pharmacological agents modulated RyRs (3Zucchi R. Ronca-Testoni S. Pharmacol. Rev. 1997; 49: 1-51PubMed Google Scholar). In addition the following oxidants have been shown to activate the channel: heavy metals, reactive disulfides, hydrogen peroxide, Fe2+/ascorbate, and Thimerosal (4Salama G. Abramson J.J. J. Biol. Chem. 1984; 259: 13363-13369Abstract Full Text PDF PubMed Google Scholar, 5Prabhu S. Salama G. Arch. Biochem. Biophys. 1990; 277: 47-55Crossref PubMed Scopus (50) Google Scholar, 6Trimm J. Salama G. Abramson J. J. Biol. Chem. 1986; 261: 16092-16098Abstract Full Text PDF PubMed Google Scholar, 7Zaidi 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, 8Stoyanovsky D.A. Salama G. Kagan V.E. Arch. Biochem. Biophys. 1994; 208: 214-221Crossref Scopus (43) Google Scholar, 9Abramson 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, 10Salama G. Menshikova E.V. Abramson J.J. Antiox. Redox Signal. 2000; 2: 5-16Crossref PubMed Scopus (46) Google Scholar). Sulfhydryl-reducing agents have been shown to reverse the effect of some oxidants to close the channel. Such studies demonstrated that skeletal and cardiac RyRs contain hyper-reactive or critical thiols of low pK avalues that could be oxidized and reduced to reversibly open and close the Ca2+-release channel (7Zaidi 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, 9Abramson 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). Heavy metals and sulfhydryl oxidants, much like Ca2+-induced Ca2+ release, exhibit biphasic effects on RyRs, where low concentrations activate and high concentrations inhibit the open probability of the channel (10Salama G. Menshikova E.V. Abramson J.J. Antiox. Redox Signal. 2000; 2: 5-16Crossref PubMed Scopus (46) Google Scholar, 11Moutin M.J. Abramson J.J. Salama G. Dupont Y. Biochim. Biophys. Acta. 1989; 984: 289-292Crossref PubMed Scopus (19) Google Scholar, 12Marengo J.J. Hidalgo C. Bull R. Biophys. J. 1998; 74: 1263-1277Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Stoyanovsky et al. (13Stoyanovsky D.A. Murphy T. Anno R.P. Kim Y.-M. Salama G. Cell Calcium. 1997; 21: 19-29Crossref PubMed Scopus (231) Google Scholar) first showed that NO donors oxidize free thiols on RyRs causing an increase in the open probability of the channel (P o) and eliciting Ca2+release from skeletal and cardiac SR vesicles. NO gas and NO donors interact with RyRs to promote channel opening by a nitrosylation of critical sulfhydryl sites involved in the modulation of channel gating, and the effect is reversed by sulfhydryl-reducing agents (e.g. reduced glutathione, GSH; dithiothreitol, DTT; andl-cysteine) (10Salama G. Menshikova E.V. Abramson J.J. Antiox. Redox Signal. 2000; 2: 5-16Crossref PubMed Scopus (46) Google Scholar, 13Stoyanovsky D.A. Murphy T. Anno R.P. Kim Y.-M. Salama G. Cell Calcium. 1997; 21: 19-29Crossref PubMed Scopus (231) Google Scholar). NO gas elicited SR Ca2+but required high concentrations both in ambient oxygen levels (200–400 μm) and deoxygenated solutions (60–200 μm). Because SR Ca2+ transport is not altered by oxygen levels this indicated that direct nitrosylation of RyRs by NO gas is not biologically significant (13Stoyanovsky D.A. Murphy T. Anno R.P. Kim Y.-M. Salama G. Cell Calcium. 1997; 21: 19-29Crossref PubMed Scopus (231) Google Scholar). Unlike authentic NO and non-thiol NO donors, nitrosothiols such asS-nitrosocysteine (cys-SNO) andS-nitroso-N-acetyl-d-l-penicillamine (SNAP) were potent at activating RyRs even in the presence of ambient oxygen levels because of a robust transnitrosylation of NO from the donor to critical thiols on RyRs (13Stoyanovsky D.A. Murphy T. Anno R.P. Kim Y.-M. Salama G. Cell Calcium. 1997; 21: 19-29Crossref PubMed Scopus (231) Google Scholar). As with other sulfhydryl oxidants, the binding of radiolabeled [3H]ryanodine to its receptor was inhibited by the NO donor cys-SNO, whereas the activity of Ca2+-ATPases was not altered by NO (13Stoyanovsky D.A. Murphy T. Anno R.P. Kim Y.-M. Salama G. Cell Calcium. 1997; 21: 19-29Crossref PubMed Scopus (231) Google Scholar). In contrast, Mészàros et al. (14Mészàros L.G. Minarovic I. Zahradnikova A. FEBS Lett. 1996; 380: 49-52Crossref PubMed Scopus (132) Google Scholar) reported that SNAP inhibited SR Ca2+ release by partially reversing caffeine-induced Ca2+ release and decreasedP o of cardiac RyRs in planar bilayers (15Zahradnikova A. Minarovic I. Venema R.C. Mészàros L.G. Cell Calcium. 1997; 22: 447-454Crossref PubMed Scopus (106) Google Scholar). Subsequent studies confirmed that sulfhydryl NO donors activate cardiac RyRs (16Xu L. Eu J.P. Meissner G. Stamler J.S. Science. 1998; 279: 234-237Crossref PubMed Scopus (860) Google Scholar) and showed that NO-like sulfhydryl oxidants and Ca2+-induced Ca2+ release elicits a biphasic response, first an activation at low NO concentrations and then an inhibition at higher concentrations (17Suko J. Drobny H. Hellmann G. Biochim. Biophys. Acta. 1999; 1451: 271-287Crossref PubMed Scopus (26) Google Scholar, 18Hart J.D. Dulhunty A.F. J. Membr. Biol. 2000; 173: 227-236Crossref PubMed Scopus (82) Google Scholar). The divergent findings of the effects of NO on Ca2+-release channel could arise from differences in the reaction conditions and/or NO concentration. Aghdasi et al. (19Aghdasi B. Zhang J.Z. Wu Y. Reid M.B. Hamilton S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) proposed that NO at classes of thiols on the NO concentration. NO concentrations oxidize thiols that inhibit channel and high NO levels oxidize thiols that activate RyRs (19Aghdasi B. Zhang J.Z. Wu Y. Reid M.B. Hamilton S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). were on the multiple classes of with which the N-ethylmaleimide (NEM) could interact (19Aghdasi B. Zhang J.Z. Wu Y. Reid M.B. Hamilton S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). In RyRs in planar bilayers, NEM (5 mm) 3 phases of channel as a function of was a in P o then was an increase and was a (19Aghdasi B. Zhang J.Z. Wu Y. Reid M.B. Hamilton S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). NEM (0.2 mm) concentrations decreased P o of the and a addition of NEM (5 mm) increased P o (19Aghdasi B. Zhang J.Z. Wu Y. Reid M.B. Hamilton S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). The phases of channel activity elicited by NEM (5 mm) to phases of alkylation to classes of sulfhydryl sites on RyRs, with for was by of [3H]ryanodine binding a and then In the sulfhydryl was shown to activate RyRs in bilayers and to RyR to RyRs by could be by NEM (5 mm), and alkylation with NEM mm) the of by that alkylation of 1 by NEM the of elicited by (19Aghdasi B. Zhang J.Z. Wu Y. Reid M.B. Hamilton S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). NEM concentrations mm) high sulfhydryl sites reducing RyR activity (P o) and not to to and 3 alkylation to ryanodine binding and in P was that NO donors to mm) like and 1 alkylation of RyRs, as an increase in ryanodine binding during 1 of NEM alkylation B. Reid M.B. Hamilton S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). NO donors also by In contrast, high concentrations of and (5 mm) RyRs by P o and B. Reid M.B. Hamilton S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). Aghdasi et al. B. Reid M.B. Hamilton S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) that NO with at classes of on RyRs a high where NO oxidants of 1 and a low where NO as an to activate RyRs and promote are to the biphasic effects of and other sulfhydryl where low concentrations first activate and higher concentrations inhibit channel activity (10Salama G. Menshikova E.V. Abramson J.J. Antiox. Redox Signal. 2000; 2: 5-16Crossref PubMed Scopus (46) Google Scholar, 18Hart J.D. Dulhunty A.F. J. Membr. Biol. 2000; 173: 227-236Crossref PubMed Scopus (82) Google Scholar). The of NO as an to other oxidants and as an of free thiols was demonstrated with but the effects were to direct interaction NO and the Salama G. G. Kagan V.E. Biochem. Biophys. 1998; PubMed Scopus Google E.V. Salama G. G. Kagan V.E. Y. 1999; PubMed Scopus Google Scholar). The the effect of NEM on channel activity for the following The of NEM as a reagent be The of ryanodine binding is not to a of channel for activation of the channel by sulfhydryl oxidants and nitrosylation of the channel. that NEM alkylates free sulfhydryl on RyRs was the that NEM with RyRs by other that could be reversed by sulfhydryl-reducing at low NEM SR were from muscle from the as by Salama and G. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). were in with mm) at and in concentrations were with the with as a Heavy SR were to RyRs by heavy SR with planar lipid bilayers for single channel recordings N.F. J. Lagenaur C. Salama G. Arch. Biochem. Biophys. PubMed Scopus (19) Google Scholar). SR were in a and were then on of a in which a was (5 of of and of The was in a at for Heavy SR were from the and of was added to the heavy SR and at for The heavy SR was in a and in Ca2+ and from SR was the of antipyrylazo at G. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). were in a Ca2+ by the SR was in a reaction the following 1 and at and an to a of and free during Ca2+ of was for each by to the reaction in the presence or of SR vesicles. Ca2+-release were the of heavy SR vesicles. SR was added to the cis an to with a planar lipid of at a of as 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, 13Stoyanovsky D.A. Murphy T. Anno R.P. Kim Y.-M. Salama G. Cell Calcium. 1997; 21: 19-29Crossref PubMed Scopus (231) Google Scholar, N.F. J. Lagenaur C. Salama G. Arch. Biochem. Biophys. PubMed Scopus (19) Google Scholar). The were in at a of and planar bilayers were across a in a channel were in the following cis side, side, and solutions were at with and of with 1 activity was at from and P were from a of 3 of recordings each with a was to were and on for of channel from the was with an to to and was by of and as (13Stoyanovsky D.A. Murphy T. Anno R.P. Kim Y.-M. Salama G. Cell Calcium. 1997; 21: 19-29Crossref PubMed Scopus (231) Google Scholar). was to that the was to cys-SNO, because act as a sulfhydryl-reducing to reverse the of RyRs by NO solutions were an antipyrylazo was from Other were from NEM is an alkylating that been to or thiol on with the that the interaction is for thiols and is As in a by J. Biochem. a of such alkylating is that reaction and the presence of reagent and for thiols be The to be in the of that with other such as and A the effect of concentrations of NEM on skeletal SR vesicles. the SR were with NEM (20–200 μm) was added to the reaction which elicited Ca2+ DTT was then added to NEM was alkylating thiols on the DTT reversed the effects of NEM (20–200 μm) in Ca2+ the SR Ca2+ addition of the Ca2+ the Ca2+ in the indicating that in DTT, the the 1 the other DTT did not reverse NEM-induced Ca2+ release when high concentrations of NEM mm) were with an alkylation of RyRs, which elicited a release of of the an of NEM mm), DTT partially reversed NEM-induced indicating that alkylation a higher of the with the interaction NEM and RyRs. the effect of NEM on SR Ca2+ release be reversed by washing the vesicles, skeletal SR were in low of NEM for and then of NEM by and in reaction SR with NEM were then for to Ca2+ with vesicles, which were in an but were with NEM in the reaction 1 that SR with NEM in the Ca2+ the addition of during an of Ca2+ transport and then release Ca2+ because of NEM-induced The of Ca2+ indicated that NEM by activating Ca2+-release rather than Ca2+ by The addition of DTT the effect of NEM in of Ca2+ by the In contrast, SR that were to NEM and then in the of Ca2+ as vesicles, which were to NEM, and DTT effect on SR Ca2+ the of with NEM μm) was fully reversed by washing the vesicles. A that SR Ca2+ release by low NEM concentrations was reversed by of the sulfhydryl-reducing agents that were The of was DTT was but did not fully inhibit NEM-induced the that release an activation of RyRs The inhibition of NEM-induced Ca2+ release by NO was by the effect of NO We NO donors at a wide range of concentrations and that the inhibition of NEM-induced Ca2+ release by NO donors was on the concentrations of the NO donor to that of The inhibition by NO was at the NEM μm) that release such that low concentrations of NO donors could be is because the higher concentrations of NO donor elicit SR that NEM μm) Ca2+ release from SR and was inhibited by the addition of NO The of of NO donors as of NEM was SNAP the NO donors effect on SR The effect of low NEM concentrations on single channel of skeletal muscle RyRs was by Ca2+-release the of heavy SR with planar channel were in solutions across the with as the 3 As in 3 NEM μm) added to the cis not trans) a increase in P o within was effect on single channel conductance at from In o increased from to in the presence of recordings showed that the presence of NEM μm) on the cis for to did not in P and an inhibition of the channel was not concentrations and μm) of NEM effect on single channel for and NEM also increased P o but with a of activation In bilayers, the effect of NEM was on the on channel activation by NEM μm) was reversed by 1 DTT, in a in P o to in 3 N-methylmaleimide effects as NEM on SR Ca2+ As shown in at SR Ca2+ release, which was fully reversed by concentrations mm) elicited release that was not reversed by Other alkylating agents like iodoacetic acid and (not also elicited SR Ca2+ release but with and were partially reversed by As for NEM, ruthenium inhibited release by alkylating but did not fully release A and with a inhibition of SR and and Google Scholar, L. Rev. Biochem. PubMed Scopus Google Scholar). with a which is structurally related to NEM, N-ethyl succinimide also elicited SR Ca2+ release at and as shown for the of release was with NEM and Ca2+ release was not reversed by DTT as expected for a non-sulfhydryl reagent and was by ruthenium with an activation of RyRs. studies have shown that sulfhydryl an in the of skeletal and cardiac muscle (10Salama G. Menshikova E.V. Abramson J.J. Antiox. Redox Signal. 2000; 2: 5-16Crossref PubMed Scopus (46) Google Scholar). been demonstrated that skeletal and cardiac RyRs contain critical thiols of low pK a that be oxidized and reduced to reversibly open and close the Ca2+-release channel. thiols on RyR also be oxidized by NO donors in an increase in the open probability of the channel and Ca2+ release from skeletal and cardiac SR (13Stoyanovsky D.A. Murphy T. Anno R.P. Kim Y.-M. Salama G. Cell Calcium. 1997; 21: 19-29Crossref PubMed Scopus (231) Google Scholar, L. Eu J.P. Meissner G. Stamler J.S. Science. 1998; 279: 234-237Crossref PubMed Scopus (860) Google Scholar, J. Drobny H. Hellmann G. Biochim. Biophys. Acta. 1999; 1451: 271-287Crossref PubMed Scopus (26) Google Scholar, 18Hart J.D. Dulhunty A.F. J. Membr. Biol. 2000; 173: 227-236Crossref PubMed Scopus (82) Google Scholar). activation most likely the transnitrosylation of critical sulfhydryl sites on RyRs in the of or by with thiols on The effects of NO and other sulfhydryl oxidants the multiple that and the to the critical on RyRs to the of NO in the of force in striated muscle (10Salama G. Menshikova E.V. Abramson J.J. Antiox. Redox Signal. 2000; 2: 5-16Crossref PubMed Scopus (46) Google Scholar). The findings of this are that low and high NEM concentrations act by concentrations of NEM (20–200 μm) activate RyRs but not the because the effect is fully reversed by washing NEM or by sulfhydryl-reducing agents. High concentrations of NEM (5 mm) also activate the channel but are not with sulfhydryl-reducing agents. is to that low NEM concentrations activate RyRs and partially inhibit because NEM effects were not fully reversed by ruthenium red. The is to be inhibited by sulfhydryl and to sulfhydryl of the of the are for activity and and Google Scholar, L. Rev. Biochem. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, B. PubMed Scopus Google Scholar). High concentrations of alkylating are required and to inhibit concentrations of alkylating agents are which is with the of Ca2+ a of the SR to NEM 1 the other of SR with low NEM concentrations partially inhibit as shown in B. The of NEM-induced Ca2+ release by DTT that the added DTT the effect of NEM by the alkylation of DTT by NEM and that NEM did not inhibit because the the release findings that at low concentrations, NEM activates RyRs by a interaction and at high concentrations by alkylating free thiols on RyRs. The NO donors and were at concentrations that effect on channel activity and were to inhibit NEM-induced Ca2+ release from SR vesicles. NO donors most likely inhibit the effect of low NEM by with NEM, because the inhibition is at low NEM concentrations with NO donor concentrations and at NEM levels that not the Thus, inhibition of NEM-induced Ca2+ release by NO be by a of on RyRs, because the of NEM does not a reaction. findings are by with other alkylating agents and with a structurally related to In elicited SR Ca2+ release, and the effects of the thiol were reversed by DTT, and as the non-thiol reagent was not reversed by The that a non-thiol reagent structurally related to NEM, elicits SR Ca2+ release that such release without the of a sulfhydryl interaction with RyRs. The of Ca2+ release by ruthenium that at RyRs. Aghdasi et al. (19Aghdasi B. Zhang J.Z. Wu Y. Reid M.B. Hamilton S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) reported phases of channel from of ryanodine binding with the that ryanodine binding is a direct of single channel The the the the channel. In is that agents that increase and channel activity increase and ryanodine binding that is not the because sulfhydryl that activate RyRs have the effect on ryanodine The heavy is a robust of RyRs (4Salama G. Abramson J.J. J. Biol. Chem. 1984; 259: 13363-13369Abstract Full Text PDF PubMed Google Scholar) and ryanodine from its high binding in seconds of the of Pharmacol. 1986; Scholar). reactive and NO activate RyRs and inhibit ryanodine binding (7Zaidi 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, 13Stoyanovsky D.A. Murphy T. Anno R.P. Kim Y.-M. Salama G. Cell Calcium. 1997; 21: 19-29Crossref PubMed Scopus (231) Google Scholar). Aghdasi et B. Zhang J.Z. Wu Y. Reid M.B. Hamilton S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) that low NEM concentrations thiols involved in 1 channel inhibition without to phases and was shown as an inhibition of RyR activity with in bilayers and a in ryanodine binding with and NEM (19Aghdasi B. Zhang J.Z. Wu Y. Reid M.B. Hamilton S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). was to that low NEM concentrations thiols or that or 1 NEM inhibited activity in single channel recordings to phases and 3 of channel In contrast, that low concentrations of NEM (0.2 mm) RyRs in and of channel inhibition in or SR In the activation of the channel by low concentrations of NEM was reversed by DTT with the of NEM-induced Ca2+ release in by washing NEM or The most likely is that DTT with NEM to NEM and NEM-induced Ca2+ high NEM concentrations (5 mm), Ca2+ release from SR was not reversed by DTT as expected for an alkylation of thiol studies have shown that NO donors (13Stoyanovsky D.A. Murphy T. Anno R.P. Kim Y.-M. Salama G. Cell Calcium. 1997; 21: 19-29Crossref PubMed Scopus (231) Google Scholar, L. Eu J.P. Meissner G. Stamler J.S. Science. 1998; 279: 234-237Crossref PubMed Scopus (860) Google Scholar, J. Drobny H. Hellmann G. Biochim. Biophys. Acta. 1999; 1451: 271-287Crossref PubMed Scopus (26) Google Scholar) or inhibited (14Mészàros L.G. Minarovic I. Zahradnikova A. FEBS Lett. 1996; 380: 49-52Crossref PubMed Scopus (132) Google Scholar, A. Minarovic I. Venema R.C. Mészàros L.G. Cell Calcium. 1997; 22: 447-454Crossref PubMed Scopus (106) Google Scholar) skeletal and cardiac Aghdasi et al. (19Aghdasi B. Zhang J.Z. Wu Y. Reid M.B. Hamilton S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) proposed that low levels of NO and high levels activate the were on the findings that NEM could interact with multiple classes of in inactivation or activation of RyR as a function of time and NEM (19Aghdasi B. Zhang J.Z. Wu Y. Reid M.B. Hamilton S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). NO and the 1 effect of NO donors, at low concentrations that have effect on channel whereas higher NO levels activate the channel (17Suko J. Drobny H. Hellmann G. Biochim. Biophys. Acta. 1999; 1451: 271-287Crossref PubMed Scopus (26) Google Scholar). The suggested that the effects of NO from interaction with show that NO donors partially reverse the effects of low of NEM, but the of this reaction also be by washing the SR or by a sulfhydryl-reducing SR were with NO donors at concentrations of NO donors, the NEM-induced Ca2+ release was inhibited but not fully In this NO to interact with NEM, its rather than causing an inhibition of NEM-induced Ca2+ release an inactivation of RyRs. The findings that NEM alkylates thiols on RyRs, but low concentrations (20–200 μm) act by a interaction indicating that 1 effects as a in ryanodine binding not to the inhibition of the channel with low NEM The of the on ryanodine binding and the that ryanodine binding is a of channel activity