Identification of a Key Determinant of Ryanodine Receptor Type 1 Required for Activation by 4-Chloro-m-cresol

4-Chloro-m-cresol (4-CmC) is a potent and specific activator of the intracellular Ca2+ release channel, the ryanodine receptor (RyR). We have previously shown that RyR1 expressed in dyspedic 1B5 myotubes is activated by 4-CmC, whereas RyR3 is not (Fessenden, J. D., Wang, Y., Moore, R. A., Chen, S. R. W., Allen, P. D., and Pessah, I. N. (2000) Biophys. J. 79, 2509–2525). To identify region(s) on RyR1 that are responsible for mediating activation by 4-CmC, we expressed RyR1-RyR3 chimeric proteins in dyspedic 1B5 myotubes and then measured 4-CmC-induced increases in intracellular Ca2+. Substitution of the C-terminal third of RyR1 into RyR3 imparted 4-CmC sensitivity to the resulting chimera, thus suggesting that determinants required for activation by 4-CmC are located in this region. We subdivided the C-terminal third of RyR1 into smaller segments and identified two overlapping regions of RyR1 (amino acids 3769–4180 and 4007–4382) that each imparted 4-CmC sensitivity to RyR3. Substitution of the 173 amino acids of RyR1 common to these two chimeras (amino acids 4007–4180) also weakly restored 4-CmC sensitivity in the resulting chimera. To confirm these findings, we created a complementary set of chimeras containing RyR3 substitutions in RyR1. Substitution of the RyR3 C terminus into RyR1 disrupted 4-CmC sensitivity in the resulting chimera. In addition, substitution of the corresponding RyR3 sequence into positions 4007–4180 of RyR1 disrupted 4-CmC sensitivity. Taken together, these results suggest that essential determinants required for activation of RyR1 by 4-CmC reside within a 173-amino acid region between residues 4007 and 4180. 4-Chloro-m-cresol (4-CmC) is a potent and specific activator of the intracellular Ca2+ release channel, the ryanodine receptor (RyR). We have previously shown that RyR1 expressed in dyspedic 1B5 myotubes is activated by 4-CmC, whereas RyR3 is not (Fessenden, J. D., Wang, Y., Moore, R. A., Chen, S. R. W., Allen, P. D., and Pessah, I. N. (2000) Biophys. J. 79, 2509–2525). To identify region(s) on RyR1 that are responsible for mediating activation by 4-CmC, we expressed RyR1-RyR3 chimeric proteins in dyspedic 1B5 myotubes and then measured 4-CmC-induced increases in intracellular Ca2+. Substitution of the C-terminal third of RyR1 into RyR3 imparted 4-CmC sensitivity to the resulting chimera, thus suggesting that determinants required for activation by 4-CmC are located in this region. We subdivided the C-terminal third of RyR1 into smaller segments and identified two overlapping regions of RyR1 (amino acids 3769–4180 and 4007–4382) that each imparted 4-CmC sensitivity to RyR3. Substitution of the 173 amino acids of RyR1 common to these two chimeras (amino acids 4007–4180) also weakly restored 4-CmC sensitivity in the resulting chimera. To confirm these findings, we created a complementary set of chimeras containing RyR3 substitutions in RyR1. Substitution of the RyR3 C terminus into RyR1 disrupted 4-CmC sensitivity in the resulting chimera. In addition, substitution of the corresponding RyR3 sequence into positions 4007–4180 of RyR1 disrupted 4-CmC sensitivity. Taken together, these results suggest that essential determinants required for activation of RyR1 by 4-CmC reside within a 173-amino acid region between residues 4007 and 4180. Skeletal muscle contraction occurs in response to depolarization of the muscle cell plasma membrane (the sarcolemma) via excitation-contraction coupling. In this process, sarcolemmal depolarization is detected by voltage-gated L-type Ca2+ channels, which, in turn, activate the intracellular Ca2+ release channel known as the ryanodine receptor (RyR). 1The abbreviations used are: RyR, ryanodine receptor; wtRyR, wild-type ryanodine receptor; MH, malignant hyperthermia; 4-CmC, 4-chloro-m-cresol; HSV, herpes simplex virus. Stimulation of this protein releases stored Ca2+ into the cytosol, which then activates the cellular contractile apparatus to initiate muscle contraction. RyRs are modulated by numerous exogenous and endogenous compounds. The principal endogenous modulator of the RyR is Ca2+, which, at micromolar levels, can activate the RyR via Ca2+-induced Ca2+ release. Moreover, studies of [3H]ryanodine binding to sarcoplasmic reticulum membranes (2Pessah I.N. Francini A.O. Scales D.J. Waterhouse A.L. Casida J.E. J. Biol. Chem. 1986; 261: 8643-8648Abstract Full Text PDF PubMed Google Scholar) and single RyRs fused into lipids bilayer (3Smith J.S. Coronado R. Meissner G. J. Gen. Physiol. 1986; 88: 573-588Crossref PubMed Scopus (306) Google Scholar) indicate that Ca2+ has a biphasic effect since millimolar concentrations of the ion can inhibit the channel activity. Other endogenous RyR modulators include magnesium (an inhibitor) (4Pessah I.N. Stambuk R.A. Casida J.E. Mol. Pharmacol. 1987; 31: 232-238PubMed Google Scholar, 5Meissner G. Henderson J.S. J. Biol. Chem. 1987; 262: 3065-3073Abstract Full Text PDF PubMed Google Scholar, 6Laver D.R. Baynes T.M. Dulhunty A.F. J. Membr. Biol. 1997; 156: 213-229Crossref PubMed Scopus (170) Google Scholar) and adenine nucleotides (activators) (4Pessah I.N. Stambuk R.A. Casida J.E. Mol. Pharmacol. 1987; 31: 232-238PubMed Google Scholar, 5Meissner G. Henderson J.S. J. Biol. Chem. 1987; 262: 3065-3073Abstract Full Text PDF PubMed Google Scholar) as well as the cellular oxidation/reduction state, which has complex effects on channel activity (7Abramson J.J. Salama G. Mol. Cell. Biochem. 1988; 82: 81-84Crossref PubMed Scopus (44) Google Scholar, 8Feng W. Liu G. Allen P.D. Pessah I.N. J. Biol. Chem. 2000; 275: 35902-35907Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). In addition, exogenous modulators that can alter RyR activity have been discovered. A number of these agents, including ryanodine, caffeine, and ruthenium red, have become specific tools used to pharmacologically identify intracellular signaling pathways involving RyRs (9Zucchi R. Ronca-Testoni S. Pharmacol. Rev. 1997; 49: 1-51PubMed Google Scholar). In addition, the RyR inhibitor dantrolene is used to treat episodes of malignant hyperthermia (MH), a human skeletal muscle disorder that can result in uncontrolled Ca2+ release and sustained muscle contraction (10Mickelson J.R. Louis C.F. Physiol. Rev. 1996; 76: 537-592Crossref PubMed Scopus (263) Google Scholar). Recently, a new RyR activator has been discovered, 4-chloro-m-cresol (4-CmC) (11Herrmann-Frank A. Richter M. Lehmann-Horn F. Biochem. Pharmacol. 1996; 52: 149-155Crossref PubMed Scopus (57) Google Scholar, 12Baur C.P. Bellon L. Felleiter P. Fiege M. Fricker R. Glahn K. Heffron J.J. Herrmann-Frank A. Jurkat-Rott K. Klingler W. Lehane M. Ording H. Tegazzin V. Wappler F. Georgieff M. Lehmann-Horn F. Anesth. Analg. 2000; 90: 200-205Crossref PubMed Scopus (41) Google Scholar, 13Tegazzin V. Scutari E. Treves S. Zorzato F. Anesthesiology. 1996; 84: 1380-1385Crossref PubMed Scopus (49) Google Scholar). Originally used as a preservative in commercial preparations of some intravenous drugs, including succinylcholine, 4-CmC can directly activate the RyR with 10–25-fold higher potency compared with the more commonly used RyR activator, caffeine (14Zorzato F. Scutari E. Tegazzini V. Clementi E. Treves S. Mol. Pharmacol. 1993; 44: 1192-1201PubMed Google Scholar, 15Herrmann-Frank A. Richter M. Sarkozi S. Mohr U. Lehmann-Horn F. Biochim. Biophys. Acta. 1996; 1289: 31-40Crossref PubMed Scopus (136) Google Scholar, 16Westerblad H. Andrade F.H. Islam M.S. Cell Calcium. 1998; 24: 105-115Crossref PubMed Scopus (45) Google Scholar). In addition, the sensitivity to activation by 4-CmC is increased for RyR1 channels containing point mutations that have been linked to MH (11Herrmann-Frank A. Richter M. Lehmann-Horn F. Biochem. Pharmacol. 1996; 52: 149-155Crossref PubMed Scopus (57) Google Scholar, 17Girard T. Cavagna D. Padovan E. Spagnoli G. Urwyler A. Zorzato F. Treves S. J. Biol. Chem. 2001; 276: 48077-48082Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 18Richter M. Schleithoff L. Deufel T. Lehmann-Horn F. Herrmann-Frank A. J. Biol. Chem. 1997; 272: 5256-5260Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Indeed, because of its higher potency relative to caffeine, 4-CmC has been recommended as a supplemental test substance in the in vitro contracture test for diagnosing MH in humans (12Baur C.P. Bellon L. Felleiter P. Fiege M. Fricker R. Glahn K. Heffron J.J. Herrmann-Frank A. Jurkat-Rott K. Klingler W. Lehane M. Ording H. Tegazzin V. Wappler F. Georgieff M. Lehmann-Horn F. Anesth. Analg. 2000; 90: 200-205Crossref PubMed Scopus (41) Google Scholar). Although little is known about the molecular mechanism of action of 4-CmC, it has been suggested that this compound acts similarly to caffeine because both compounds increase the sensitivity of the RyR to activation by Ca2+ (15Herrmann-Frank A. Richter M. Sarkozi S. Mohr U. Lehmann-Horn F. Biochim. Biophys. Acta. 1996; 1289: 31-40Crossref PubMed Scopus (136) Google Scholar). In addition, studies on isolated RyR1 channels indicate that 4-CmC acts preferentially on the luminal side of the channel (15Herrmann-Frank A. Richter M. Sarkozi S. Mohr U. Lehmann-Horn F. Biochim. Biophys. Acta. 1996; 1289: 31-40Crossref PubMed Scopus (136) Google Scholar). 4-CmC can cause contractures of both ventricular (19Choisy S. Huchet-Cadiou C. Leoty C. J. Pharmacol. Exp. Ther. 1999; 290: 578-586PubMed Google Scholar) and skeletal (20Choisy S. Huchet-Cadiou C. Leoty C. J. Pharmacol. Exp. Ther. 2000; 294: 884-893PubMed Google Scholar) muscles, thus suggesting that it can activate at least two of the three RyR isoforms, RyR1 (skeletal) and RyR2 (cardiac). Interestingly, 4-CmC can also activate RyR3 with an EC50 of ∼1.5 mm (21Matyash M. Matyash V. Nolte C. Sorrentino V. Kettenmann H. FASEB J. 2002; 16: 84-86Crossref PubMed Scopus (96) Google Scholar), thus indicating that 4-CmC can activate this isoform, albeit with a 10-fold lower potency compared with its ability to activate RyR1. These findings have been confirmed by recent studies indicating that concentrations of 4-CmC up to 0.5 mm can fully activate Ca2+ release in dyspedic 1B5 myotubes expressing recombinant RyR1 and RyR2, but not RyR3 (1Fessenden J.D. Wang Y. Moore R.A. Chen S.R.W. Allen P.D. Pessah I.N. Biophys. J. 2000; 79: 2509-2525Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 22Lee E.H. Protasi F. Pessah I.N. Kim D.H. Allen P.D. Biophys. Soc. Annu. Meet. Abstr. 2002; 82: 374Google Scholar). One hypothesis that may explain these findings is that sequence divergence within RyR3 could lower the affinity and/or efficacy for activation by 4-CmC compared with RyR1 and RyR2. To determine the site(s) on RyR1 necessary for activation by 4-CmC, we have taken advantage of these isoform-specific differences in 4-CmC sensitivity and constructed RyR1-RyR3 chimeric proteins. If 4-CmC acts on a discrete region of RyR1, then substitution of this region into RyR3 should restore 4-CmC sensitivity to the chimeric protein. In contrast, substitution of RyR3 into this region of RyR1 should 4-CmC-induced activation of RyR1. the of a of chimeric we have identified a 173-amino acid in RyR1 that activation by the of RyR1 and RyR3 have previously been into thus the of required for of 1B5 myotubes Y. C. Protasi F. Moore R.A. J.D. Pessah I.N. A. Allen P.D. J. Physiol. 2000; PubMed Google Scholar). The chimeras constructed via of between the as RyR3 sequence is in and an to a new as a via in the are chimeras as and and and and and and and and and and chimeras as and and and and and and and The of the RyR1 used to the by as and and and The of the RyR3 used to the by are as and and and and into and confirmed Cell myotubes as (1Fessenden J.D. Wang Y. Moore R.A. Chen S.R.W. Allen P.D. Pessah I.N. Biophys. J. 2000; 79: 2509-2525Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, R.A. H. J. Pessah I.N. Allen P.D. J. Cell Biol. 1998; PubMed Scopus Google Scholar). 1B5 in with and mm at at a and by the to with and as at at myotubes with the RyR of Y. C. Protasi F. Moore R.A. J.D. Pessah I.N. A. Allen P.D. J. Physiol. 2000; PubMed Google Scholar) containing the the a at in the and the myotubes for to of the RyRs in intracellular Ca2+ in 1B5 myotubes measured the Ca2+ 1B5 myotubes in the of for at in mm mm mm mm and mm with with in intracellular Ca2+ an and a a set at the 1B5 myotubes measured an and on a at the of and in of regions of stored in and with with of mm mm caffeine, and 0.5 mm 4-CmC in These to the myotubes a a RyR are as a single by of 1B5 myotubes with that the of the at the of the Ca2+ by the RyR of relative of Ca2+ in response to each RyR by the in of the then to the of the cell to of the of with RyR by expressing the of each response as a of the of the mm Ca2+ the These each RyR then compared with the the 4-CmC-induced of with A at and membrane preparations used for [3H]ryanodine binding studies and as previously C.F. A. Pessah I.N. Allen P.D. Biophys. J. 84: Full Text Full Text PDF PubMed Scopus Google Scholar). with RyR 1B5 myotubes in mm mm mm mm and mm and at The myotubes then in of mm and mm with mm and mm a cell the at for the at for at The resulting in and mm into and in The RyRs in these membrane preparations of membranes 1B5 myotubes with and on a to a which then with and proteins activity of binding of [3H]ryanodine to sarcoplasmic reticulum preparations the by Pessah (4Pessah I.N. Stambuk R.A. Casida J.E. Mol. Pharmacol. 1987; 31: 232-238PubMed Google Scholar). The ability of recombinant and skeletal muscle sarcoplasmic reticulum preparations to [3H]ryanodine activity of [3H]ryanodine by skeletal sarcoplasmic reticulum in of mm mm and Ca2+ concentrations with the and Biochem. PubMed Scopus Google Scholar). to at for with then by on and with of mm and The of [3H]ryanodine to each by complementary to regions of sequence required for 4-CmC activation of RyR1. we segments of RyR3 into the corresponding regions of RyR1 and identified substitutions that to 4-CmC to depolarization a of activation by 4-CmC to determinants required for to confirm the of regions identified this we created a set of chimeras that RyR1 substitutions in RyR3 and which RyR1 substitutions 4-CmC activation of RyR3. these two should identify regions in RyR1 that are essential for mediating activation by and chimeric RyR into 1B5 myotubes Y. C. Protasi F. Moore R.A. J.D. Pessah I.N. A. Allen P.D. J. Physiol. 2000; PubMed Google Scholar). These myotubes then with and with three test myotubes with mm a that Ca2+ release in 1B5 myotubes expressing RyR1 (1Fessenden J.D. Wang Y. Moore R.A. Chen S.R.W. Allen P.D. Pessah I.N. Biophys. J. 2000; 79: 2509-2525Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, R.A. H. J. Pessah I.N. Allen P.D. J. Cell Biol. 1998; PubMed Scopus Google Scholar). RyR1 not can excitation-contraction in 1B5 myotubes (1Fessenden J.D. Wang Y. Moore R.A. Chen S.R.W. Allen P.D. Pessah I.N. Biophys. J. 2000; 79: 2509-2525Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, C.F. A. Pessah I.N. Allen P.D. Biophys. J. 84: Full Text Full Text PDF PubMed Scopus Google Scholar), this depolarization of the used to the of the chimeric receptor RyR3 then with mm caffeine to that the expressed chimeric protein myotubes with 0.5 mm 4-CmC, a that activates RyR1, but has little effect on RyR3 (1Fessenden J.D. Wang Y. Moore R.A. Chen S.R.W. Allen P.D. Pessah I.N. Biophys. J. 2000; 79: 2509-2525Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). we the 4-CmC is located in the region the of the We constructed two chimeric RyRs of a RyR1 with the C-terminal amino acids the of the with the RyR3 and the of of a RyR3 containing the corresponding C-terminal amino acids RyR1. RyR Ca2+ release myotubes with these compared with that myotubes expressing and expressing to both and caffeine, thus that the chimeric receptor and that it a RyR1 4-CmC-induced Ca2+ in myotubes relative to in these myotubes to mm caffeine by an to 4-CmC-induced in 1B5 myotubes We then expressing this not to depolarization to the to Interestingly, these myotubes to 4-CmC, and the of these to that of the by myotubes expressing Taken together, these results suggest that the essential required for 4-CmC activation of the RyR reside in the C-terminal third of the protein. To the 4-CmC we the C-terminal third of RyR1 into three of and then the corresponding RyR3 into these three to chimeras Although chimeras to and caffeine compared with amino acids with RyR3 to 4-CmC that compared with whereas amino acids and amino acids amino acids with RyR3 containing a RyR3 substitution in an overlapping region between chimeras and also to 4-CmC Taken together, these studies indicate that determinants required for activation by 4-CmC reside between RyR1 amino acids and the RyR1 region with RyR3 sequence in chimeras and To confirm these findings, we created a of chimeras in which the regions of RyR1 into RyR3 substitution of RyR3 sequence into the C-terminal amino acids of RyR1 not 4-CmC-induced Ca2+ we the of chimeras and of and restored to 4-CmC that compared with 4-CmC in myotubes expressing is since substitution of the corresponding RyR3 into the and regions of 4-CmC activation of RyR1 and in 4-CmC activation between myotubes expressing and To the 4-CmC subdivided into and of these RyR3 into RyR1 Substitution of RyR3 into the of the RyR1 region RyR1 amino acids in a chimeric RyR with to 4-CmC to of and substitution of RyR3 into the C-terminal of RyR1 amino acids 4007–4180 to 4-CmC to caffeine is since the region of the overlapping between chimeras and both of which not to of this region into and in proteins with to 4-CmC that not in compared with of To determine the and/or of could 4-CmC activation to we created the and to 0.5 mm 4-CmC, myotubes expressing but not to 4-CmC that of myotubes expressing The of Ca2+ by 4-CmC in myotubes of that of the thus suggesting that essential determinants required for 4-CmC-induced activation of RyR1 channels reside in the region (amino acids are required to fully restore the 4-CmC in RyR1. of the 4-CmC by the ability of 4-CmC to affinity binding of [3H]ryanodine to and chimeras and We sarcoplasmic reticulum skeletal muscle a of 1B5 myotubes expressing The molecular and of these proteins confirmed not We then measured the of [3H]ryanodine binding to these RyRs at two Ca2+ In the of Ca2+ mm 4-CmC binding of [3H]ryanodine to to of [3H]ryanodine of protein. Interestingly, 4-CmC to [3H]ryanodine binding to isolated myotubes expressing a with in In addition, 4-CmC binding of [3H]ryanodine to isolated myotubes expressing to of [3H]ryanodine of protein. To determine the effect of Ca2+ on [3H]ryanodine binding to these we also a set of binding in the of Ca2+ 10-fold higher Ca2+ the of [3H]ryanodine binding for 4-CmC [3H]ryanodine binding to both and in the of Ca2+, 4-CmC also [3H]ryanodine binding to membrane preparations containing and and These results suggest that the ability of 4-CmC to [3H]ryanodine binding to the RyR is on Ca2+ a that is with (15Herrmann-Frank A. Richter M. Sarkozi S. Mohr U. Lehmann-Horn F. Biochim. Biophys. Acta. 1996; 1289: 31-40Crossref PubMed Scopus (136) Google Scholar). In of and expressed in 1B5 differences between these (1Fessenden J.D. Wang Y. Moore R.A. Chen S.R.W. Allen P.D. Pessah I.N. Biophys. J. 2000; 79: 2509-2525Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). RyR1 not could Ca2+ release RyR3 more to activation by caffeine compared with RyR1. at the of 4-CmC this compound could activate RyR1, but not RyR3. We advantage of this isoform-specific in 4-CmC sensitivity to regions of RyR1 required for 4-CmC two complementary we have that a principal 4-CmC in the sequence is located within a of 173 amino acids located between positions 4007 and 4180. The RyR1-RyR3 RyRs have in within the RyR sequence in has been used to identify regions of RyR1 that excitation-contraction C.F. A. Pessah I.N. Allen P.D. Biophys. J. 84: Full Text Full Text PDF PubMed Scopus Google Scholar, J. N. Allen P.D. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, F. C. J. C. Allen P.D. Biophys. J. 2002; Full Text Full Text PDF PubMed Scopus (79) Google Scholar), Ca2+ sensitivity D.H. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J. L. L. C. Meissner G. 1999; PubMed Scopus Google Scholar), and caffeine sensitivity D.H. J. 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A advantage to chimeras is that substitution of regions between proteins should result in of the protein compared with thus more specific and in In addition, the of chimeric can by smaller a region a specific between two isoforms, then its this can as a point for to the of the the of RyR1-RyR3 chimeric we have identified a 173-amino acid RyR1 that 4-CmC activation of are required since chimeras containing segments of RyR1 sequence and more fully 4-CmC activation compared with These of RyR1 sequence are required to the for channel activation by One in the of chimeric proteins is that an exogenous amino acid sequence its into a new protein. is protein segments are as smaller and smaller regions are the increases that the amino acid can its The 173-amino acid region we have identified chimeric RyRs may the of this in this specific and 4-CmC [3H]ryanodine binding to an to the 4-CmC Interestingly, binding studies at Ca2+, a that the intracellular Ca2+ in results that well with findings RyR3 and for which 4-CmC in not of [3H]ryanodine binding in the of In addition, of ryanodine binding for and a that cell at Ca2+, a that increases channel activity and [3H]ryanodine ryanodine binding for including and result is not that 4-CmC and Ca2+ are of the of Ca2+ the RyR more to as caffeine, and adenine nucleotides (4Pessah I.N. Stambuk R.A. Casida J.E. Mol. Pharmacol. 1987; 31: 232-238PubMed Google Scholar). In the of RyR3 and Ca2+ may similarly to these channels to activation by 4-CmC can directly activate albeit with efficacy and/or is by studies in indicating that 4-CmC can activate RyR3 with an EC50 of ∼1.5 mm (21Matyash M. Matyash V. Nolte C. Sorrentino V. Kettenmann H. FASEB J. 2002; 16: 84-86Crossref PubMed Scopus (96) Google Scholar). In of RyR1 and RyR3 expressed in 1B5 myotubes (1Fessenden J.D. Wang Y. Moore R.A. Chen S.R.W. Allen P.D. Pessah I.N. Biophys. J. 2000; 79: 2509-2525Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), we not Ca2+ in by 4-CmC concentrations up to 0.5 we not test 4-CmC concentrations higher 0.5 mm at these levels, 4-CmC to Ca2+ in 1B5 Other 4-CmC mutations in the MH sensitivity region of can increase (11Herrmann-Frank A. Richter M. Lehmann-Horn F. Biochem. Pharmacol. 1996; 52: 149-155Crossref PubMed Scopus (57) Google Scholar, 17Girard T. Cavagna D. Padovan E. Spagnoli G. Urwyler A. Zorzato F. Treves S. J. Biol. Chem. 2001; 276: 48077-48082Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 18Richter M. Schleithoff L. Deufel T. Lehmann-Horn F. Herrmann-Frank A. J. Biol. Chem. 1997; 272: 5256-5260Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) H. D.H. Biochem. J. 2001; PubMed Scopus Google Scholar) 4-CmC thus suggesting that this also a 4-CmC these mutations also alter sensitivity to RyR as caffeine and thus suggesting a more effect on RyR activity. Indeed, myotubes expressing RyR1 channels containing MH mutations have of Ca2+ J.R. Allen P.D. L. D. 1988; PubMed Scopus Google Scholar, G. J. Gen. Physiol. 2001; PubMed Scopus Google Scholar, J.R. J. N. Allen P.D. Anesthesiology. 2000; PubMed Scopus Google Scholar). Ca2+ can alter the sensitivity of the RyR to 4-CmC, it that the differences in 4-CmC activation of these MH are to this in intracellular Ca2+ to a in MH sensitivity region mechanism by which 4-CmC activates the RyR to One is that the 173-amino acid in this the and that differences in amino acid between RyR1 and RyR3 result in affinity of 4-CmC binding to this A is that 4-CmC can to both RyR1 and RyR3 with but that this binding is more into channel activation for RyR1. In this the region determinants that 4-CmC binding to channel A is that 4-CmC to an protein on the RyR, which then activates the the we between these since is that 4-CmC directly to the RyR to of its proteins. Although 4-CmC can activate isolated RyR1 proteins in single channel studies and also activate [3H]ryanodine binding to RyR1 in (15Herrmann-Frank A. Richter M. Sarkozi S. Mohr U. Lehmann-Horn F. Biochim. Biophys. Acta. 1996; 1289: 31-40Crossref PubMed Scopus (136) Google Scholar), the that 4-CmC could to a protein. If this is the then the between this protein and the RyR the region in this of the C C-terminal third of the RyR is to a number of including the complex F. J. K. M. Meissner G. D.H. J. Biol. Chem. Full Text PDF PubMed Google the Ca2+ M. P. L. Chen S.R.W. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google and for Ca2+ S.R.W. K. L. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus (108) Google Scholar, H. L. L. Meissner G. 1998; PubMed Scopus Google Scholar), caffeine D.H. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), H. S. T. H. K. N. H. M. M. T. S. PubMed Scopus Google Scholar), and ryanodine C. A. Wang D.H. C. Y. T. J. Biol. Chem. Full Text PDF PubMed Google Scholar), In addition, this region amino acids G. J. Gen. Physiol. 2001; PubMed Scopus Google Scholar) and also the amino acid of which is between the three RyR that the 4-CmC is also located in the C this not with the but to it on the Interestingly, the region a of between RyR1 and with amino acid Moreover, within the the amino acid is with amino is that a well region should in a that is between RyR1 and RyR3. this of should in the amino acids that are required to activation of the RyR by the of RyR1-RyR3 chimeric we have identified a 173-amino acid of RyR1 that can 4-CmC sensitivity to RyR3. These findings suggest that a on RyR1 required for activation by 4-CmC between amino acids 4007 and 4180.