Ryanodine Sensitizes the Ca2+ Release Channel (Ryanodine Receptor) to Ca2+ Activation

Ryanodine, a plant alkaloid, is one of the most widely used pharmacological probes for intracellular Ca2+ signaling in a variety of muscle and non-muscle cells. Upon binding to the Ca2+ release channel (ryanodine receptor), ryanodine causes two major changes in the channel: a reduction in single-channel conductance and a marked increase in open probability. The molecular mechanisms underlying these alterations are not well understood. In the present study, we investigated the gating behavior and Ca2+ dependence of the wild type (wt) and a mutant cardiac ryanodine receptor (RyR2) after being modified by ryanodine. Single-channel studies revealed that the ryanodine-modified wt RyR2 channel was sensitive to inhibition by Mg2+ and to activation by caffeine and ATP. In the presence of Mg2+, the ryanodine-modified single wt RyR2 channel displayed a sigmoidal Ca2+ dependence with an EC50 value of 110 nm, whereas the ryanodine-unmodified single wt channel exhibited an EC50 of 120 µm for Ca2+ activation, indicating that ryanodine is able to increase the sensitivity of the wt RyR2 channel to Ca2+activation by ∼1,000-fold. Furthermore, ryanodine is able to restore Ca2+ activation and ligand response of the E3987A mutant RyR2 channel that has been shown to exhibit ∼1,000-fold reduction in Ca2+ sensitivity to activation. The E3987A mutation, however, affects neither [3H]ryanodine binding to nor the stimulatory and inhibitory effects of ryanodine on the RyR2 channel. These results demonstrate that ryanodine does not “lock” the RyR channel into an open state as generally believed; rather, it sensitizes dramatically the channel to activation by Ca2+. Ryanodine, a plant alkaloid, is one of the most widely used pharmacological probes for intracellular Ca2+ signaling in a variety of muscle and non-muscle cells. Upon binding to the Ca2+ release channel (ryanodine receptor), ryanodine causes two major changes in the channel: a reduction in single-channel conductance and a marked increase in open probability. The molecular mechanisms underlying these alterations are not well understood. In the present study, we investigated the gating behavior and Ca2+ dependence of the wild type (wt) and a mutant cardiac ryanodine receptor (RyR2) after being modified by ryanodine. Single-channel studies revealed that the ryanodine-modified wt RyR2 channel was sensitive to inhibition by Mg2+ and to activation by caffeine and ATP. In the presence of Mg2+, the ryanodine-modified single wt RyR2 channel displayed a sigmoidal Ca2+ dependence with an EC50 value of 110 nm, whereas the ryanodine-unmodified single wt channel exhibited an EC50 of 120 µm for Ca2+ activation, indicating that ryanodine is able to increase the sensitivity of the wt RyR2 channel to Ca2+activation by ∼1,000-fold. Furthermore, ryanodine is able to restore Ca2+ activation and ligand response of the E3987A mutant RyR2 channel that has been shown to exhibit ∼1,000-fold reduction in Ca2+ sensitivity to activation. The E3987A mutation, however, affects neither [3H]ryanodine binding to nor the stimulatory and inhibitory effects of ryanodine on the RyR2 channel. These results demonstrate that ryanodine does not “lock” the RyR channel into an open state as generally believed; rather, it sensitizes dramatically the channel to activation by Ca2+. ryanodine receptor wild type 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Krebs-Ringer-Hepes Ryanodine, a plant alkaloid, binds specifically with high affinity to and alters the function of intracellular Ca2+ release channels (ryanodine receptors, RyRs).1 Because of its high affinity and specificity, ryanodine has been widely used as a specific ligand for the identification, purification, cloning, and functional characterization of RyRs (1Coronado R. Morrissette J. Sukhareva M. Vaughan D.M. Am. J. Physiol. 1994; 266: C1485-C1504Crossref PubMed Google Scholar, 2Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (840) Google Scholar, 3Ogawa Y. Crit. Rev. Biochem. Mol. Biol. 1994; 29: 229-274Crossref PubMed Scopus (228) Google Scholar, 4Franzini-Armstrong C. Protasi F. Physiol. Rev. 1997; 77: 699-729Crossref PubMed Scopus (591) Google Scholar, 5Zucchi R. Ronca-Testoni S. Pharmacol. Rev. 1997; 49: 1-51PubMed Google Scholar, 6Sutko J.L. Airey J.A. Welch W. Ruest L. Pharmacol. Rev. 1997; 49: 53-98PubMed Google Scholar, 7Shoshan-Barmatz V. Ashley R.H. Int. Rev. Cytol. 1998; 183: 185-270Crossref PubMed Google Scholar). The unique and specific action of ryanodine on RyR function also has made it an invaluable pharmacological probe for intracellular Ca2+ signaling in a variety of cells (8Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6157) Google Scholar, 9Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2261) Google Scholar), and for understanding the mechanisms of ion conduction and channel gating of RyRs (10Lindsay A.R. Tinker A. Williams A.J. J. Gen. Physiol. 1994; 104: 425-447Crossref PubMed Scopus (55) Google Scholar, 11Xu L. Tripathy A. Pasek D.A. Meissner G. Ann. N. Y. Acad. Sci. 1998; 853: 130-148Crossref PubMed Scopus (5) Google Scholar). Ryanodine has dual actions on the Ca2+ release channel depending on its concentration. Ca2+ flux studies using sarcoplasmic reticulum vesicles revealed that, at nanomolar to micromolar concentrations, ryanodine stimulates Ca2+release from sarcoplasmic reticulum vesicles, whereas, at higher concentrations (micromolar to millimolar range), ryanodine inhibits Ca2+ release (12Meissner G. J. Biol. Chem. 1986; 261: 6300-6306Abstract Full Text PDF PubMed Google Scholar, 13Fleischer S. Ogunbunmi E.M. Dixon M.C. Fleer E.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7256-7259Crossref PubMed Scopus (306) Google Scholar, 14Pessah I.N. Waterhouse A.L. Casida J.E. Fleischer S. Ogunbunmi E.M. Dixon M.C. Fleer E.A. Biochem. Biophys. Res. Commun. 1985; 128: 449-456Crossref PubMed Scopus (227) Google Scholar, 15Lattanzio Jr., F.A. Schlatterer R.G. Nicar M. Campbell K.P. Sutko J.L. J. Biol. Chem. 1987; 262: 2711-2718Abstract Full Text PDF PubMed Google Scholar). Consistent with these Ca2+ release studies, [3H]ryanodine binding analysis established the existence of low and high affinity ryanodine binding sites (16Pessah I.N. Zimanyi I. Mol. Pharmacol. 1991; 39: 679-689PubMed Google Scholar, 17Lai F.A. Erickson H.P. Rousseau E. Liu Q.Y. Meissner G. Nature. 1988; 331: 315-319Crossref PubMed Scopus (68) Google Scholar, 18Inui M. Saito A. Fleischer S. J. Biol. Chem. 1987; 262: 1740-1747Abstract Full Text PDF PubMed Google Scholar, 19Wang J.P. Needleman D.H. Hamilton S.L. J. Biol. Chem. 1993; 268: 20974-20982Abstract Full Text PDF PubMed Google Scholar, 20McGrew S.G. Wolleben C. Siegl P. Inui M. Fleischer S. Biochemistry. 1989; 28: 1686-1691Crossref PubMed Scopus (77) Google Scholar). Binding of ryanodine to the high affinity site was correlated with channel activation, whereas occupation of the low affinity ryanodine binding site was related to channel inhibition (19Wang J.P. Needleman D.H. Hamilton S.L. J. Biol. Chem. 1993; 268: 20974-20982Abstract Full Text PDF PubMed Google Scholar, 20McGrew S.G. Wolleben C. Siegl P. Inui M. Fleischer S. Biochemistry. 1989; 28: 1686-1691Crossref PubMed Scopus (77) Google Scholar, 21Lai F.A. Misra M. Xu L. Smith H.A. Meissner G. J. Biol. Chem. 1989; 264: 16776-16785Abstract Full Text PDF PubMed Google Scholar). These opposite effects of ryanodine have been clearly demonstrated at the single-channel level. Addition of micromolar concentrations of ryanodine caused the RyR channel to enter into a long-lived open state with a reduced single-channel conductance (22Rousseau E. Smith J.S. Meissner G. Am. J. Physiol. 1987; 253: C364-C368Crossref PubMed Google Scholar,23Smith J.S. Imagawa T. Ma J. Fill M. Campbell K.P. Coronado R. J. Gen. Physiol. 1988; 92: 1-26Crossref PubMed Scopus (351) Google Scholar). Exposure to high concentrations (submillimolar to millimolar) of ryanodine led to a persistent blockade of single RyR channels (11Xu L. Tripathy A. Pasek D.A. Meissner G. Ann. N. Y. Acad. Sci. 1998; 853: 130-148Crossref PubMed Scopus (5) Google Scholar,21Lai F.A. Misra M. Xu L. Smith H.A. Meissner G. J. Biol. Chem. 1989; 264: 16776-16785Abstract Full Text PDF PubMed Google Scholar). The molecular mechanisms underlying these complex changes in channel conductance and gating behavior of RyR upon ryanodine modification are not well understood. [3H]Ryanodine binding studies have suggested that complete blockade of the RyR channel by high concentrations of ryanodine may result from the occlusion of the channel conduction pore by ryanodine (19Wang J.P. Needleman D.H. Hamilton S.L. J. Biol. Chem. 1993; 268: 20974-20982Abstract Full Text PDF PubMed Google Scholar). Detailed characterization of the effects of ryanodine or ryanodine derivatives on ion handling by RyR has been reported. These studies have led to the proposal that reduction in channel conductance upon ryanodine modification is likely to result from allosteric alterations in ion permeation and ion binding, and/or from partial block by ryanodine (10Lindsay A.R. Tinker A. Williams A.J. J. Gen. Physiol. 1994; 104: 425-447Crossref PubMed Scopus (55) Google Scholar, 24Tinker A. Sutko J.L. Ruest L. Deslongchamps P. Welch W. Airey J.A. Gerzon K. Bidasee K.R. Besch Jr., H.R. Williams A.J. Biophys. J. 1996; 70: 2110-2119Abstract Full Text PDF PubMed Scopus (51) Google Scholar). Little is known about the molecular mechanism by which ryanodine increases the open probability (Po) of the RyR channels. Ryanodine is thought to “lock” the RyR channel into a subconductance open state upon binding, and the ryanodine-modified RyR channel is thought to be insensitive to modulation by other ligands such as Ca2+, Mg2+, and ruthenium red (5Zucchi R. Ronca-Testoni S. Pharmacol. Rev. 1997; 49: 1-51PubMed Google Scholar, 11Xu L. Tripathy A. Pasek D.A. Meissner G. Ann. N. Y. Acad. Sci. 1998; 853: 130-148Crossref PubMed Scopus (5) Google Scholar,12Meissner G. J. Biol. Chem. 1986; 261: 6300-6306Abstract Full Text PDF PubMed Google Scholar, 22Rousseau E. Smith J.S. Meissner G. Am. J. Physiol. 1987; 253: C364-C368Crossref PubMed Google Scholar). The view that ryanodine locks the RyR channel in an open substate has recently been contested by Tanna et al. (25Tanna B. Welch W. Ruest L. Sutko J.L. Williams A.J. J. Gen. Physiol. 1998; 112: 55-69Crossref PubMed Scopus (42) Google Scholar), who observed that the Po of the channel before ryanodine modification influenced the Po of the ryanodine-modified state. Low Po channels displayed more closing events than high Pochannels after modified by ryanodine. This observation suggests that ryanodine does not simply lock the channel into an open state. This observation also implies that activation of the RyR channel by ryanodine would be finite and regulatable. To test these possibilities, we have examined the gating properties and the Ca2+dependence of the ryanodine-modified wild type cardiac ryanodine receptor (RyR2) and a mutant RyR2 that exhibits a reduced sensitivity to activation by Ca2+. results demonstrate that ryanodine-modified channels are sensitive to and that ryanodine increases dramatically the sensitivity of the RyR2 channel to activation by Ca2+. Ryanodine was from [3H]Ryanodine was from was from and from cells for after using Ca2+ with and in the by in and a and on for was after the by in at for wt and E3987A mutant RyR2 used for single-channel from by as P. J. Gen. Physiol. PubMed Scopus Google Scholar). and in in a and in of at a of of a in a two The was to the of an The was at and was used for of the wt or mutant RyR2 was to the channel was for sensitivity to and Ca2+, about the Ca2+ in the and of the channel. made to that in which the of the of the channel. This to the of the Ca2+ release channel. at The using Ca2+ concentrations using the of and A. F. J. Physiol. Scholar). Ca2+ in cells was using the Ca2+ as P. J. Gen. Physiol. PubMed Scopus Google with cells and with in and and µm used for the of the E. J. F. J. 1994; PubMed Scopus Google with or µm ryanodine at for of cells with and in of and The cells to of in a of at was in an with at [3H]ryanodine binding to was as P. J. Gen. Physiol. PubMed Scopus Google Scholar). binding of and the was at for To [3H]ryanodine binding to the mutant and caffeine in the binding P. J. Gen. Physiol. PubMed Scopus Google Scholar). The binding after of was into of and with or µm and was at for The of [3H]ryanodine that was by using with The and the with the by binding was by [3H]ryanodine binding in the presence of µm ryanodine. binding in single-channel studies that ryanodine the open probability (Po) of the RyR channel to and that, at micromolar Ca2+ concentrations, the ryanodine-modified channels by Mg2+ and ruthenium red (22Rousseau E. Smith J.S. Meissner G. Am. J. Physiol. 1987; 253: C364-C368Crossref PubMed Google Scholar). These have led to the that ryanodine-modified RyR channels are insensitive to modulation (11Xu L. Tripathy A. Pasek D.A. Meissner G. Ann. N. Y. Acad. Sci. 1998; 853: 130-148Crossref PubMed Scopus (5) Google Scholar). for the partial inhibition is that, at micromolar ryanodine activation may be to be by Mg2+ and ruthenium To test we examined the of Mg2+ on ryanodine-modified single RyR2 channels at nanomolar that a single wt RyR2 channel displayed and at Addition of ryanodine the channel into a state with long-lived and a marked increase and a reduced single-channel conductance a and The was reduced to by of which in a reduction of however, of the ryanodine-modified channel The ryanodine-modified RyR2 channel be by of caffeine and by of and in the presence of low concentrations of Ca2+, the ryanodine-modified RyR2 the ryanodine-unmodified are sensitive to modulation by channel To the mechanism of ryanodine activation, we examined the Ca2+ dependence of ryanodine-modified single wt RyR2 channels. single RyR2 channel was modified by ryanodine in the presence of and Mg2+ The Ca2+ was reduced to by of in the ryanodine-modified single RyR2 channel was at Ca2+ and was at Ca2+, a sigmoidal Ca2+ response of the Ca2+ dependence using the an EC50 of and a of in the presence of Mg2+ the other the ryanodine-unmodified channel in the presence of Mg2+ was by Ca2+ at micromolar concentrations and was at The Ca2+ response of the ryanodine-unmodified channel in the presence of be by an EC50 of µm and a of for Ca2+ activation, and an of and a of for Ca2+ These demonstrate that ryanodine is able to the sensitivity of single RyR2 channels to Ca2+ activation by ∼1,000-fold In the of Mg2+, the EC50 for Ca2+ activation of single wt RyR2 channels is µm P. J. Gen. Physiol. PubMed Scopus Google Scholar). on the of the of ryanodine activation, single wt RyR2 channels upon modification by ryanodine would be to be at Ca2+ concentrations in the of shown in the ryanodine-modified single wt RyR2 channels in the of Mg2+ sensitive to Ca2+ activation. channel be the was reduced to as low as and channels at a Ca2+ than and These however, be by a of these that ryanodine dramatically increases the Po of the RyR channel by the channel to To demonstrate the of ryanodine on Ca2+ activation, we a mutant of have shown that a single E3987A reduced the sensitivity to Ca2+ activation of single RyR2 channels by as by single-channel in P. J. Gen. Physiol. PubMed Scopus Google Scholar). that, ryanodine is able to the sensitivity of the wt RyR2 channel to Ca2+ activation by ryanodine would be able to restore Ca2+activation of the E3987A mutant RyR2 channel to a to that of the To we examined the Ca2+ dependence of single E3987A mutant RyR2 channels after being modified by ryanodine. shown in upon modification by a single E3987A mutant RyR2 channel was to a state with a reduced single-channel conductance and a as observed with the wt channel P. J. Gen. Physiol. PubMed Scopus Google Scholar). is to that the ryanodine-modified single E3987A mutant channels open to the ryanodine-unmodified conductance level. In from the ryanodine-unmodified mutant the ryanodine-modified single E3987A mutant channel was sensitive to Ca2+ activation. The Ca2+ dependence of ryanodine-modified single E3987A mutant channels be by an EC50 of µm and a of These are to of single wt RyR2 channels in the of ryanodine P. J. Gen. Physiol. PubMed Scopus Google Scholar). ryanodine is able to restore Ca2+ activation of the E3987A mutant RyR2 channels. Furthermore, that observed with the ryanodine-modified wt RyR2 ryanodine-modified E3987A mutant channels at Ca2+ These however, be by of Mg2+ These that, upon ryanodine the to Ca2+activation of the wt and E3987A mutant RyR2 channels is from that the E3987A mutant channels the wt channel with to activation. To single E3987A mutant channels also to channel in a as does the we the of Mg2+ on the Ca2+dependence of the mutant channels. shown in in the presence of single E3987A mutant channels at µm Ca2+ and at micromolar be that the ryanodine-unmodified single E3987A mutant channels exhibited in the presence of not of the Ca2+dependence that the EC50 and the µm and of the single E3987A mutant channels are to of the ryanodine-unmodified wt channels µm and in the presence of Mg2+ and the single E3987A mutant channels exhibit Ca2+ and Mg2+ to of the ryanodine-unmodified wt channels. In other ryanodine the Ca2+ and Mg2+ of the E3987A mutant RyR2 channel. of the EC50 of the ryanodine-modified wt and E3987A mutant channels in the presence of Mg2+ and also that the of single E3987A mutant and wt RyR2 channels by ∼1,000-fold. This is in with that by single-channel in the of ryanodine in the presence of and The E3987A mutant channels also sensitive to The Ca2+ dependence of the E3987A mutant channels was by caffeine to the by from µm to µm that the ryanodine-unmodified E3987A mutant channels in the presence of caffeine and Mg2+ ryanodine is able to restore the ligand response of single E3987A mutant channels. is that ryanodine is able to restore the function and of single E3987A mutant channels in To ryanodine is able to restore function of the E3987A mutant in the we cells the wt and E3987A mutant RyR2 channels with or µm ryanodine and Ca2+ release in these cells. of wt cells with ryanodine Ca2+ release a and whereas with ryanodine of E3987A cells the mutant channel from to and have also examined the of ryanodine on the caffeine response of cells the wt and the mutant in ryanodine the caffeine response of the cells and that of the wt cells ryanodine is able to restore the of the of RyR2 and of in the of cells. concentrations of ryanodine are known to block the RyR channel by binding to the low affinity binding µm ryanodine not to block the of RyR2 or in cells These a of the E3987A mutant RyR2 channel is sensitive to blockade by high concentrations of ryanodine. To we investigated the of high concentrations of ryanodine on the of single E3987A mutant channels into that a single E3987A mutant channel by and caffeine was modified by µm ryanodine into a reduced subconductance state with after modification the ryanodine-modified E3987A mutant channel was and These that single E3987A mutant channels are sensitive to block or occlusion by ryanodine at high has been shown also that binding of ryanodine to the low affinity binding site the of [3H]ryanodine from the high affinity binding site (19Wang J.P. Needleman D.H. Hamilton S.L. J. Biol. Chem. 1993; 268: 20974-20982Abstract Full Text PDF PubMed Google Scholar, 20McGrew S.G. Wolleben C. Siegl P. Inui M. Fleischer S. Biochemistry. 1989; 28: 1686-1691Crossref PubMed Scopus (77) Google Scholar, 21Lai F.A. Misra M. Xu L. Smith H.A. Meissner G. J. Biol. Chem. 1989; 264: 16776-16785Abstract Full Text PDF PubMed Google Scholar). have demonstrated that the E3987A mutant RyR2 is of binding [3H]ryanodine with binding affinity to that of the wt RyR2 P. J. Gen. Physiol. PubMed Scopus Google Scholar). To demonstrate that the E3987A mutant channels also the low affinity ryanodine binding we examined the of ryanodine on the of that in the of [3H]ryanodine to wt and E3987A mutant RyR2 with an of and Addition of ryanodine the of [3H]ryanodine from the wt and E3987A mutant RyR2 channels The for the wt and E3987A mutant RyR2 channels in the presence of µm ryanodine to and these that the E3987A mutant RyR2 the the high and low affinity ryanodine binding is generally that, upon binding, ryanodine locks the RyR channel into an open and that the ryanodine-modified RyR is insensitive to modulation (5Zucchi R. Ronca-Testoni S. Pharmacol. Rev. 1997; 49: 1-51PubMed Google Scholar, 11Xu L. Tripathy A. Pasek D.A. Meissner G. Ann. N. Y. Acad. Sci. 1998; 853: 130-148Crossref PubMed Scopus (5) Google Scholar). These however, have not been examined in and have recently been (25Tanna B. Welch W. Ruest L. Sutko J.L. Williams A.J. J. Gen. Physiol. 1998; 112: 55-69Crossref PubMed Scopus (42) Google Scholar). In the present study, we demonstrate that ryanodine is able to increase the sensitivity of single wt RyR2 channels to Ca2+ activation by ∼1,000-fold. In we that ryanodine is able to restore Ca2+ response of the mutant which has been shown to exhibit ∼1,000-fold reduction in Ca2+ Furthermore, the ryanodine-modified wt and ryanodine-modified E3987A mutant RyR2 channels are sensitive to modulation by channel such as Mg2+ and These results that ryanodine does not lock the RyR channel into an open rather, it dramatically the channel by the channel to Ca2+ activation. results also that Ca2+ activation and ryanodine binding are Ca2+ activation has been shown to ryanodine binding to the RyR channel I.N. Casida J.E. Mol. Pharmacol. 1987; Google A. M. M.J. K. Hamilton S.L. Mol. Pharmacol. Google Scholar), present that ryanodine binding to the channel in Ca2+ activation. The molecular underlying functional however, has to be understood. ryanodine binds to the open state of the it is that ryanodine Ca2+ activation by the open state or the state of the channel. ryanodine binding may changes that result in an increase in the affinity of the channel for Ca2+, the sensitivity of the channel to Ca2+ activation. In it is of to that the ryanodine-modified wt and E3987A mutant channels insensitive to of sensitive to of that the Ca2+ from the activation site in the ryanodine-modified channels and to be by be by for these is that the Ca2+ may have been in the Ca2+ activation site in the ryanodine-modified channels. on the of the ryanodine binding and Ca2+ activation sites may into the mechanism ryanodine binding and the high and low affinity ryanodine binding sites have been to a at of RyR T. P. M.J. Campbell K.P. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, C. A. J.P. Needleman D.H. C. Y. T. A.R. Hamilton S.L. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). we have shown that in the in RyR2 or [3H]ryanodine binding, channel M. P. L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), that the is the major of ryanodine have shown also that E3987A dramatically the Ca2+ sensitivity to activation of does not ryanodine binding to the high or low affinity site P. J. Gen. Physiol. PubMed Scopus Google These that is not in ryanodine binding, and that the sites for ryanodine binding and Ca2+ activation are ryanodine most likely affects Ca2+ activation an allosteric such a mechanism does not the that the ryanodine binding and Ca2+ activation are in in the of would be of to the ryanodine binding and Ca2+ activation the of of the of its may such J. P. T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). observation that ryanodine is able to restore the other ligand of the RyR2 is in with the results of a of the in L. Y. C. C. I.N. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus (42) Google Scholar). was shown that the the of to by and Ca2+. with ryanodine the of the mutant to these the as a are and that by et al. is with the single-channel conductance of the ryanodine-modified mutant channels. was that single mutant channels exhibited gating to the open state single-channel conductance to that of the wt channels L. Y. C. C. I.N. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus (42) Google Scholar). In in study, we have observed gating to the open state of the wt in the ryanodine-modified E3987A mutant RyR2 channels. In upon modification by single-channel of the wt and E3987A mutant RyR2 channels reduced and the modified channels the and subconductance The for is not is that the may have effects on and RyR2 in the of the mutant and the E3987A mutant RyR2 to blockade by high concentrations of ryanodine. The observation that of cells with µm ryanodine not response to led et al. to that the mutant channel is insensitive to blockade by high concentrations of ryanodine. also have observed that of the E3987A mutant cells with µm ryanodine the caffeine response single-channel and [3H]ryanodine binding studies clearly that the E3987A mutant RyR2 channel is sensitive to block by high concentrations of and that the E3987A mutant channel the high and low affinity ryanodine binding sites and to ryanodine in a to that of the wt and of complete blockade of RyR channel by high concentrations of ryanodine in cells may not that the channel is insensitive to ryanodine is not single mutant channels in be modified by ryanodine and be by high concentrations of ryanodine. is of to that of wt cells with caffeine µm ryanodine in a Ca2+ L. Y. C. C. I.N. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus (42) Google Scholar). ryanodine at µm able to block the wt one would a This observation suggests that, these µm ryanodine was to block the wt channel in it has been shown from single-channel studies in that the wt channel be by high concentrations of ryanodine (11Xu L. Tripathy A. Pasek D.A. Meissner G. Ann. N. Y. Acad. Sci. 1998; 853: 130-148Crossref PubMed Scopus (5) Google Scholar). The for the of complete inhibition of the mutant or E3987A mutant RyR2 channels in cells by high concentrations of ryanodine are not The of blockade by high concentrations of ryanodine in cells be from that in ryanodine sensitizes the wt and E3987A mutant RyR2 channels to Ca2+ activation, the of ryanodine on wt and E3987A mutant cells be dramatically In the of E3987A mutant ryanodine is able to the Ca2+ sensitivity of the mutant channel to a to that of the and the ligand response of the cells. the other the ryanodine-modified wt RyR2 channel has high sensitivity to Ca2+ activation. the the ryanodine-modified wt RyR2 channel would be and to by the dramatically observed with the wt and E3987A mutant cells with or ryanodine are most likely to be the of marked in Ca2+ sensitivity to activation, than the result of actions of ryanodine on the wt and E3987A mutant RyR2 channels. In we have demonstrated that ryanodine sensitizes the RyR channel to Ca2+ activation and that ryanodine-modified channel is regulatable. of the Ca2+activation and ryanodine binding sites in the and on the of RyR to a understanding of the functional Ca2+activation and ryanodine ryanodine does not the as thought ryanodine or ryanodine be used as specific of the RyR channel. In ryanodine derivatives that have and are in Ca2+ would be for Ca2+ R. and the of on and for and M. for the of

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