Ca2+ Inactivation Sites Are Located in the COOH-terminal Quarter of Recombinant Rabbit Skeletal Muscle Ca2+ Release Channels (Ryanodine Receptors)

Ca2+ activation of skeletal (RyR1) and cardiac (RyR2) muscle Ca2+ release channels (ryanodine receptors) occurs with EC50 values of about 1 μm. Ca2+ inactivation occurs with an IC50 value of about 3.7 mm for RyR1 , but RyR2 shows little inactivation, even at >100 mm Ca2+. In an attempt to localize the low affinity Ca2+ binding sites responsible for Ca2+ inactivation in RyR1, chimeric RyR1/RyR2 molecules were constructed. Because [3H]ryanodine binds only to open channels, and because channel opening and closing are Ca2+-dependent, the Ca2+ dependence of [3H]ryanodine binding was used as an indirect measurement of Ca2+ release channel opening and closing. IC50 values for [3H]ryanodine binding suggested that Ca2+ affinity for the low affinity Ca2+ inactivation sites was unchanged in a chimera in which a glutamate-rich sequence (amino acids 1743–1964) in RyR1 was replaced with the corresponding, less acidic sequence from RyR2. Ca2+ affinity (IC50) for low affinity Ca2+ inactivation sites was intermediate in RyR1/RyR2 chimeras containing RyR2 amino acids 3726–4186 (RF9), 4187–4628 (RF10), or 4629–5037 (RF11), was closer to RyR2 values in RyR1 chimeras with longer RyR2 replacements (RF9/10 or RF10/11), and was indistinguishable from RyR2 in RyR1 containing all three RyR2 replacements (RF9/10/11). These data suggest that multiple low affinity Ca2+ binding sites or multiple components of a low affinity Ca2+ binding site are located between amino acids 3726 and 5037 and that their effects on Ca2+ inactivation of the release channel are cooperative. Measurement of Ca2+activation of [3H]ryanodine binding showed that chimeras RF10, RF9/10, and RF9/10/11 were more sensitive to Ca2+than was either RyR1 or RyR2. Measurement of caffeine activation of Ca2+ release in vivo showed that chimeras RF9, RF10, RF9/10, RF10/11, and RF9/10/11 were more sensitive to caffeine than wild-type RyR1. These results suggest that Ca2+ and caffeine activation sites also involve COOH-terminal sequences in RyR1 and RyR2. Ca2+ activation of skeletal (RyR1) and cardiac (RyR2) muscle Ca2+ release channels (ryanodine receptors) occurs with EC50 values of about 1 μm. Ca2+ inactivation occurs with an IC50 value of about 3.7 mm for RyR1 , but RyR2 shows little inactivation, even at >100 mm Ca2+. In an attempt to localize the low affinity Ca2+ binding sites responsible for Ca2+ inactivation in RyR1, chimeric RyR1/RyR2 molecules were constructed. Because [3H]ryanodine binds only to open channels, and because channel opening and closing are Ca2+-dependent, the Ca2+ dependence of [3H]ryanodine binding was used as an indirect measurement of Ca2+ release channel opening and closing. IC50 values for [3H]ryanodine binding suggested that Ca2+ affinity for the low affinity Ca2+ inactivation sites was unchanged in a chimera in which a glutamate-rich sequence (amino acids 1743–1964) in RyR1 was replaced with the corresponding, less acidic sequence from RyR2. Ca2+ affinity (IC50) for low affinity Ca2+ inactivation sites was intermediate in RyR1/RyR2 chimeras containing RyR2 amino acids 3726–4186 (RF9), 4187–4628 (RF10), or 4629–5037 (RF11), was closer to RyR2 values in RyR1 chimeras with longer RyR2 replacements (RF9/10 or RF10/11), and was indistinguishable from RyR2 in RyR1 containing all three RyR2 replacements (RF9/10/11). These data suggest that multiple low affinity Ca2+ binding sites or multiple components of a low affinity Ca2+ binding site are located between amino acids 3726 and 5037 and that their effects on Ca2+ inactivation of the release channel are cooperative. Measurement of Ca2+activation of [3H]ryanodine binding showed that chimeras RF10, RF9/10, and RF9/10/11 were more sensitive to Ca2+than was either RyR1 or RyR2. Measurement of caffeine activation of Ca2+ release in vivo showed that chimeras RF9, RF10, RF9/10, RF10/11, and RF9/10/11 were more sensitive to caffeine than wild-type RyR1. These results suggest that Ca2+ and caffeine activation sites also involve COOH-terminal sequences in RyR1 and RyR2. ryanodine receptor 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid monoclonal antibody human embryonic kidney. Ca2+ release channels from the sarcoplasmic reticulum of skeletal and cardiac muscle (ryanodine receptors, RyRs)1 are modulated by endogenous and exogenous modulators such as ATP, Ca2+, calmodulin, Mg2+, ruthenium red, and ryanodine (1Coronado R. Morrissette J. Sukhareva M. Vaughan D.M. Am. J. Physiol. 1994; 266: C1485-C1504Crossref PubMed Google Scholar, 2Zucchi R. Ronca-Testoni S. Pharmacol. Rev. 1997; 49: 53-98PubMed Google Scholar). The Ca2+ release channels from both skeletal (RyR1) and cardiac (RyR2) muscles are activated by micromolar Ca2+, but only RyR1 is inactivated by millimolar Ca2+ (1Coronado R. Morrissette J. Sukhareva M. Vaughan D.M. Am. J. Physiol. 1994; 266: C1485-C1504Crossref PubMed Google Scholar, 2Zucchi R. Ronca-Testoni S. Pharmacol. Rev. 1997; 49: 53-98PubMed Google Scholar, 3Meissner G. J. Biol. Chem. 1984; 259: 2365-2374Abstract Full Text PDF PubMed Google Scholar, 4Pessah I.N. Waterhouse A.L. Casida J.E. Biochem. Biophys. Res. Commun. 1985; 128: 449-456Crossref PubMed Scopus (226) Google Scholar, 5Smith J.S. Coronado R. Meissner G. J. Gen. Physiol. 1986; 88: 573-588Crossref PubMed Scopus (304) Google Scholar, 6Michalak M. Dupraz P. Shoshan-Barmatz V. Biochim. Biophys. Acta. 1988; 939: 587-594Crossref PubMed Scopus (91) Google Scholar, 7Chu A. Fill M. Stefani E. Entman M.L. J. Membr. Biol. 1993; 135: 49-59Crossref PubMed Scopus (92) Google Scholar, 8Laver D.R. Roden L.D. Ahern G.P. Eager K.R. Junankar P.R. Dulhunty A.F. J. Membr. Biol. 1995; 147: 7-22Crossref PubMed Scopus (150) Google Scholar). [3H]Ryanodine binds only to activated Ca2+release channels, making [3H]ryanodine binding a useful assay for the activation state of the channels (1Coronado R. Morrissette J. Sukhareva M. Vaughan D.M. Am. J. Physiol. 1994; 266: C1485-C1504Crossref PubMed Google Scholar, 2Zucchi R. Ronca-Testoni S. Pharmacol. Rev. 1997; 49: 53-98PubMed Google Scholar). High concentrations of Ca2+, which inhibit [3H]ryanodine binding to CHAPS-solubilized recombinant RyR1, do not inhibit [3H]ryanodine binding to CHAPS-solubilized recombinant RyR2 under identical conditions (9Du G.G. Imredy J.P. MacLennan D.H. J. Biol. Chem. 1998; 273: 33259-33266Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). These results suggest that RyR1 has high affinity Ca2+binding sites for Ca2+ activation and low affinity Ca2+ binding sites for inactivation of channel function, whereas RyR2 has only high affinity sites for Ca2+activation (1Coronado R. Morrissette J. Sukhareva M. Vaughan D.M. Am. J. Physiol. 1994; 266: C1485-C1504Crossref PubMed Google Scholar, 7Chu A. Fill M. Stefani E. Entman M.L. J. Membr. Biol. 1993; 135: 49-59Crossref PubMed Scopus (92) Google Scholar, 9Du G.G. Imredy J.P. MacLennan D.H. J. Biol. Chem. 1998; 273: 33259-33266Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar).The location of the low affinity Ca2+ binding site(s) in RyR1 is not known. A glutamate-rich sequence lying between residues 1872 and 1923 (D3) is a potential low affinity Ca2+ binding site, based on the amino acid sequence deduced from a cDNA sequence (10Zorzato F. Fujii J. Otsu K. Phillips M. Green N.M. Lai F.A. Meissner G. MacLennan D.H. J. Biol. Chem. 1990; 265: 2244-2256Abstract Full Text PDF PubMed Google Scholar). This potential Ca2+ binding site is also one of the three most divergent sequences between RyR1 and RyR2, which include RyR1 amino acids 1342–1403 (D2) and 4254–4631 (D1) (11Sorrentino V. Volpe P. Trends Pharmacol. Sci. 1993; 14: 98-103Abstract Full Text PDF PubMed Scopus (272) Google Scholar). Three regions in the COOH terminus of RyR1 and two regions in the middle of RyR2 score highly as potential EF-hand structures for high affinity Ca2+ binding (10Zorzato F. Fujii J. Otsu K. Phillips M. Green N.M. Lai F.A. Meissner G. MacLennan D.H. J. Biol. Chem. 1990; 265: 2244-2256Abstract Full Text PDF PubMed Google Scholar, 12Takeshima H. Nishimura S. Matsumoto T. Ishida H. Kangawa K. Minamino N. Matsuo H. Ueda M. Hanaoka M. Hirose T. Numa S. Nature. 1989; 339: 439-445Crossref PubMed Scopus (857) Google Scholar, 13Otsu K. Willard H.F. Khanna V.K. Zorzato F. Green N.M. MacLennan D.H. J. Biol. Chem. 1990; 265: 13472-13483Abstract Full Text PDF PubMed Google Scholar, 14Nakai J. Imagawa T. Hakamat Y. Shigekawa M. Takeshima H. Numa S. FEBS Lett. 1990; 271: 169-177Crossref PubMed Scopus (287) Google Scholar). Two potential EF-hand sequences detected in lobster RyR1 were shown to have homology with similar sequences in mammalian RyR1 and RyR2 and were proposed to be involved in Ca2+ inactivation (15Xiong H. Feng X. Gao L. Xu L. Pasek D.A. Seok J.H. Meissner G. Biochemistry. 1998; 37: 4804-4814Crossref PubMed Scopus (53) Google Scholar). In RyR1, Ca2+binding and ruthenium red binding sites have been mapped to several locations, including three in the COOH terminus of RyR1 (16Chen S.R. Zhang L. MacLennan D.H. J. Biol. Chem. 1992; 267: 23318-23326Abstract Full Text PDF PubMed Google Scholar, 17Chen S.R. Zhang L. MacLennan D.H. J. Biol. Chem. 1993; 268: 13414-13421Abstract Full Text PDF PubMed Google Scholar, 18Chen S.R. MacLennan D.H. J. Biol. Chem. 1994; 269: 22698-22704Abstract Full Text PDF PubMed Google Scholar). High affinity Ca2+ binding sites have also been identified in hydrophobic sequences (19Feng W. Shoshan-Barmatz V. Mol. Membr. Biol. 1996; 13: 85-93Crossref PubMed Scopus (9) Google Scholar, 20Chen S.R.W. Ebisawa K. Li X. Zhang L. J. Biol. Chem. 1998; 273: 14675-14678Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 21Du G.G. MacLennan D.H. J. Biol. Chem. 1998; 273: 31867-31872Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The relationship of any of these Ca2+ binding sites to Ca2+ inactivation of channel function is undefined.We have explored the question of whether the glutamate-rich region (D3) and the COOH-terminal region are responsible for Ca2+inactivation in RyR1 by constructing a series of RyR1/RyR2 chimeras in which the glutamate-rich D3 sequence and three other sequences (3726–4186 (F9), 4187–4628 (F10), and 4629–5037 (F11)) in RyR1 were replaced separately and in groups by the corresponding sequences in RyR2. We tested the Ca2+ dependence of high affinity [3H]ryanodine binding to the chimeras, and we measured in vivo Ca2+ release induced by caffeine in Ca2+ photometry. We found that the low affinity Ca2+ inactivation site is not affected by exchange of the D3 sequence but that Ca2+ inactivation is affected to different degrees by multiple exchanges of fragments at the COOH terminus of RyR1. Our results suggest that multiple Ca2+inactivation sites or multiple components of a single Ca2+inactivation site in RyR1 are located between amino acids 3726 and 5037. Ca2+ release channels from the sarcoplasmic reticulum of skeletal and cardiac muscle (ryanodine receptors, RyRs)1 are modulated by endogenous and exogenous modulators such as ATP, Ca2+, calmodulin, Mg2+, ruthenium red, and ryanodine (1Coronado R. Morrissette J. Sukhareva M. Vaughan D.M. Am. J. Physiol. 1994; 266: C1485-C1504Crossref PubMed Google Scholar, 2Zucchi R. Ronca-Testoni S. Pharmacol. Rev. 1997; 49: 53-98PubMed Google Scholar). The Ca2+ release channels from both skeletal (RyR1) and cardiac (RyR2) muscles are activated by micromolar Ca2+, but only RyR1 is inactivated by millimolar Ca2+ (1Coronado R. Morrissette J. Sukhareva M. Vaughan D.M. Am. J. Physiol. 1994; 266: C1485-C1504Crossref PubMed Google Scholar, 2Zucchi R. Ronca-Testoni S. Pharmacol. Rev. 1997; 49: 53-98PubMed Google Scholar, 3Meissner G. J. Biol. Chem. 1984; 259: 2365-2374Abstract Full Text PDF PubMed Google Scholar, 4Pessah I.N. Waterhouse A.L. Casida J.E. Biochem. Biophys. Res. Commun. 1985; 128: 449-456Crossref PubMed Scopus (226) Google Scholar, 5Smith J.S. Coronado R. Meissner G. J. Gen. Physiol. 1986; 88: 573-588Crossref PubMed Scopus (304) Google Scholar, 6Michalak M. Dupraz P. Shoshan-Barmatz V. Biochim. Biophys. Acta. 1988; 939: 587-594Crossref PubMed Scopus (91) Google Scholar, 7Chu A. Fill M. Stefani E. Entman M.L. J. Membr. Biol. 1993; 135: 49-59Crossref PubMed Scopus (92) Google Scholar, 8Laver D.R. Roden L.D. Ahern G.P. Eager K.R. Junankar P.R. Dulhunty A.F. J. Membr. Biol. 1995; 147: 7-22Crossref PubMed Scopus (150) Google Scholar). [3H]Ryanodine binds only to activated Ca2+release channels, making [3H]ryanodine binding a useful assay for the activation state of the channels (1Coronado R. Morrissette J. Sukhareva M. Vaughan D.M. Am. J. Physiol. 1994; 266: C1485-C1504Crossref PubMed Google Scholar, 2Zucchi R. Ronca-Testoni S. Pharmacol. Rev. 1997; 49: 53-98PubMed Google Scholar). High concentrations of Ca2+, which inhibit [3H]ryanodine binding to CHAPS-solubilized recombinant RyR1, do not inhibit [3H]ryanodine binding to CHAPS-solubilized recombinant RyR2 under identical conditions (9Du G.G. Imredy J.P. MacLennan D.H. J. Biol. Chem. 1998; 273: 33259-33266Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). These results suggest that RyR1 has high affinity Ca2+binding sites for Ca2+ activation and low affinity Ca2+ binding sites for inactivation of channel function, whereas RyR2 has only high affinity sites for Ca2+activation (1Coronado R. Morrissette J. Sukhareva M. Vaughan D.M. Am. J. Physiol. 1994; 266: C1485-C1504Crossref PubMed Google Scholar, 7Chu A. Fill M. Stefani E. Entman M.L. J. Membr. Biol. 1993; 135: 49-59Crossref PubMed Scopus (92) Google Scholar, 9Du G.G. Imredy J.P. MacLennan D.H. J. Biol. Chem. 1998; 273: 33259-33266Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The location of the low affinity Ca2+ binding site(s) in RyR1 is not known. A glutamate-rich sequence lying between residues 1872 and 1923 (D3) is a potential low affinity Ca2+ binding site, based on the amino acid sequence deduced from a cDNA sequence (10Zorzato F. Fujii J. Otsu K. Phillips M. Green N.M. Lai F.A. Meissner G. MacLennan D.H. J. Biol. Chem. 1990; 265: 2244-2256Abstract Full Text PDF PubMed Google Scholar). This potential Ca2+ binding site is also one of the three most divergent sequences between RyR1 and RyR2, which include RyR1 amino acids 1342–1403 (D2) and 4254–4631 (D1) (11Sorrentino V. Volpe P. Trends Pharmacol. Sci. 1993; 14: 98-103Abstract Full Text PDF PubMed Scopus (272) Google Scholar). Three regions in the COOH terminus of RyR1 and two regions in the middle of RyR2 score highly as potential EF-hand structures for high affinity Ca2+ binding (10Zorzato F. Fujii J. Otsu K. Phillips M. Green N.M. Lai F.A. Meissner G. MacLennan D.H. J. Biol. Chem. 1990; 265: 2244-2256Abstract Full Text PDF PubMed Google Scholar, 12Takeshima H. Nishimura S. Matsumoto T. Ishida H. Kangawa K. Minamino N. Matsuo H. Ueda M. Hanaoka M. Hirose T. Numa S. Nature. 1989; 339: 439-445Crossref PubMed Scopus (857) Google Scholar, 13Otsu K. Willard H.F. Khanna V.K. Zorzato F. Green N.M. MacLennan D.H. J. Biol. Chem. 1990; 265: 13472-13483Abstract Full Text PDF PubMed Google Scholar, 14Nakai J. Imagawa T. Hakamat Y. Shigekawa M. Takeshima H. Numa S. FEBS Lett. 1990; 271: 169-177Crossref PubMed Scopus (287) Google Scholar). Two potential EF-hand sequences detected in lobster RyR1 were shown to have homology with similar sequences in mammalian RyR1 and RyR2 and were proposed to be involved in Ca2+ inactivation (15Xiong H. Feng X. Gao L. Xu L. Pasek D.A. Seok J.H. Meissner G. Biochemistry. 1998; 37: 4804-4814Crossref PubMed Scopus (53) Google Scholar). In RyR1, Ca2+binding and ruthenium red binding sites have been mapped to several locations, including three in the COOH terminus of RyR1 (16Chen S.R. Zhang L. MacLennan D.H. J. Biol. Chem. 1992; 267: 23318-23326Abstract Full Text PDF PubMed Google Scholar, 17Chen S.R. Zhang L. MacLennan D.H. J. Biol. Chem. 1993; 268: 13414-13421Abstract Full Text PDF PubMed Google Scholar, 18Chen S.R. MacLennan D.H. J. Biol. Chem. 1994; 269: 22698-22704Abstract Full Text PDF PubMed Google Scholar). High affinity Ca2+ binding sites have also been identified in hydrophobic sequences (19Feng W. Shoshan-Barmatz V. Mol. Membr. Biol. 1996; 13: 85-93Crossref PubMed Scopus (9) Google Scholar, 20Chen S.R.W. Ebisawa K. Li X. Zhang L. J. Biol. Chem. 1998; 273: 14675-14678Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 21Du G.G. MacLennan D.H. J. Biol. Chem. 1998; 273: 31867-31872Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The relationship of any of these Ca2+ binding sites to Ca2+ inactivation of channel function is undefined. We have explored the question of whether the glutamate-rich region (D3) and the COOH-terminal region are responsible for Ca2+inactivation in RyR1 by constructing a series of RyR1/RyR2 chimeras in which the glutamate-rich D3 sequence and three other sequences (3726–4186 (F9), 4187–4628 (F10), and 4629–5037 (F11)) in RyR1 were replaced separately and in groups by the corresponding sequences in RyR2. We tested the Ca2+ dependence of high affinity [3H]ryanodine binding to the chimeras, and we measured in vivo Ca2+ release induced by caffeine in Ca2+ photometry. We found that the low affinity Ca2+ inactivation site is not affected by exchange of the D3 sequence but that Ca2+ inactivation is affected to different degrees by multiple exchanges of fragments at the COOH terminus of RyR1. Our results suggest that multiple Ca2+inactivation sites or multiple components of a single Ca2+inactivation site in RyR1 are located between amino acids 3726 and 5037.