The Cytoplasmic Loops between Domains II and III and Domains III and IV in the Skeletal Muscle Dihydropyridine Receptor Bind to a Contiguous Site in the Skeletal Muscle Ryanodine Receptor

Abstract

Excitation-contraction coupling in skeletal muscle is a result of the interaction between the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (ryanodine receptor or RyR1) and the skeletal muscle L-type Ca2+ channel (dihydropyridine receptor or DHPR). Interactions between RyR1 and DHPR are critical for the depolarization-induced activation of Ca2+ release from the sarcoplasmic reticulum, enhancement of DHPR Ca2+ channel activity, and repolarization-induced inactivation of RyR1. The DHPR III–IV loop was fused to glutathione S-transferase (GST) or His-peptide and used as a protein affinity column for35S-labeled, in vitro translated fragments from the N-terminal three-fourths of RyR1. RyR1 residues Leu922–Asp1112 bound specifically to the DHPR III–IV loop column, but the corresponding fragment from the cardiac ryanodine receptor (RyR2) did not. Construction of chimeras between RyR1 and RyR2 showed that amino acids Lys954–Asp1112 retained full binding activity, whereas Leu922–Phe1075 had no binding activity. The RyR1 sequence Arg1076–Asp1112, previously shown to interact with the DHPR II–III loop (Leong, P., and MacLennan, D., H. (1998) J. Biol. Chem. 273, 7791–7794), bound to DHPR III–IV loop columns, but with only half the efficiency of binding of the longer RyR1 sequence, Lys954–Asp1112. These data suggest that the site of DHPR III–IV loop interaction contains elements from both the Lys954–Phe1075 and Arg1076–Asp1112 fragments. The presence of 4 ± 0.4 μm GST-DHPR II–III or 5 ± 0.1 μm His-peptide-DHPR III–IV was required for half-maximal co-purification of 35S-labeled RyR1 Leu922–Asp1112 on glutathione-Sepharose or Ni2+-nitrilotriacetic acid. Dose-dependent inhibition of 35S-labeled RyR1 Leu922–Asp1112 binding to GST-DHPR II–III and GST-DHPR III–IV by His10-DHPR II–III and His-peptide-DHPR III–IV was observed. These studies indicate that the DHPR II–III and III–IV loops bind to contiguous and possibly overlapping sites on RyR1 between Lys954 and Asp1112. Excitation-contraction coupling in skeletal muscle is a result of the interaction between the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (ryanodine receptor or RyR1) and the skeletal muscle L-type Ca2+ channel (dihydropyridine receptor or DHPR). Interactions between RyR1 and DHPR are critical for the depolarization-induced activation of Ca2+ release from the sarcoplasmic reticulum, enhancement of DHPR Ca2+ channel activity, and repolarization-induced inactivation of RyR1. The DHPR III–IV loop was fused to glutathione S-transferase (GST) or His-peptide and used as a protein affinity column for35S-labeled, in vitro translated fragments from the N-terminal three-fourths of RyR1. RyR1 residues Leu922–Asp1112 bound specifically to the DHPR III–IV loop column, but the corresponding fragment from the cardiac ryanodine receptor (RyR2) did not. Construction of chimeras between RyR1 and RyR2 showed that amino acids Lys954–Asp1112 retained full binding activity, whereas Leu922–Phe1075 had no binding activity. The RyR1 sequence Arg1076–Asp1112, previously shown to interact with the DHPR II–III loop (Leong, P., and MacLennan, D., H. (1998) J. Biol. Chem. 273, 7791–7794), bound to DHPR III–IV loop columns, but with only half the efficiency of binding of the longer RyR1 sequence, Lys954–Asp1112. These data suggest that the site of DHPR III–IV loop interaction contains elements from both the Lys954–Phe1075 and Arg1076–Asp1112 fragments. The presence of 4 ± 0.4 μm GST-DHPR II–III or 5 ± 0.1 μm His-peptide-DHPR III–IV was required for half-maximal co-purification of 35S-labeled RyR1 Leu922–Asp1112 on glutathione-Sepharose or Ni2+-nitrilotriacetic acid. Dose-dependent inhibition of 35S-labeled RyR1 Leu922–Asp1112 binding to GST-DHPR II–III and GST-DHPR III–IV by His10-DHPR II–III and His-peptide-DHPR III–IV was observed. These studies indicate that the DHPR II–III and III–IV loops bind to contiguous and possibly overlapping sites on RyR1 between Lys954 and Asp1112. excitation-contraction ryanodine receptor dihydropyridine receptor Ni2+-nitrilotriacetic acid polymerase chain reaction glutathioneS-transferase polyacrylamide gel electrophoresis bovine serum albumin. Excitation-contraction (E-C)1 coupling describes the events leading from electrical stimulation of muscle to the release of Ca2+ from the sarcoplasmic reticulum. Closure of the Ca2+ release channel and activation of the sarco(endo)plasmic reticulum Ca2+ATPase (SERCA) return intracellular Ca2+ to resting levels (1Lytton J. MacLennan D.H. Fozzard H.A. Haber E. Jennings R.B. Katz A.M. Morgan H.E. The Heart and Cardiovascular System: Scientific Foundations. 2. Raven Press, New York1992: 1203-1222Google Scholar). Extracellular Ca2+ is not necessary for E-C coupling in skeletal muscle, but is required in cardiac muscle, distinguishing the mechanism of E-C coupling in these muscles (2Catterall W.A. Cell. 1991; 64: 871-874Abstract Full Text PDF PubMed Scopus (148) Google Scholar). E-C coupling in RYR1 knockout mice can be restored in primary cultures of myotubes isolated from these mice by injection with RYR1 cDNA (3Nakai J. Dirksen R.T. Nguyen H.T. Pessah I.N. Beam K.G. Allen P.D. Nature. 1996; 380: 72-75Crossref PubMed Scopus (398) Google Scholar). During E-C coupling, Ca2+ release from the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (ryanodine receptor or RyR1) is activated by the L-type Ca2+ channel of skeletal muscle (dihydropyridine receptor or DHPR) (4Rios E. Brum G. Nature. 1987; 325: 717-720Crossref PubMed Scopus (650) Google Scholar). A retrograde signal from RyR1, but not from the cardiac ryanodine receptor (RyR2), enhances DHPR Ca2+ channel activity (3Nakai J. Dirksen R.T. Nguyen H.T. Pessah I.N. Beam K.G. Allen P.D. Nature. 1996; 380: 72-75Crossref PubMed Scopus (398) Google Scholar, 5Nakai J. Ogura T. Protasi F. Franzini-Armstrong C. Allen P.D. Beam K.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1019-1022Crossref PubMed Scopus (91) Google Scholar). In addition, skeletal DHPR is critical for terminating Ca2+ release through RyR1 upon membrane repolarization (6Suda N. Penner R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5725-5729Crossref PubMed Scopus (29) Google Scholar, 7Suda N. J. Physiol. (Lond.). 1995; 486: 105-112Crossref Scopus (11) Google Scholar, 8Suda N. Heinemann C. Pfluegers Arch. Eur. J. Physiol. 1996; 432: 948-951Crossref PubMed Scopus (3) Google Scholar). Regions of RyR1 important for interactions with DHPR, leading to activation of Ca2+ release during E-C coupling and for retrograde enhancement of DHPR Ca2+ channel activity by RyR1, have been identified through the study of RyR1/RyR2 chimeras in RYR1 knockout mice (9Nakai J. Sekiguchi N. Rando T.A. Allen P.D. Beam K.G. J. Biol. Chem. 1998; 273: 13403-13406Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). The complex physiology involving the multiple interactions between RyR1 and DHPR suggests that the structural interaction between RyR1 and DHPR may involve more than one site in the two proteins. The importance of the DHPR II–III loop in E-C coupling has been demonstrated by Tanabe et al. (10Tanabe T. Beam K.G. Powell J.A. Numa S. Nature. 1988; 336: 134-139Crossref PubMed Scopus (582) Google Scholar, 11Tanabe T. Beam K.G. Adams B.A. Niidome T. Numa S. Nature. 1990; 346: 567-569Crossref PubMed Scopus (492) Google Scholar) through the study of dysgenic myotubes lacking DHPR. Skeletal-type E-C coupling can be reconstituted in these myotubes by injecting cDNA encoding chimeras between skeletal and cardiac DHPRs with only the II–III loop retaining the skeletal sequence. The DHPR II–III loop has been shown to activate the skeletal muscle ryanodine receptor in studies measuring Ca2+ currents across planar lipid bilayer carried by RyR1 (12Lu X. Xu L. Meissner G. J. Biol. Chem. 1994; 269: 6511-6516Abstract Full Text PDF PubMed Google Scholar, 13Lu X. Xu L. Meissner G. J. Biol. Chem. 1995; 270: 18459-18464Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Ca2+ release has also been elicited from muscle triad vesicle preparations by the addition of the DHPR II–III loop peptide (14el-Hayek R. Antoniu B. Wang J. Hamilton S.L. Ikemoto N. J. Biol. Chem. 1995; 270: 22116-22118Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). A protein affinity chromatography approach was used to identify an interaction site on RyR1 for the DHPR II–III loop (15Leong P. MacLennan D.H. J. Biol. Chem. 1998; 273: 7791-7794Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Malignant hyperthermia is an abnormality of Ca2+ regulation that is linked to RYR1 on human chromosome 19 in at least 50% of cases (16MacLennan D.H. Phillips M.S. Science. 1992; 256: 789-794Crossref PubMed Scopus (417) Google Scholar). A mutation in the DHPR III–IV loop has also been linked to susceptibility to malignant hyperthermia (17Monnier N. Procaccio V. Stieglitz P. Lunardi J. Am. J. Hum. Genet. 1997; 60: 1316-1325Abstract Full Text PDF PubMed Scopus (356) Google Scholar). Thus, the DHPR III–IV loop may play a role in E-C coupling, and a mutation in this loop could modify the interaction between the dihydropyridine and ryanodine receptors, mimicking the effects of mutations of RyR1 previously found to be causal for malignant hyperthermia susceptibility. The studies in dysgenic mice (11Tanabe T. Beam K.G. Adams B.A. Niidome T. Numa S. Nature. 1990; 346: 567-569Crossref PubMed Scopus (492) Google Scholar) that identify the DHPR II–III loop as being critical for RyR1 activation neither rule out nor implicate the III–IV loop in E-C coupling. In this study, we used protein affinity chromatography to demonstrate that the DHPR III–IV loop can interact with RyR1. All chemicals were of molecular biology grade. Ni2+-NTA resin was purchased from QIAGEN Inc., and glutathione-Sepharose 4B and translational grade [35S]Met were from Amersham Pharmacia Biotech. The coupled in vitro transcription and translation kit (TNT Quick) was from Promega. Rabbit skeletal and cardiac muscle cDNAs were purified from λ phage cDNA libraries (18Zorzato F. Fujii J. Otsu K. Phillips M. Green N.M. Meissner G. MacLennan D.H. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar, K. F. Green N.M. MacLennan D.H. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar) from QIAGEN The Ca2+ channel III–IV loop in was a from A. of The ryanodine receptor cDNA has been previously J.A. R. MacLennan D.H. PubMed Scopus Google Scholar). encoding the skeletal muscle DHPR II–III were and the polymerase chain reaction as previously (15Leong P. MacLennan D.H. J. Biol. Chem. 1998; 273: 7791-7794Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). encoding of the DHPR III–IV were with the skeletal muscle cDNA as and of the cardiac DHPR III–IV loop were with the cardiac cDNA as and the cDNA sequence for the loops were with and sites for the or the Pharmacia and sites for of the RYR1 sequence encoding amino acid residues the were to used in of RyR1 All fragments were by sequence The glutathioneS-transferase (GST) with residues to has been (15Leong P. MacLennan D.H. J. Biol. Chem. 1998; 273: 7791-7794Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The and were used to residues to the of the DHPR II–III loop in the GST-DHPR II–III protein by of the two at the sites the sequence, and the site of was used for the of proteins. His-peptide in and were purified with Ni2+-NTA and were purified with glutathione-Sepharose 4B R. J.A. K. in Inc., New Scholar). the His-peptide were with in were with glutathione and were by and and by was with was by the II–III was purified by binding to of Ni2+-NTA was from II–III by in the presence of of His10-DHPR II–III was from Ni2+-NTA by in His10-DHPR had not been from was by with for in vitro transcription and translation were from J.A. R. MacLennan D.H. PubMed Scopus Google Scholar) of a sequence M. 60: PubMed Scopus Google Scholar) and an for translation (15Leong P. MacLennan D.H. J. Biol. Chem. 1998; 273: 7791-7794Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). RyR1 and RyR1 were by from between RyR1 and RyR2 were and through the of sites by as previously (15Leong P. MacLennan D.H. J. Biol. Chem. 1998; 273: 7791-7794Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The are as with on and RyR1 fragments were by coupled in vitro transcription and translation (TNT Quick) and by acid and R. J.A. K. in Inc., New of RyR1/RyR2 to the skeletal DHPR III–IV RyR1/RyR2 as previously (15Leong P. MacLennan D.H. J. Biol. Chem. 1998; 273: 7791-7794Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The a in RyR1 two and acid to RyR1 residues in the of in vitro translated RyR1/RyR2 fragments of and of fragments from and skeletal DHPR III–IV loop affinity binding of in vitro translated RyR1/RyR2 fragments to the skeletal DHPR loop affinity columns, by and as the ± from at least binding was as binding to His-peptide-DHPR III–IV protein binding to The binding was from that of and MacLennan (15Leong P. MacLennan D.H. J. Biol. Chem. 1998; 273: 7791-7794Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). purified His-peptide or was bound to of Ni2+-NTA or glutathione-Sepharose to the of protein of The of protein bound to affinity was by a of the affinity with the protein by and with protein was by to were bound were with of and with of in of column μm and and with 0.4 of in of column fragments of by coupled in vitro transcription and translation (TNT were of column and through affinity by The were with of column retained on the affinity were with of was used to of the in vitro translation of the column and of the of the column The were in of and acid for The signal was with and the were and to of corresponding to the molecular of the RyR1 fragments were by a binding was as binding binding to or or Ni2+-NTA resin was with of in of column and with 0.4 of in of column GST-DHPR II–III or His-peptide-DHPR III–IV fragments at a of μm in of column were for with of vitro and translated RyR1 fragments and through the glutathione-Sepharose or Ni2+-NTA The were with of column RyR1 loop retained on the resin were with of and by and the 35S-labeled RyR1 fragments were a as The of DHPR loop protein required for half-maximal of co-purification of RyR1 fragments was GST-DHPR II–III and GST-DHPR III–IV affinity were as and with of in of column and with 0.4 of in of column The RyR1 fragment was for with μm purified His10-DHPR II–III or III–IV in of column and through of GST-DHPR II–III and GST-DHPR III–IV affinity The were with of column RyR1 loop protein retained on the resin were with of and by and the 35S-labeled RyR1 fragment was a as The of DHPR loop required for half-maximal inhibition of RyR1 fragment binding was in vitro translated RyR1 fragments for interactions with the DHPR III–IV loop on a column as a His-peptide protein found that ± of in vitro translated fragment acids was retained on the DHPR III–IV loop affinity column and the RyR1 fragment was in to amino residues the of the in vitro translated fragment retained on DHPR III–IV loop at ± that the binding to RyR1 and fragments was of column with for the RyR1 fragments the that in vitro fragments may with the affinity proteins. DHPR II–III DHPR III–IV or we the to be in of to molecular showed that of the did not molecular GST-DHPR II–III was to be to the molecular of a of two GST-DHPR II–III and GST-DHPR III–IV was to be to the molecular of a of two GST-DHPR III–IV proteins. are to in J. P. J. PubMed Scopus Google Scholar). of the DHPR II–III and III–IV loop was was to be to molecular of In addition, no molecular were GST-DHPR II–III and or GST-DHPR III–IV and His-peptide were in a the of the RyR1 interaction with DHPR, we in vitro translated fragments of RyR2 the affinity did not binding of to to the skeletal DHPR III–IV loop affinity column in the of the RyR1 fragment in of of the in vitro translated we the of the RyR1 sequence that to the DHPR III–IV loop by chimeras between RyR1 and as in A. The did not bind to the III–IV but ± of the and ± of the were retained on a DHPR III–IV loop affinity column and These indicate that the RyR1 amino acids Arg1076–Asp1112, previously shown to interact with the DHPR II–III loop (15Leong P. MacLennan D.H. J. Biol. Chem. 1998; 273: 7791-7794Abstract Full Text Full Text PDF PubMed Scopus (99) Google not by the binding site for both the DHPR II–III and III–IV but not rule out the that to the binding site for the DHPR III–IV RyR1 residues between to the binding site for the DHPR III–IV than of two RyR1 and was also retained on the His-peptide-DHPR III–IV column with for of the fragments of RyR1 between amino acids and A of interactions is to two in and to the two proteins. found that ± of 35S-labeled with His-peptide-DHPR III–IV loops on whereas of 35S-labeled RyR1 and of 35S-labeled RyR1 also that ± of 35S-labeled with GST-DHPR on but of 35S-labeled RyR1 RyR1 with GST-DHPR 35S-labeled with the DHPR II–III and III–IV loops to the of DHPR loops in required for half-maximal co-purification of of in of II–III on glutathione-Sepharose was at 4 ± 0.4 μm GST-DHPR II–III of III–IV on Ni2+-NTA was at 5 ± 0.1 μm His-peptide-DHPR III–IV that did not with DHPR loops was found in the not the was not to to between and GST-DHPR loop in a of the two proteins. of RyR1 to a GST-DHPR II–III affinity column was in a by both His10-DHPR II–III and His-peptide-DHPR III–IV at a of μm 5 binding RyR1 to a GST-DHPR III–IV affinity column was in a by μm His10-DHPR II–III or His-peptide-DHPR III–IV 5 The of His10-DHPR II–III required to binding of to GST-DHPR II–III and GST-DHPR III–IV was 5 as was the of His-peptide-DHPR III–IV required to binding to GST-DHPR II–III and GST-DHPR III–IV 5 A protein affinity column of the skeletal muscle DHPR III–IV loop retained ± of the of vitro translated RyR1 the corresponding cardiac muscle DHPR III–IV loop retained ± and the Ca2+ channel III–IV loop retained 5 ± and and cardiac muscle DHPR III–IV loops are in of amino The that only of amino acids are between the III–IV loop and the skeletal DHPR III–IV loop is with the binding of to III–IV In this study, we a binding site for the skeletal muscle DHPR III–IV loop to a acid sequence between and in in and translated fragments of RyR1 and of this fragment to a DHPR III–IV loop affinity column was bound also binding of of fragments and to the between the DHPR II–III or III–IV loop and fragment or were in and that bind the DHPR co-purification of was for both the II–III and III–IV loops but co-purification of or was Thus, RyR1 fragments and may have been retained on His-peptide-DHPR III–IV to interactions with of His-peptide-DHPR III–IV RyR1 was to interact with to DHPR, the that RyR1 can interact with proteins. The interaction between and DHPR loops is not to the of of that molecular by of GST-DHPR and GST-DHPR III–IV protein or in with the of the fragment in of in vitro and His-peptide of RyR1 fragments than amino acids were that the sequence a structural of in (18Zorzato F. Fujii J. Otsu K. Phillips M. Green N.M. Meissner G. MacLennan D.H. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar, K. F. Green N.M. MacLennan D.H. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar, J. H. K. 1992; PubMed Scopus Google Scholar) to RyR1/RyR2 chimeras retained binding to the DHPR III–IV loop column, and retained full both the acid sequence Arg1076–Asp1112, previously identified as being important for interaction with the DHPR II–III loop (15Leong P. MacLennan D.H. J. Biol. Chem. 1998; 273: 7791-7794Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). the sequence Arg1076–Asp1112 was from did not bind the DHPR III–IV The RyR1 sequence is in to the DHPR III–IV loop interaction site and may of a structural and be important for of the affinity for of the DHPR II–III and III–IV loops can be from the of DHPR loops required for half-maximal co-purification of with GST-DHPR II–III on glutathione-Sepharose and with His-peptide-DHPR III–IV on Ni2+-NTA The of the DHPR II–III loop required was 4 ± 0.4 and the of the DHPR III–IV loop was 5 ± 0.1 the affinity was not to of to that the RyR1 that we identified as with the DHPR II–III loop also of the interaction site of the DHPR III–IV loop through the of a binding the complex between the DHPR II–III loop and was from binding to a DHPR II–III loop affinity column, and the complex between the DHPR III–IV loop and was from binding to the DHPR III–IV loop affinity also the inhibition of binding of the complex between His-peptide-DHPR III–IV and to a GST-DHPR II–III affinity column, and the inhibition of binding of the complex between His10-DHPR II–III and to a DHPR III–IV loop These can be in two is that the DHPR II–III loop to a site on that with the binding site for the DHPR III–IV the of a GST-DHPR loop on protein affinity to binding site on RyR1 bound to a DHPR loop in may be by the of the bound to is that the of the contains amino The between the of the DHPR II–III loop required for half-maximal inhibition of binding to GST-DHPR II–III or GST-DHPR III–IV 5 suggests that in between the DHPR II–III loop and These are to the 4 μm DHPR II–III loop required for half-maximal of binding to RyR1 in the co-purification studies A and The of the DHPR III–IV loop required for half-maximal inhibition of binding to GST-DHPR II–III and GST-DHPR III–IV affinity was also 5 These are also to the 5 μm DHPR III–IV loop required for half-maximal of of between the DHPR III–IV loop and and skeletal and cardiac DHPR III–IV loops are was not that bound to protein affinity from both the skeletal and cardiac DHPR III–IV loops The in binding to a protein of the loop and of the Ca2+ channel to the in amino acid with the DHPR III–IV et al. T. H. M. M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) that of the of RyR1 to skeletal E-C coupling, E-C coupling is the sequence is to the RyR2 sequence. The of may the of and interactions with DHPR. is with the that the of the sequence to the corresponding RyR2 sequence not E-C coupling the between RyR1 and RyR2 is to T. H. M. M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). et al. (9Nakai J. Sekiguchi N. Rando T.A. Allen P.D. Beam K.G. J. Biol. Chem. 1998; 273: 13403-13406Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar) used chimeras between RyR1 and RyR2 to that is for cardiac E-C coupling and that and are for enhancement of DHPR Ca2+ channel activity. is that the sites in these studies of RyR1/RyR2 chimeras may an interaction from the of that amino acids that are in the sequence. Thus, the that we used to for interactions may not of the amino acids required for a affinity interaction The levels of binding of RyR1 fragments to RyR1 and RyR1 may binding interactions with DHPR The that RyR1 fragments may interaction with DHPR, may also binding et al. (17Monnier N. Procaccio V. Stieglitz P. Lunardi J. Am. J. Hum. Genet. 1997; 60: 1316-1325Abstract Full Text PDF PubMed Scopus (356) Google Scholar) have demonstrated that an mutation in the loop DHPR and in the of skeletal muscle DHPR can be linked to susceptibility to malignant A mutation in DHPR that the of Ca2+ release through RyR1 upon repolarization N. J. Physiol. (Lond.). 1995; 486: 105-112Crossref Scopus (11) Google Scholar, E. J. 1987; Full Text PDF PubMed Scopus Google Scholar, J. Physiol. (Lond.). 1988; Scopus Google Scholar, E. G. Physiol. 1991; PubMed Scopus Google Scholar) the as a mutation in RyR1 that the regulation of Physiol. 1996; PubMed Scopus Google Scholar, K. K. T. M. T. M. J. Biol. 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Nature. 1990; 346: 567-569Crossref PubMed Scopus (492) Google Scholar) the II–III loop as being critical to E-C coupling were not used to rule out or to implicate the III–IV loop in E-C coupling. Thus, we that a from the interaction between and the DHPR III–IV loop is to Ca2+ release through RyR1 during A peptide from amino acids to of skeletal DHPR has been shown to the Ca2+ release of RyR1 Wang B. F. N. Hamilton S.L. Am. J. Physiol. 1997; PubMed Google but the RyR1 interaction site is not is that amino acids in the of skeletal DHPR in with amino acids in the DHPR III–IV The we have identified may be important in the DHPR interaction with RyR1 at the between and the of the sarcoplasmic reticulum. In skeletal muscle, but not cardiac muscle, DHPRs are in of and are in with RyR1 that DHPR is to one of the RyR1 B.A. T. Franzini-Armstrong C. J. Biol. 1988; PubMed Scopus Google Scholar, C. Protasi F. Physiol. 1997; PubMed Scopus Google Scholar). studies of mice with a of the skeletal have shown that the of DHPRs of is on the presence of RyR1 H. L. Tanabe T. Beam K.G. Franzini-Armstrong C. J. 1994; Full Text PDF PubMed Scopus Google Scholar). Thus, for the interaction between and the DHPR III–IV loop that we have identified be of DHPRs of in skeletal The of interaction with DHPR that we with the RyR2 sequence corresponding to be in with this of and ryanodine receptor that the Ca2+ and the that are important for regulation of channel are between and The that we have identified as an interaction site between RyR1 and both the DHPR II–III and III–IV loops is not on T. S. A. K. N. K. Nature. PubMed Scopus Google Scholar, J. MacLennan D.H. Am. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) and is a to that be important for E-C coupling. of residues between and that between RyR1 and RyR2 is to on the amino acids that the interaction site between the two proteins. of these or structural studies of these The of RyR1/RyR2 chimeras lacking RyR1 the importance of the interaction of the DHPR II–III loop with and of the DHPR III–IV loop with The of receptor chimeras lacking RyR1 of a to A. of for the of B. for with and S. for are to A. for the of the Ca2+ channel cDNA