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• W2012247789 abstract "Ryanodine receptors (RyRs) are present in the endoplasmic reticulum of virtually every cell type and serve critical roles, including excitation-contraction (EC) coupling in muscle cells. In skeletal muscle the primary control of RyR-1 (the predominant skeletal RyR isoform) occurs via an interaction with plasmalemmal dihydropyridine receptors (DHPRs), which function as both voltage sensors for EC coupling and as l-type Ca2+ channels (Rios, E., and Brum, G. (1987) Nature325, 717–720). In addition to “receiving” the EC coupling signal from the DHPR, RyR-1 also “transmits” a retrograde signal that enhances the Ca2+ channel activity of the DHPR (Nakai, J., Dirksen, R. T., Nguyen, H. T., Pessah, I. N., Beam, K. G., and Allen, P. D. (1996) Nature380, 72–76). A similar kind of retrograde signaling (from RyRs tol-type Ca2+ channels) has also been reported in neurons (Chavis, P., Fagni, L., Lansman, J. B., and Bockaert, J. (1996) Nature382, 719–722). To investigate the molecular mechanism of reciprocal signaling, we constructed cDNAs encoding chimeras of RyR-1 and RyR-2 (the predominant cardiac RyR isoform) and expressed them in dyspedic myotubes, which lack an endogenous RyR-1. We found that a chimera that contained residues 1,635–2,636 of RyR-1 both mediated skeletal-type EC coupling and enhanced Ca2+channel function, whereas a chimera containing adjacent RyR-1 residues (2,659–3,720) was only able to enhance Ca2+ channel function. These results demonstrate that two distinct regions are involved in the reciprocal interactions of RyR-1 with the skeletal DHPR. Ryanodine receptors (RyRs) are present in the endoplasmic reticulum of virtually every cell type and serve critical roles, including excitation-contraction (EC) coupling in muscle cells. In skeletal muscle the primary control of RyR-1 (the predominant skeletal RyR isoform) occurs via an interaction with plasmalemmal dihydropyridine receptors (DHPRs), which function as both voltage sensors for EC coupling and as l-type Ca2+ channels (Rios, E., and Brum, G. (1987) Nature325, 717–720). In addition to “receiving” the EC coupling signal from the DHPR, RyR-1 also “transmits” a retrograde signal that enhances the Ca2+ channel activity of the DHPR (Nakai, J., Dirksen, R. T., Nguyen, H. T., Pessah, I. N., Beam, K. G., and Allen, P. D. (1996) Nature380, 72–76). A similar kind of retrograde signaling (from RyRs tol-type Ca2+ channels) has also been reported in neurons (Chavis, P., Fagni, L., Lansman, J. B., and Bockaert, J. (1996) Nature382, 719–722). To investigate the molecular mechanism of reciprocal signaling, we constructed cDNAs encoding chimeras of RyR-1 and RyR-2 (the predominant cardiac RyR isoform) and expressed them in dyspedic myotubes, which lack an endogenous RyR-1. We found that a chimera that contained residues 1,635–2,636 of RyR-1 both mediated skeletal-type EC coupling and enhanced Ca2+channel function, whereas a chimera containing adjacent RyR-1 residues (2,659–3,720) was only able to enhance Ca2+ channel function. These results demonstrate that two distinct regions are involved in the reciprocal interactions of RyR-1 with the skeletal DHPR. Dihydropyridine receptors (DHPRs) 1The abbreviations used are: DHPR, dihydropyridine receptor; RyR, ryanodine receptor; EC, excitation-contraction; PCR, polymerase chain reaction; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′,-tetraacetic acid tetraacetoxymethyl ester; Fluo-3, 1-[2-amino-5-(2,7-dichloro6-hydroxy-3-oxo-3H-xanthen-9-yl)]-2-(2′-amino-5′-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid pentaacetoxymethyl ester. and ryanodine receptors (RyRs) are essential for excitation-contraction (EC) coupling in skeletal muscle (1Rios E. Brum G. Nature. 1987; 325: 717-720Crossref PubMed Scopus (650) Google Scholar, 4Beam K.G. Knudson C.M. Powell J.A. Nature. 1986; 320: 168-170Crossref PubMed Scopus (183) Google Scholar, 5Tanabe T. Beam K.G. Powell J.A. Numa S. Nature. 1988; 336: 134-139Crossref PubMed Scopus (582) Google Scholar, 6Takeshima H. Iino M. Takekura H. Nishi M. Kuno J. Minowa O. Takano H. Noda T. Nature. 1994; 369: 556-559Crossref PubMed Scopus (326) Google Scholar). The DHPRs represent voltage-sensing elements in the plasmalemma (1Rios E. Brum G. Nature. 1987; 325: 717-720Crossref PubMed Scopus (650) Google Scholar), and the RyRs function as Ca2+ release channels in the sarcoplasmic reticulum (7Imagawa T. Smith J.S. Coronado R. Campbell K.P. J. Biol. Chem. 1987; 262: 16636-16643Abstract Full Text PDF PubMed Google Scholar, 8Lai F.A. Erickson H.P. Rousseau E. Liu Q.-Y. Meissner G. Nature. 1988; 331: 315-319Crossref PubMed Scopus (68) Google Scholar, 9Hymel L. Inui M. Fleischer S. Schindler H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 441-445Crossref PubMed Scopus (177) Google Scholar). In response to depolarization, the DHPRs undergo conformational changes that produce membrane-bound charge movements (10Schneider M.F. Chandler W.K. Nature. 1973; 242: 244-246Crossref PubMed Scopus (650) Google Scholar). As one consequence of these conformational changes, a signal is transmitted to the RyRs, causing them to release Ca2+ from the sarcoplasmic reticulum. These conformational changes also control a slowly activating l-type Ca2+ current, which mediates the entry of extracellular Ca2+ across the plasmalemma. However, the slow l-type Ca2+ current is not important for skeletal muscle-type EC coupling because this coupling persists under conditions that prevent the entry of extracellular Ca2+ (11Armstrong C.M. Bezanilla F.M. Horowicz P. Biochem. Biophys. Acta. 1972; 267: 605-608Crossref PubMed Scopus (299) Google Scholar). The use of dysgenic myotubes (12Gluecksohn-Waelsch S. Science. 1963; 142: 1269-1276Crossref PubMed Scopus (32) Google Scholar), which lack the endogenous α1 subunit of the skeletal DHPR (α1S), together with expression of cDNAs encoding chimeric combinations of α1S and α1C (the cardiac isoform of the DHPR α1 subunit), has revealed (13Tanabe T. Beam K.G. Adams B.A. Niidome T. Numa S. Nature. 1990; 346: 567-569Crossref PubMed Scopus (492) Google Scholar) that the loop linking homology repeats II and III is critical for transmitting the orthograde, EC coupling signal from the skeletal DHPR to RyR-1, the predominant skeletal isoform of the RyR. Recently, cultured myotubes from dyspedic mice (6Takeshima H. Iino M. Takekura H. Nishi M. Kuno J. Minowa O. Takano H. Noda T. Nature. 1994; 369: 556-559Crossref PubMed Scopus (326) Google Scholar), which lack a functional RyR-1 gene, have provided a skeletal muscle system that makes it possible to express and functionally analyze cDNAs encoding RyRs. Results with dyspedic myotubes indicate that in addition to the “orthograde” (EC coupling) signal from DHPRs to RyRs in skeletal muscle, there also seems to be a “retrograde” signal from RyRs to DHPRs (2Nakai J. Dirksen R.T. Nguyen H.T. Pessah I.N. Beam K.G. Allen P.D. Nature. 1996; 380: 72-76Crossref PubMed Scopus (398) Google Scholar). Specifically, DHPRs appear to be present in the plasmalemma of dyspedic myotubes and able to undergo the voltage-driven conformational changes producing charge movement; however, the magnitude of slow l-type Ca2+ current is decreased in relationship to that of charge movement, indicating that the probability of channel opening is reduced and/or the channels open to less than the full conductance level. Expression in dyspedic myotubes of cDNA encoding RyR-1 causes the magnitude of thel-type Ca2+ current to return toward normal and also restores skeletal-type EC coupling. Expression in dyspedic myotubes of cDNA encoding RyR-2, the predominant cardiac RyR, fails to restore either orthograde signaling (skeletal-type EC coupling) or retrograde signaling (increased density of l-type Ca2+ current) (14Nakai 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). To identify regions of RyR-1 important for reciprocal interactions with the skeletal DHPR, we have expressed cDNAs encoding chimeras of RyR-1 and RyR-2. We find that two distinct regions of RyR-1 appear to be important for reciprocal interactions with the DHPR. Because the RyR-1 plasmid (pRyR/Hygro) used in our previous experiments (2Nakai J. Dirksen R.T. Nguyen H.T. Pessah I.N. Beam K.G. Allen P.D. Nature. 1996; 380: 72-76Crossref PubMed Scopus (398) Google Scholar) lacked convenient restriction sites at the ends of the cDNA insert, we constructed a new RyR-1 plasmid (pCIneoRyR-1) as follows. MluI andXbaI sites were created before the Kozak initiation sequence (GCCGCC) and after the translation termination codon (15,112–15,114) of pRyR/Hygro, respectively, by means of polymerase chain reaction (PCR). The MluI-SalI (PCR-547) fragment amplified from pRyR/Hygro, the SalI-ClaI (547–14,313) fragment from pRyR/Hygro, and the ClaI-XbaI (14,313-PCR) fragment amplified from pRyR/Hygro were ligated to theMluI/XbaI sites of pCIneo (Promega) to yield pCIneoRyR-1. Because pRyR/Hygro and pCIneoRyR-1 behaved similarly, data from both clones are illustrated. The construction of pCIneoRyR-2 was described previously (14Nakai 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). Chimeric plasmids included the following sequences (PL designates the polylinker, and an asterisk designates a restriction site introduced by means of PCR). pCIneoR1:MluI-NdeI (PL-Sk 11,173) from pCIneoRyR-1 andNdeI-MluI (Ca 11,071-PL) from pCIneoRyR-2;pCIneoR2: MluI-BamHI (PL-Sk 4,894) from pCIneoRyR-1 and BamHI-MluI (Ca 4,867-PL) from pCIneoRyR-2; pCIneoR4: BamHI-NdeI (Sk 4, 894–11,173) from pCIneoRyR-1 andNdeI-BamHI (Ca 11,071–4,867) from pCIneoRyR-2;pCIneoR6: EcoRI-NdeI (Sk 2, 396–11,173) from pCIneoRyR-1 and NdeI-EcoRI (Ca 11,071–2,429*) from pCIneoRyR-2; pCIneoR9:AflII-NdeI (Sk 7,922*-11, 173) from pCIneoRyR-1 and NdeI-AflII (Ca 11,071–7,820) from pCIneoRyR-2; pCIneoR10: BamHI-AflII (Sk 4,894–7,922*) from pCIneoRyR-1 andAflII-BamHI (Ca 7,820–4,867) from pCIneoRyR-2. PCR was used to create EcoRI (Ca 2,429) and AflII (Sk 7,922) sites (by the mutations G2430A and C7923T, respectively) without altering the amino acid code. All fragments amplified by PCR were sequenced. The procedures for primary culture of dyspedic myotubes and for nuclear injection of plasmid DNA were as described previously (2Nakai J. Dirksen R.T. Nguyen H.T. Pessah I.N. Beam K.G. Allen P.D. Nature. 1996; 380: 72-76Crossref PubMed Scopus (398) Google Scholar, 5Tanabe T. Beam K.G. Powell J.A. Numa S. Nature. 1988; 336: 134-139Crossref PubMed Scopus (582) Google Scholar, 15Rando T.A. Blau H.M. J. Cell Biol. 1994; 125: 1275-1287Crossref PubMed Scopus (804) Google Scholar). RyR cDNAs (0.5 μg/μl except 0.1–0.5 μg/μl for pCIneoRyR-2) were coinjected with CD8 cDNA (0.1 μg/μl) (16Jurman M.E. Boland L.M. Liu Y. Yellen G. BioTechniques. 1994; 17: 876-881PubMed Google Scholar). Cells expressing the injected plasmids were identified on the basis of contraction and/or binding of CD8 antibody-coated beads (16Jurman M.E. Boland L.M. Liu Y. Yellen G. BioTechniques. 1994; 17: 876-881PubMed Google Scholar) and were analyzed 1–4 days after plasmid injection. Ca2+ currents were measured with the whole cell patch clamp technique (2Nakai J. Dirksen R.T. Nguyen H.T. Pessah I.N. Beam K.G. Allen P.D. Nature. 1996; 380: 72-76Crossref PubMed Scopus (398) Google Scholar). In some instances, a 5-min exposure of cells to 0.1 mm BAPTA-AM (at room temperature) was used to abolish spontaneous contractions. The patch pipette contained (mM) 140 cesium aspartate, 5 MgCl2, 10 Cs2EGTA (20 nm free Ca2+), 5 Na2ATP, and 10 HEPES (pH 7.4 with CsOH) with or without 0.2 or 0.4 pentapotassium Fluo-3. The bath solution contained (mM) 145 tetraethylammonium+, 165 Cl−, 10 HEPES (pH 7.4 with CsOH), 0.003 tetrodotoxin, and 10 Ca2+. The voltage clamp command sequence consisted of stepping from the holding potential (−80 mV) to −30 mV for 1 s, to −50 mV for 25–30 ms, to the test potential for 200 ms, to −50 mV for 25–30 ms, and then back to the holding potential. To analyze intracellular Ca2+ transients in response to electrical stimulation, myotubes were loaded with Fluo-3 AM, and fluorescence changes (in arbitrary units) were measured as described previously (17GarcıÖa J. Beam K.G. J. Gen. Physiol. 1994; 103: 107-123Crossref PubMed Scopus (65) Google Scholar). The cells were bathed in either normal rodent Ringer containing (mM) 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES (pH 7.4 with NaOH), or in Ca2+-free Ringer (made by equimolar substitution of Mg2+ for Ca2+ in the normal rodent Ringer). Cells were stimulated with a 10-ms pulse applied via an extracellular pipette (5Tanabe T. Beam K.G. Powell J.A. Numa S. Nature. 1988; 336: 134-139Crossref PubMed Scopus (582) Google Scholar, 17GarcıÖa J. Beam K.G. J. Gen. Physiol. 1994; 103: 107-123Crossref PubMed Scopus (65) Google Scholar). Temperature was 20–22 °C. Based on both hydropathy profile (6Takeshima H. Iino M. Takekura H. Nishi M. Kuno J. Minowa O. Takano H. Noda T. Nature. 1994; 369: 556-559Crossref PubMed Scopus (326) Google Scholar, 18Takeshima H. Nishimura N. 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 (867) Google Scholar, 19Zorzato F. Fujii J. Otsu K. Green N.M. Lai F.A. Meissner G. MacLennan D.H. J. Biol. Chem. 1990; 265: 2244-2256Abstract Full Text PDF PubMed Google Scholar, 20Nakai J. Imagawa T. Hakamata Y. Shigekawa M. Takeshima H. Numa S. FEBS Lett. 1990; 271: 169-177Crossref PubMed Scopus (288) Google Scholar, 21Otsu 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, 22Hakamata Y. Nakai J. Takeshima H. Imoto K. FEBS Lett. 1992; 312: 229-235Crossref PubMed Scopus (351) Google Scholar) and comparison of sequence with the inositol 1,4,5-trisphosphate receptor, the other intracellular Ca2+ release channel (23Furuichi T. Yoshikawa S. Miyawaki A. Wada K. Maeda N. Mikoshiba K. Nature. 1989; 342: 32-38Crossref PubMed Scopus (825) Google Scholar), RyRs are predicted to have two main regions: a cytoplasmic “foot” structure representing the amino-terminal nine-tenths of the protein and a channel region comprising the carboxyl-terminal tenth. Additional support for this general architecture is provided by the recent observation that even after removal of the majority of the amino-terminal (~80%), the remaining carboxyl-terminal portion of RyR-1 is still able to form functional Ca2+ release channels (24Bhat M.B. Zhao J. Takeshima H. Ma J. Biophys. J. 1997; 73: 1329-1336Abstract Full Text PDF PubMed Scopus (116) Google Scholar). Because the foot bridges the gap between the sarcoplasmic reticulum and the sarcolemma (25Takekura H. Nishi M. Noda T. Takeshima H. Franzini-Armstrong C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3381-3385Crossref PubMed Scopus (115) Google Scholar), it would seem to be the part of the RyR most likely to participate in reciprocal interactions with the DHPR. To identify regions of RyR-1 (18Takeshima H. Nishimura N. 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 (867) Google Scholar, 19Zorzato F. Fujii J. Otsu K. Green N.M. Lai F.A. Meissner G. MacLennan D.H. J. Biol. Chem. 1990; 265: 2244-2256Abstract Full Text PDF PubMed Google Scholar) critical for these reciprocal interactions, we constructed six cDNAs encoding chimeric RyRs in which a varying portion of the foot region of RyR-2 (20Nakai J. Imagawa T. Hakamata Y. Shigekawa M. Takeshima H. Numa S. FEBS Lett. 1990; 271: 169-177Crossref PubMed Scopus (288) Google Scholar, 21Otsu 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) was replaced with the corresponding portion of RyR-1 (Fig.1). The chimeric RyRs were expressed in myotubes obtained from dyspedic mice, which lack an intact RyR-1 gene (2Nakai J. Dirksen R.T. Nguyen H.T. Pessah I.N. Beam K.G. Allen P.D. Nature. 1996; 380: 72-76Crossref PubMed Scopus (398) Google Scholar). This approach is based on previous work with dyspedic myotubes, which demonstrated that (a) expression of RyR-1 both restored skeletal-type EC coupling (i.e. not requiring entry of extracellular Ca2+) and enhanced the Ca2+channel activity of the DHPR (2Nakai J. Dirksen R.T. Nguyen H.T. Pessah I.N. Beam K.G. Allen P.D. Nature. 1996; 380: 72-76Crossref PubMed Scopus (398) Google Scholar) and (b) expression of RyR-2 neither restored skeletal-type EC coupling nor enhancedl-type Ca2+ channel activity (14Nakai 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). The RyR expression plasmids were co-injected with a plasmid encoding CD8 T-cell antigen into nuclei of dyspedic myotubes. Myotubes selected for analysis displayed spontaneous and/or electrically evoked contractions together with decoration by CD8 antibody-coated beads (16Jurman M.E. Boland L.M. Liu Y. Yellen G. BioTechniques. 1994; 17: 876-881PubMed Google Scholar), which had been added to the bathing medium. Fig. 2 illustrates whole cell Ca2+ currents and Ca2+ transients recorded from myotubes expressing wild-type or chimeric RyRs. In order to minimize the amount of exogenous Ca2+ buffering, the Ca2+ transients were measured in intact myotubes loaded with Fluo-3 AM. Some of the chimeric RyRs caused spontaneous oscillatory contractions like those previously described for RyR-2 (14Nakai 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,26Yamazawa T. Takeshima H. Sakurai T. Endo M. Iino M. EMBO J. 1996; 15: 6172-6177Crossref PubMed Scopus (51) Google Scholar). Therefore, BAPTA-AM was used in some experiments to suppress the contractions before measurement of Ca2+ currents (see legend to Fig. 2). Control experiments on normal myotubes demonstrated that the Ca2+ current density was slightly lower in BAPTA-AM-treated cells (11.3 ± 2.7 pA/pF, n = 10) than in nontreated cells (16.8 ± 4.1 pA/pF, n = 15). As reported previously, dyspedic myotubes had a small Ca2+ current density (2Nakai J. Dirksen R.T. Nguyen H.T. Pessah I.N. Beam K.G. Allen P.D. Nature. 1996; 380: 72-76Crossref PubMed Scopus (398) Google Scholar, 27Fleig A. Takeshima H. Penner R. J. Physiol. 1996; 496: 339-345Crossref PubMed Scopus (29) Google Scholar) and lacked EC coupling (2Nakai J. Dirksen R.T. Nguyen H.T. Pessah I.N. Beam K.G. Allen P.D. Nature. 1996; 380: 72-76Crossref PubMed Scopus (398) Google Scholar, 6Takeshima H. Iino M. Takekura H. Nishi M. Kuno J. Minowa O. Takano H. Noda T. Nature. 1994; 369: 556-559Crossref PubMed Scopus (326) Google Scholar), both of which were restored toward normal after expression of RyR-1 (Fig. 2) (2Nakai J. Dirksen R.T. Nguyen H.T. Pessah I.N. Beam K.G. Allen P.D. Nature. 1996; 380: 72-76Crossref PubMed Scopus (398) Google Scholar). The restored EC coupling was skeletal-type because the depolarization-evoked Ca2+ transient was observed even in the absence of extracellular Ca2+. Also as reported previously, expression of RyR-2 restored neither l-type Ca2+ current (14Nakai 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) nor depolarization-induced Ca2+ release (even when external Ca2+ was present) (14Nakai 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, 26Yamazawa T. Takeshima H. Sakurai T. Endo M. Iino M. EMBO J. 1996; 15: 6172-6177Crossref PubMed Scopus (51) Google Scholar). The chimera R1, in which the majority of the amino-terminal portion of RyR-2 was replaced with RyR-1 sequence (1Rios E. Brum G. Nature. 1987; 325: 717-720Crossref PubMed Scopus (650) Google Scholar, 2Nakai J. Dirksen R.T. Nguyen H.T. Pessah I.N. Beam K.G. Allen P.D. Nature. 1996; 380: 72-76Crossref PubMed Scopus (398) Google Scholar, 3Deleted in proofGoogle Scholar, 720), restored both Ca2+ current density and skeletal-type EC coupling. Therefore, the foot portion of RyR-1 is important for reciprocal interactions with the skeletal DHPR. To localize more precisely the RyR regions critical for reciprocal interaction with the skeletal DHPR, we next examined chimeras that contained segments of RyR-1 sequence smaller than in R1. The chimera R2, which contained the RyR-1 sequence (1–1,631) corresponding only to the amino-terminal half of that in R1, restored neither Ca2+ current nor EC coupling. Despite being unable to interact reciprocally with the DHPR, R2 did encode a functional protein because myotubes expressing R2 cDNA displayed spontaneous, oscillatory contractions and could release Ca2+ in response to application of 0.1 mm caffeine (data not shown). Because R1 (RyR-1: 1–3, 720) could reciprocally interact with the skeletal DHPR but R2 (RyR-1: 1–1, 631) could not, we next examined chimeras R6 (RyR-1: 812–3,720), R4 (RyR-1: 1,635–3, 720), and R9 (RyR-1: 2,659–3,720), in which successively longer portions of the RyR-1 sequence at the amino terminus of R1 were replaced with RyR-2 sequence. The R6 and R4 chimeras were able both to increase Ca2+current density and to restore skeletal-type EC coupling. Chimera R9 increased Ca2+ current density and restored a depolarization-induced Ca2+ transient that was presentonly when there was extracellular Ca2+. It is unlikely that Ca2+ entering via the enhanced, slowl-type Ca2+ current was by itself sufficient to produce this Ca2+ transient because even a large, rapidly activating Ca2+ current (resulting from heterologously expressed l-channels) produces only a small change in myoplasmic Ca2+ in dyspedic myotubes (14Nakai 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). Thus, R9 appears capable of supporting Ca2+-induced Ca2+ release but cannot mediate skeletal-type EC coupling. Because R4 (RyR-1: 1,635–3,720) supported skeletal-type coupling and R9 (RyR-1: 2,659–3,720) did not, we next examined R10 (RyR-1: 1,635–2,636). R10 both supported skeletal-type coupling and increased the density ofl-type Ca2+ current (Fig. 2). Three regions, which have been designated D1, D2, and D3 (28Sorrentino V. Volpe P. Trends Pharmacol. Sci. 1993; 14: 98-103Abstract Full Text PDF PubMed Scopus (274) Google Scholar), are particularly divergent between RyR-1 (18Takeshima H. Nishimura N. 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 (867) Google Scholar, 19Zorzato F. Fujii J. Otsu K. Green N.M. Lai F.A. Meissner G. MacLennan D.H. J. Biol. Chem. 1990; 265: 2244-2256Abstract Full Text PDF PubMed Google Scholar) and RyR-2 (20Nakai J. Imagawa T. Hakamata Y. Shigekawa M. Takeshima H. Numa S. FEBS Lett. 1990; 271: 169-177Crossref PubMed Scopus (288) Google Scholar, 21Otsu 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). Yamazawa et al. reported that deletion of D2 (RyR-1: 1,342–1,403) abolishes the ability of RyR-1 to mediate skeletal-type EC coupling, although EC coupling is preserved when the sequence of the D2 region is converted to RyR-2 sequence (29Yamazawa T. Takeshima H. Shimuta M. Iino M. J. Biol. Chem. 1997; 272: 8161-8164Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Our data indicate that neither D2 nor D1 (RyR-1: 4,254–4,631) is important for the difference between RyR-1 and RyR-2 in mediating skeletal-type EC coupling and enhanced Ca2+ channel activity of the DHPR (Fig. 1). However, the D3 region (RyR-1: 1,872–1,923), which contains a cluster of acidic residues not present in RyR-2, could have specific importance for the ability of RyR-1 to mediate skeletal-type EC coupling because the R10 chimera includes the D3 region. Fig. 3 is a schematic model of interactions between the DHPR and RyR in skeletal muscle. The skeletal DHPR is an l-type Ca2+ channel with voltage-sensing elements that have been adapted to control the gating of the slow l-type Ca2+ current and also to initiate EC coupling by triggering the opening of the Ca2+release channel (i.e. RyR-1). Previous work has shown that the putative, intracellular loop connecting repeats II and III of the DHPR is critical for skeletal-type EC coupling (13Tanabe T. Beam K.G. Adams B.A. Niidome T. Numa S. Nature. 1990; 346: 567-569Crossref PubMed Scopus (492) Google Scholar, 30Lu X. Xu L. Meissner G. J. Biol. Chem. 1994; 269: 6511-6516Abstract Full Text PDF PubMed Google Scholar, 31Lu X. Xu L. Meissner G. J. Biol. Chem. 1995; 270: 18459-18464Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 32El-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). The demonstration here that chimera R10 restores skeletal-type EC coupling suggests the possibility that the II-III loop triggers Ca2+release by means of contact with RyR-1 in a region delimited by amino acids 1,635–2,636. Because l-type Ca2+ current density was increased by both R10 and R9, some of the residues between 1,635 and 2,636, as well as residues between 2,659 and 3,720, may represent sites of contact for retrograde signaling whereby RyR-1 enhances Ca2+ channel activity of the DHPR (2Nakai J. Dirksen R.T. Nguyen H.T. Pessah I.N. Beam K.G. Allen P.D. Nature. 1996; 380: 72-76Crossref PubMed Scopus (398) Google Scholar). Interestingly, R10 and R9 are contained within two calpain digestion fragments of RyR-1 (1,401–2,843 and 2,844–4,685), which appear to be linked by an intrasubunit disulfide bridge (33Wu Y. Aghdasi B. Dou S.J. Zhang J.Z. Liu S.Q. Hamilton S.L. J. Biol. Chem. 1997; 272: 25051-25061Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). No matter what the actual folding structure of RyRs, the demonstration that R9 enhancesl-type Ca2+ channel activity without restoring skeletal-type EC coupling indicates that the structures of RyR-1 involved in retrograde (channel-enhancing) signaling are not identical to those involved in orthograde (EC coupling) signaling. Because RyR-1 is expressed in brain (22Hakamata Y. Nakai J. Takeshima H. Imoto K. FEBS Lett. 1992; 312: 229-235Crossref PubMed Scopus (351) Google Scholar, 34Kuwajima G. Futatsugi A. Niinobe M. Nakanishi S. Mikoshiba K. Neuron. 1992; 9: 1133-1142Abstract Full Text PDF PubMed Scopus (125) Google Scholar, 35Furuichi T. Furutama D. Hakamata Y. Nakai J. Takeshima H. Mikoshiba K. J. Neurosci. 1994; 14: 4794-4805Crossref PubMed Google Scholar, 36Giannini G. Conti A. Mammarella S. Scrobogna M. Sorrentino V. J. Cell Biol. 1995; 128: 893-904Crossref PubMed Scopus (486) Google Scholar), it will be important to determine whether the regions we have identified here are also involved in reciprocal signaling in neurons. We thank Dr. N. Suda for helpful discussions and K. Lopez for technical help." @default.
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• W2012247789 title "Two Regions of the Ryanodine Receptor Involved in Coupling withl-Type Ca2+ Channels" @default.
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