Maurocalcine and Domain A of the II-III Loop of the Dihydropyridine Receptor Cav 1.1 Subunit Share Common Binding Sites on the Skeletal Ryanodine Receptor
2005; Elsevier BV; Volume: 280; Issue: 6 Linguagem: Inglês
10.1074/jbc.c400433200
ISSN1083-351X
AutoresXavier Altafaj, Weijun Cheng, É. Estève, Julie Urbani, Didier Grünwald, Jean‐Marc Sabatier, Roberto Coronado, Michel De Waard, Michel Ronjat,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoMaurocalcine is a scorpion venom toxin of 33 residues that bears a striking resemblance to the domain A of the dihydropyridine voltage-dependent calcium channel type 1.1 (Cav1.1) subunit. This domain belongs to the II-III loop of Cav1.1, which is implicated in excitation-contraction coupling. Besides the structural homology, maurocalcine also modulates RyR1 channel activity in a manner akin to a synthetic peptide of domain A. Because of these similarities, we hypothesized that maurocalcine and domain A may bind onto an identical region(s) of RyR1. Using a set of RyR1 fragments, we demonstrate that peptide A and maurocalcine bind onto two discrete RyR1 regions: fragments 3 and 7 encompassing residues 1021–1631 and 3201–3661, respectively. The binding onto fragment 7 is of greater importance and was thus further investigated. We found that the amino acid region 3351–3507 of RyR1 (fragment 7.2) is sufficient for these interactions. Proof that peptide A and maurocalcine bind onto the same site is provided by competition experiments in which binding of fragment 7.2 to peptide A is inhibited by preincubation with maurocalcine. Moreover, when expressed in COS-7 cells, RyR1 carrying a deletion of fragment 7 shows a loss of interaction with both peptide A and maurocalcine. At the functional level, this deletion abolishes the maurocalcine induced stimulation of [3H]ryanodine binding onto microsomes of transfected COS-7 cells without affecting the caffeine and ATP responses. Maurocalcine is a scorpion venom toxin of 33 residues that bears a striking resemblance to the domain A of the dihydropyridine voltage-dependent calcium channel type 1.1 (Cav1.1) subunit. This domain belongs to the II-III loop of Cav1.1, which is implicated in excitation-contraction coupling. Besides the structural homology, maurocalcine also modulates RyR1 channel activity in a manner akin to a synthetic peptide of domain A. Because of these similarities, we hypothesized that maurocalcine and domain A may bind onto an identical region(s) of RyR1. Using a set of RyR1 fragments, we demonstrate that peptide A and maurocalcine bind onto two discrete RyR1 regions: fragments 3 and 7 encompassing residues 1021–1631 and 3201–3661, respectively. The binding onto fragment 7 is of greater importance and was thus further investigated. We found that the amino acid region 3351–3507 of RyR1 (fragment 7.2) is sufficient for these interactions. Proof that peptide A and maurocalcine bind onto the same site is provided by competition experiments in which binding of fragment 7.2 to peptide A is inhibited by preincubation with maurocalcine. Moreover, when expressed in COS-7 cells, RyR1 carrying a deletion of fragment 7 shows a loss of interaction with both peptide A and maurocalcine. At the functional level, this deletion abolishes the maurocalcine induced stimulation of [3H]ryanodine binding onto microsomes of transfected COS-7 cells without affecting the caffeine and ATP responses. In skeletal muscle cells, the activation of the dihydropyridine receptor (DHPR) 1The abbreviations used are: DHPR, dihydropyridine receptor; Cav1.1, voltage-dependent calcium channel type 1.1; PBS, phosphate-buffered saline; MCa, maurocalcine; aa, amino acids; contig, group of overlapping clones.1The abbreviations used are: DHPR, dihydropyridine receptor; Cav1.1, voltage-dependent calcium channel type 1.1; PBS, phosphate-buffered saline; MCa, maurocalcine; aa, amino acids; contig, group of overlapping clones. induces, through the ryanodine receptor (RyR1), a massive release of the calcium stored in the sarcoplasmic reticulum. The bi-directional communication between RyR1 and DHPR (i.e. orthograde and retrograde signals) has been shown to be enabled by the physical interactions of the two Ca2+ channels (1Schneider M.F. Chandler W.K. Nature. 1973; 242: 747-751Crossref Scopus (647) Google Scholar, 2Tanabe T. Beam K.G. Powell J.A. Numa S. Nature. 1988; 336: 134-139Crossref PubMed Scopus (580) Google Scholar, 3Nakai J. Dirksen R.T. Nguyen H.T. Pessah I.N. Beam K.G. Allen P.D. Nature. 1996; 380: 72-75Crossref PubMed Scopus (394) Google Scholar, 4Chavis P. Fagni L. Lansman J.B. Bockaert J. Nature. 1996; 382: 719-722Crossref PubMed Scopus (271) Google Scholar). The DHPR α1 subunit forms the pore region, carries the voltage sensitivity of the channel, and is also directly involved in the functional coupling of the DHPR with RyR1 (2Tanabe T. Beam K.G. Powell J.A. Numa S. Nature. 1988; 336: 134-139Crossref PubMed Scopus (580) Google Scholar, 5Catterall W.A. Science. 1991; 253: 1499-1500Crossref PubMed Scopus (135) Google Scholar). Indeed, the cytoplasmic loop linking domains II and III of the α1 subunit has been proposed to be responsible for the mechanical coupling between the DHPR and the RyR1 (6Tanabe T. Beam K.G. Adams B.A. Nicodome T. Numa S. Nature. 1990; 346: 567-569Crossref PubMed Scopus (488) Google Scholar, 7Nakai J. Tanabe T. Konno T. Adams B. Beam K.G. J. Biol. Chem. 1998; 273: 24983-24986Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Within this loop, two regions (domains A and C) have been shown to regulate ryanodine binding and channel gating of RyR1 in vitro (8El-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). RyR1 activity is regulated in vitro by a number of chemical compounds such as ATP, Ca2+, Mg2+, and proteins such as calmodulin and FK506-binding protein (FKBP12) (9Melzer W. Herrmann-Frank A. Luttgau Ch.H Biochim. Biophys. Acta. 1995; 1241: 59-116Crossref PubMed Scopus (480) Google Scholar, 10MacKrill J.J. Biochem. J. 1999; 337: 345-361Crossref PubMed Scopus (192) Google Scholar). Nevertheless, due to (i) the extremely large size of RyR1, (ii) the small number of high affinity effectors, and (iii) the absence of atomic resolution structure, the mapping of the functional sites of RyR1 is still far from completion. As part of the search for molecules able to strongly modify RyR1 properties, toxins isolated from scorpion venoms have been described as the most active effectors of RyR1. Among them, imperatoxin A and maurocalcine (MCa) have been extensively characterized both in terms of their effects on RyR1 and their three-dimensional structure (11Gurrola G.B. Arévalo C. Sreekumar R. Lokuta A.J. Walker J.W. Valdivia H.H. J. Biol. Chem. 1999; 274: 7879-7886Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 12Moshba A. Kharrat R. Fajloun Z. Renisio J.G. Blanc E. Sabatier J.M. Al Ayeb M. Darbon H. Proteins Struct. Funct. Genet. 2000; 40: 436-442Crossref PubMed Scopus (80) Google Scholar, 13Chen L. Estève E. Sabatier J.M. Ronjat M. De Waard M. Allen P.D. Pessah I.N. J. Biol. Chem. 2003; 278: 16095-16106Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 14Esteve E. Smida-Rezgui S. Sarkozi S. Szegedi C. Regaya I. Chen L Altafaj X Rochat H. Allen P.D. Pessah I.N. Marty I. Sabatier J.M. Jona I. De Waard M. Ronjat M. J. Biol. Chem. 2003; 278: 37822-37831Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 15Green D. Pace S. Curtis S.M. Sakowska M. Lamb G.D. Dulhunty A.F. Casaroto M.G. Biochem. J. 2003; 370: 517-527Crossref PubMed Scopus (26) Google Scholar, 16Lee C.W. Lee E.H. Takeuchi K. Takahashi H. Shimada I. Sato K. Shin S.Y. Kim D.H. Kim J.I. Biochem. J. 2004; 377: 385-394Crossref PubMed Scopus (29) Google Scholar). These two toxins present some amino acid sequence homology with the domain A of DHPR α1 subunit, and structural studies strongly suggest that their β-sheet structure could mimic that of the domain A (15Green D. Pace S. Curtis S.M. Sakowska M. Lamb G.D. Dulhunty A.F. Casaroto M.G. Biochem. J. 2003; 370: 517-527Crossref PubMed Scopus (26) Google Scholar, 16Lee C.W. Lee E.H. Takeuchi K. Takahashi H. Shimada I. Sato K. Shin S.Y. Kim D.H. Kim J.I. Biochem. J. 2004; 377: 385-394Crossref PubMed Scopus (29) Google Scholar). In a previous work, we demonstrated that nanomolar concentrations of MCa induces a dramatic conformational change of RyR1, witnessed by the increase in [3H]ryanodine binding (7-fold) and the induction of long lasting channel openings (13Chen L. Estève E. Sabatier J.M. Ronjat M. De Waard M. Allen P.D. Pessah I.N. J. Biol. Chem. 2003; 278: 16095-16106Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 14Esteve E. Smida-Rezgui S. Sarkozi S. Szegedi C. Regaya I. Chen L Altafaj X Rochat H. Allen P.D. Pessah I.N. Marty I. Sabatier J.M. Jona I. De Waard M. Ronjat M. J. Biol. Chem. 2003; 278: 37822-37831Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The observed homologies in amino acid sequences and in three-dimensional solution structure have prompted different authors to make the hypothesis that imperatoxin A and MCa could share a common binding site with the domain A of the DHPR α1 subunit on the RyR1. We have therefore undertaken the identification of the amino acid region of RyR1 that carries the binding site for MCa and/or the domain A of the skeletal DHPR α1 subunit. In this work, a set of polypeptides covering the entire sequence of RyR1 was expressed in vitro and tested individually for their ability to interact with either MCa or peptide Ask (a synthetic peptide corresponding to the domain A of the skeletal DHPR α1 subunit). We identified a single domain of RyR1 interacting with both MCa and peptide Ask. We also showed that MCa and peptide Ask compete for the interaction on this domain. To asses the functional relevance of this domain for the effect of MCa on RyR1, we expressed a RyR1 channel that lacks the MCa binding domain in COS-7 cells and measured the effect of this deletion on the interaction of RyR1 with both MCa and peptide Ask as well as on the activation of ryanodine binding by MCa. Taken together, our results show that MCa and domain A of the DHPR α1 subunit share a common binding site on RyR1. Purification of RyR1—Skeletal heavy SR vesicles and purified RyR1 were prepared as described previously (17Marty I. Robert M. Villaz M. De Jongh K.S. Lai Y. Catterall W.A. Ronjat M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2270-2274Crossref PubMed Scopus (137) Google Scholar, 18Lai F.A. Erickson H.P. Rousseau E. Liu Q.Y. Meissner G. Nature. 1988; 331: 315-319Crossref PubMed Scopus (68) Google Scholar). Peptide Synthesis—Peptides were synthesized as described previously (14Esteve E. Smida-Rezgui S. Sarkozi S. Szegedi C. Regaya I. Chen L Altafaj X Rochat H. Allen P.D. Pessah I.N. Marty I. Sabatier J.M. Jona I. De Waard M. Ronjat M. J. Biol. Chem. 2003; 278: 37822-37831Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 19O'Reilly F. Robert M. Jona I. Szegedi C. Albrieux M. Geib S. De Waard M. Villaz M. Ronjat M. Biophys. J. 2002; 82: 145-155Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), with the addition of an exogenous biotin on the C-terminal amino acid of (i) MCa synthetic peptide, (ii) peptide Ask and peptide Csk corresponding to residues Thr671–Lys690 and Glu724–Pro760 of the DHPR α1s subunit, respectively. Pull-down Experiments—Polystyrene magnetic beads (500 μg) coated with streptavidine (Dynal) were incubated for 30 min at room temperature in the presence of biotinylated peptide (100 μg/ml in buffer A (150 mm NaCl, 2 mm EGTA, 2 mm CaCl2, pCa5, and 20 mm HEPES, pH 7.4)). Beads were then washed three times with buffer A and incubated for 2 h at room temperature with purified RyR1 (100 nm in buffer A). After washing twice with buffer A, bound proteins were eluted and analyzed by Western blot using antibodies directed against RyR1 (20Marty I Villaz M Arlaud G. Bally I. Ronjat M. Biochem. J. 1994; 298: 743-749Crossref PubMed Scopus (27) Google Scholar). Cloning and Expression of RyR1 Fragments—RyR1 cDNA (GenBank™ accession number X15750) was subcloned in pSG5 vector (Stratagene) by a PCR-based method (21Cheng W. Carbonneau L. Keys L. Altafaj X. Ronjat M. Coronado R. Biophys. J. 2004; 86: 220aGoogle Scholar), leading to the following fragments: fragment F1 (aa 1–526), F2 (aa 527–1021), F3 (aa 1022–1631), F4 (aa 1632–2169), F5 (aa 2170–2697), F6 (aa 2698–3200), F7 (aa 3201–3661), F8 (aa 3662–4244). Fragment F9 (aa 4007–5037) was produced by cutting the original pCIneo-RyR1 plasmid (kindly provided by Dr. Allen) with XhoI, followed by self-ligation. 35S-Labeled RyR1 fragments were expressed using a coupled in vitro transcription and translation system (TNT™ kit, Promega). For pull-down assays, 5 μlof the translation product were incubated with polystyrene magnetic beads coated with the peptide of interest as described above. After overnight incubation at 4 °C, beads were washed with 1 volume of buffer A. Bound proteins were eluted in denaturing buffer and analyzed by autoradiography after separation on SDS-PAGE. Expression and Purification of Fusion Proteins—RyR1-F7 was subcloned into F7.1, F7.2, and F7.3 and the constructs inserted in His-tag pMR78 vector (22Arnaud N. Cheynet V. Oriol G. Mandrand B. Mallet F. Gene (Amst.). 1997; 199: 149-156Crossref PubMed Scopus (19) Google Scholar). Escherichia coli BL21 pLys(DE3) bacteria were transformed with each construct and protein synthesis induced for 3 h by addition of 1 mm isopropyl β-d-thiogalactopyranoside. Bacteria were collected by centrifugation and resuspended in MCAC buffer (20 mm Tris/HCl, 500 mm NaCl, 10% v/v glycerol, pH 8.0) supplemented with protease inhibitors, Triton X-100 (0.1% v/v), DNase I (50 μg), and MgCl2 (10 mm). After sonication, insoluble material was removed by centrifugation (15 min at 20,000 × g and 4 °C). Fusion proteins were immobilized onto a nickel-nitrilotriacetic acid-agarose column (Qiagen) and washed with 4 volumes of MCAC buffer containing 60 mm imidazole. Retained proteins were eluted with MCAC buffer containing 250 mm imidazole and detected by Western blot with anti-His antibodies (dilution 1:10,000, Sigma). Transfection of COS-7 Cells with RyR1 Constructs and Cytoimmunofluorescence—Wild-type RyR1 (RyR1wt) construct was cloned into pCI-neo vector (Promega). RyR1ΔF7 construct was generated from the pCI-neo/RyR1 clone by cutting out the sequence between nucleotides 9730 and 10983. COS-7 cells were transfected with RyR1wt or RyR1ΔF7 constructs using the jetPEI™ reagent (Qbiogene). Immunofluorescence analysis was performed 48 h after transfection. Cells were fixed in 4% paraformaldehyde for 10 min, washed three times with PBS and permeabilized with 0.1% Triton X-100, and incubated for 1 h at room temperature with 1% bovine serum albumin and 1% goat serum before adding anti-RyR1 antibodies (dilution 1:200 in PBS-0.1% Tween 20, PBS-T) (20Marty I Villaz M Arlaud G. Bally I. Ronjat M. Biochem. J. 1994; 298: 743-749Crossref PubMed Scopus (27) Google Scholar). After 2 h, cells were washed four times with PBS-T and incubated for 30 min at room temperature with Alexa488-conjugated anti-rabbit IgG (dilution 1:800 in PBS-T). Cells were washed four times with PBS-T, mounted, and analyzed by confocal laser scanning microscopy using a Leica TCS-SP2 operating system. Microsomes Preparation—COS-7 cells were harvested 48 h after transfection in cold buffer containing 320 mm sucrose, 5 mm HEPES, pH 7.4, and protease inhibitors. Cells were homogenized and centrifuged for 5 min at 7000 × g at 4 °C. Microsomes were collected by centrifugation at 100,000 × g for 1 h at 4 °C, resuspended in the same buffer and stored in liquid nitrogen. [3H]Ryanodine Binding Assay—Microsomes (100 μg of protein) were incubated 1 h at room temperature with 1 μm recombinant FKBP12 in 150 mm NaCl, 2 mm EGTA and CaCl2 (pCa5), 20 mm HEPES, pH 7.4, buffer. After 150-min incubation at 37 °C in the presence of [3H]ryanodine (10 nm) and caffeine (0 or 6 mm), ATP (0 or 6 mm) or MCa (0 or 30 nm), free and bound [3H]ryanodine were separated by filtration of the microsomes on Whatmann GF/B filters and [3H]ryanodine measured by liquid scintillation. Nonspecific [3H]ryanodine binding was measured in the presence of 10 μm unlabeled ryanodine. Fig. 1A shows the primary structure of MCa, domain A and domain C of the II-III loop of the skeletal muscle α1 DHPR. Biotinylated peptides, named MCab, peptide Ask (pAsk), and peptide Csk (pCsk), corresponding to these different sequences were synthesized and immobilized on polystyrene beads coated with streptavidine. Fig. 1B shows the SDS-PAGE analysis of the bound RyR1 on beads covered with each of the peptide. A polypeptide band corresponding to RyR1 is only observed in the bound fraction obtained with beads covered with MCab or peptide Ask. No signal was detected when beads were covered with peptide Csk or biotin. These results confirm the specificity of the interaction between the skeletal sequence of domain A and RyR1 and represent the first biochemical evidence of a direct interaction of MCa with RyR1. To identify the amino acid sequences of RyR1 involved in the interaction with MCab and peptide Ask, we generated a contig of nine clones encompassing the full-length RyR1 cDNA (Fig. 1C). Each clone was expressed separately in an in vitro cell-free translation system in the presence of radiolabeled [35S]methionine. RyR1 fragments (F1–F9) were incubated in the presence of beads covered either with MCab, peptide Ask, peptide Csk, or biotin. The fragments interacting with the different peptides were detected by autoradiography after separation on SDS-PAGE. Fig. 1C shows that among the nine translated fragments, the F7 fragment encompassing RyR1 residues 3201–3661 interacts strongly with MCab and with peptide Ask. Fragment F3 encompassing RyR1 residues 1021–1631 consistently shows a weak interaction with both MCab and peptide Ask. In agreement with what is observed with purified RyR1, no significant interaction with peptide Csk was observed with any of the nine RyR1 fragments. Similarly, no interaction was observed of any fragment with beads covered with biotin (data not shown). Statistical analysis of the amount of complex formed with each of the fragment shows that about 50% of the F7 fragment interacts with MCab or peptide Ask, while only about 15% of the F3 fragment was retained by MCab or peptide Ask. For all other fragments, the amount of complex formed with MCab or peptide Ask was not significantly different from what observed with peptide Csk and represented less than 5% of the input. To further define the RyR1 sequence that interacts with MCab and peptide Ask, the cDNA corresponding to F7 was subcloned into three smaller fragments, named F7.1, F7.2, and F7.3, encompassing RyR1 residues 3201–3350, 3351–3507, and 3508–3661, respectively (Fig. 2A). Corresponding polypeptides were then expressed in E. coli, affinity-purified trough their histidine tag, and tested for their interaction with MCab or peptide Ask. Interacting fragments were detected by immunolabeling with antibodies directed against the His-tag, after separation on SDS-PAGE. Fig. 2A shows that only the fragment F7.2, encompassing residues 3351–3507, retains the ability to interact with both MCab and peptide Ask. No interaction was observed with fragment F7.1 or F7.3. The specificity of the interaction of the F7.2 fragment with MCab and peptide Ask was confirmed by the lack of signal with beads covered with peptide Csk or biotin (data not shown). We therefore investigated the ability of MCab to inhibit the interaction of fragment F7.2 with peptide Ask. Incubation of fragment F7.2 for 2 h in the presence of non-biotinylated MCa induces a complete loss of interaction of this fragment with immobilized peptide Ask (Fig. 2B) strongly suggesting that MCa and peptide Ask interact on the same site on the F7.2 fragment. We next investigated the importance of the F7 region in the sensitivity of RyR1 to MCa. For this purpose, COS-7 cells were co-transfected with two plasmids encoding the DsRed protein and the entire RyR1 (RyR1wt) or the RyR1 deleted of residues 3241–3661 (RyR1ΔF7) (Fig. 3). Immunolabeling of DsRed-positive cells with antibodies directed against the C-terminal domain of RyR1 demonstrates that RyR1wt and RyR1ΔF7 exhibit a similar distribution within the cell. This distribution essentially consists in a cytoplasmic labeling most likely representing the endoplasmic reticulum (Fig. 3A). Fluorescence analysis showed that about 10% of cells co-express DsRed and RyRwt or DsRed and RyRΔF7. A weak labeling with anti-RyR1 antibodies was also observed in cells that do not express DsRed protein, suggesting that some cells could express exogenous RyR1 without DsRed or that they possess a low level of endogenous RyR1 expression (however barely detected by Western blot analysis, Fig. 3B). To establish the functional relevance of the F7 region in RyR1 regulation by MCa or peptide Ask, we first studied the ability of expressed RyR1wt and RyR1ΔF7 to interact with MCa or peptide Ask. For this purpose, we prepared microsomes from cells transfected with either RyR1wt or RyR1ΔF7. Immunoblot analysis of the different microsome preparations using antibodies directed against RyR1 (Fig. 3B, top left panel) supports the localization of expressed RyR1wt and RyR1ΔF7 in reticulum membrane of transfected cells. These results also show that RyR1wt and RyR1ΔF7 are expressed at a similar level and remain stable in transfected cells. Finally, endogenous RyR1, if present, is barely measurable. Using these microsome preparations, we performed pull-down experiments with beads covered with either MCa or peptide Ask. Fig. 3B (top right panel) shows that in contrast to RyR1wt, RyR1ΔF7 almost completely loss its ability to interact with both MCa and peptide Ask. We then characterized the [3H]ryanodine binding on RyR1wt or RyR1ΔF7 and tested the effects of caffeine, ATP, and MCa on this binding. No significant specific [3H]ryanodine binding was observed on microsomes obtained from non-transfected cells (Fig. 3B, bottom panel). In addition, no effect of caffeine, ATP, or MCa was observed on these control microsomes. These data confirmed immunological and Western blot data showing a low level if any of endogenous RyR1 in COS-7 cells. In contrast, [3H]ryanodine binding was observed on microsomes populations obtained from COS-7 cells transfected with RyR1 constructs. Specific binding was 116 ± 57 fmol/mg and 113 ± 31 fmol/mg for microsomes obtained from RyR1wt and RyR1ΔF7 transfected cells, respectively. Taken together with the Western blot analysis, these results indicate a similar [3H]ryanodine binding ability of expressed RyR1wt and RyR1ΔF7. Activation of [3H]ryanodine binding by caffeine, ATP, and MCa is presented in Fig. 3B (bottom panel). In the presence of 6 mm caffeine or ATP, similar increase of the [3H]ryanodine binding was observed on RyR1wt and RyR1ΔF7 microsomes. In contrast, 30 nm MCa induced a 202 ± 28% increase of [3H]ryanodine binding on RyR1wt microsomes but no significant change of [3H]ryanodine binding on RyR1ΔF7 microsomes (107 ± 3%). These results demonstrate that deletion of RyR1 residues 3241–3661, included in fragment F7, induces a complete loss of RyR1 sensitivity to MCa without significantly modify its sensitivity to caffeine or ATP. In this work, we identified two sequences of RyR1, fragment F3 encompassing residues 1021–1631 and fragment F7 comprising the residues 3201–3661, that are able to interact in vitro with both MCa and domain A of the II-III loop of the DHPR α1 subunit. Among these two sequences, fragment F7 displays the strongest interaction with MCa and peptide Ask. We then generated a RyR1 construct that lacks the amino acid region corresponding to fragment F7. This RyR1ΔF7 construct displays an expression profile and a sensitivity to caffeine or ATP similar to the wild-type RyR1. In contrast, deletion of the F7 region abolished the interaction of RyR1 with both MCa and peptide Ask as well as the stimulatory effect of MCa on ryanodine binding, highlighting the critical role of this domain for MCa regulation. A molecular dissection of fragment F7 allowed us to identify a discrete domain corresponding to residues 3351–3507 (fragment F7.2) responsible for the interaction of MCa and peptide Ask with RyR1. These results demonstrate that MCa and domain A interact with the same RyR1 region within the 3351–3507 stretch of residues. Binding of MCa and peptide Ask to the same RyR1 site was confirmed by the complete inhibition of the interaction of peptide Ask with the fragment F7.2 in the presence of an excess of MCa. Using an affinity chromatography approach, Leong and MacLennan (23Leong P. MacLennan D.H. J. Biol. Chem. 1998; 273: 29958-29964Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) identified a domain of RyR1 that interacts with the entire II-III loop of the DHPR α1 subunit. This sequence (residues 1076–1112) is included within fragment F3 that we found to interact more weakly with both peptide Ask and MCa. In contrast, these authors did not observe any significant interaction of the entire II-III loop with a polypeptide comprising residues 3158–3724 (including F7). The results presented here, together with our previous results, suggest that domain Ask is the only sequence of II-III loop interacting with RyR1 (19O'Reilly F. Robert M. Jona I. Szegedi C. Albrieux M. Geib S. De Waard M. Villaz M. Ronjat M. Biophys. J. 2002; 82: 145-155Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The difference between the two sets of results may reflect an effect of the distal part of the II-III loop on its conformation thereby possibly modifying the accessibility of domain Ask to RyR1. Several studies using either RyR1/RyR2 or RyR1/RyR3 chimeras led to the identification of different domains of RyR1 involved in functional and structural interaction of DHPR with RyR1 (24Protasi F. Paolini C. Nakai J. Beam K.G. Franzini-Armstrong C. Allen P.D. Biophys. J. 2002; 83: 3230-3244Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 25Perez C.F. Mukherjee S. Allen P. J. Biol. Chem. 2003; 278: 39644-39652Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Interestingly, fragment F7 carrying the MCa and peptide Ask binding domain is included in one of these domains formally defined as domain R9 or Ch-9. Similarly, the deletion of residues 1272–1455 of RyR1 (included in F3 fragment) has been shown to induce a loss of depolarization-induced calcium release (25Perez C.F. Mukherjee S. Allen P. J. Biol. Chem. 2003; 278: 39644-39652Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Therefore, regardless of the approach, the two regions of RyR1 spanning fragments F3 and F7 have been shown to play important roles in the functional communication between RyR1 and DHPR. In agreement with previous studies suggesting that noncontiguous regions of RyR1 may act together to control the functional coupling between RyR1 and DHPR, the F3 and F7 fragments may form a unique functional domain. This possibility is supported by three-dimensional reconstruction of RyR1 indicating that regions including the F3 and F7 fragments could be close from each other in the RyR1 tetramer (26Wagenknecht T. Samso M. Front. Biosci. 2002; 7: 464-474Crossref Google Scholar). In conclusion, we identified the binding domains of MCa and domain A on RyR1. We also introduce the first biochemical evidence that MCa and domain A of the DHPR α1 subunit interact with identical sequences on RyR1. In view of the important effects of MCa on RyR1 gating properties (13Chen L. Estève E. Sabatier J.M. Ronjat M. De Waard M. Allen P.D. Pessah I.N. J. Biol. Chem. 2003; 278: 16095-16106Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 14Esteve E. Smida-Rezgui S. Sarkozi S. Szegedi C. Regaya I. Chen L Altafaj X Rochat H. Allen P.D. Pessah I.N. Marty I. Sabatier J.M. Jona I. De Waard M. Ronjat M. J. Biol. Chem. 2003; 278: 37822-37831Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), the RyR1 sequences that we identified in this report ought to play an important function in RyR1 calcium channel behavior. The fact that domain A of the II-III loop of DHPR binds onto the same RyR1 sequences re-opens the question of the role of the domain A in the control of RyR1 calcium channel activity in the context of the DHPR/RyR1 complex. Our results suggest that the two sequences of RyR1 (F3 and F7), which are involved in the binding of MCa, could fold together to create a unique MCa binding site. Cryoelectron microscopy studies of the RyR1-MCa complex should permit to test this hypothesis and to refine the correspondence between primary structure and three-dimensinoal reconstruction images of RyR1.
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