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• W1970571883 abstract "Specific interactions between adjacent ryanodine receptor (RyR) molecules to form ordered two-dimensional arrays in the membrane have been demonstrated using electron microscopy both in situ, in tissues and cells, and in vitro, with the purified protein. RyR interoligomeric association has also been inferred from observations of simultaneous channel gating during multi-RyR channel recordings in lipid bilayers. In this study, we report experiments designed to identify the region(s) of the RyR molecule, participating in this reciprocal interaction. Using epitope-specific antibodies, we identified a RyR tryptic fragment that specifically bound the intact immobilized RyR. Three overlapping RyR fragments encompassing this epitope, expressed using an in vitro mammalian expression system, were immunoprecipitated by RyR. To refine the binding regions, smaller RyR fragments were expressed as glutathione S-transferase (GST) fusion proteins, and their binding to RyR was monitored using a “sandwich” enzyme-linked immunosorbent assay. Three GST-RyR fusion proteins demonstrated specific binding, dependent upon ionic strength. Binding was greatest at 50–150 mm NaCl for two GST-RyR constructs, and a third GST-RyR construct demonstrated maximum binding between 150 and 450 mm NaCl. The binding at high NaCl concentration suggested involvement of a hydrophobic interaction. In silico analysis of secondary structure showed evidence of coil regions in two of these RyR fragment sequences, which might explain these data. In GST pull-down assays, these same three fragments captured RyR2, and two of them retained RyR1. These results identify a region at the center of the linear RyR (residues 2540–3207 of human RyR2) which is able to bind to the RyR oligomer. This region may constitute a specific subdomain participating in RyR-RyR interaction. Specific interactions between adjacent ryanodine receptor (RyR) molecules to form ordered two-dimensional arrays in the membrane have been demonstrated using electron microscopy both in situ, in tissues and cells, and in vitro, with the purified protein. RyR interoligomeric association has also been inferred from observations of simultaneous channel gating during multi-RyR channel recordings in lipid bilayers. In this study, we report experiments designed to identify the region(s) of the RyR molecule, participating in this reciprocal interaction. Using epitope-specific antibodies, we identified a RyR tryptic fragment that specifically bound the intact immobilized RyR. Three overlapping RyR fragments encompassing this epitope, expressed using an in vitro mammalian expression system, were immunoprecipitated by RyR. To refine the binding regions, smaller RyR fragments were expressed as glutathione S-transferase (GST) fusion proteins, and their binding to RyR was monitored using a “sandwich” enzyme-linked immunosorbent assay. Three GST-RyR fusion proteins demonstrated specific binding, dependent upon ionic strength. Binding was greatest at 50–150 mm NaCl for two GST-RyR constructs, and a third GST-RyR construct demonstrated maximum binding between 150 and 450 mm NaCl. The binding at high NaCl concentration suggested involvement of a hydrophobic interaction. In silico analysis of secondary structure showed evidence of coil regions in two of these RyR fragment sequences, which might explain these data. In GST pull-down assays, these same three fragments captured RyR2, and two of them retained RyR1. These results identify a region at the center of the linear RyR (residues 2540–3207 of human RyR2) which is able to bind to the RyR oligomer. This region may constitute a specific subdomain participating in RyR-RyR interaction. Acting as sentinels to the sarcoplasmic reticulum (SR) 1The abbreviations used are: SR, sarcoplasmic reticulum; Ab, antibody; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; BSA, bovine serum albumin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DTT, dithiothreitol; ELISA, enzyme-linked immunosorbent assay; FKBP12, FK506-binding protein; GST, glutathione S-transferase; hRyR, human ryanodine receptor; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; RyR, ryanodine receptor. 1The abbreviations used are: SR, sarcoplasmic reticulum; Ab, antibody; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; BSA, bovine serum albumin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DTT, dithiothreitol; ELISA, enzyme-linked immunosorbent assay; FKBP12, FK506-binding protein; GST, glutathione S-transferase; hRyR, human ryanodine receptor; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; RyR, ryanodine receptor. Ca2+ store in muscle cells are ryanodine receptors (RyR), the ion channels that mediate release of SR Ca2+ into the cytosol (1Campbell K. Franzini-Armstrong C. Shamoo A. Biochim. Biophys. Acta. 1980; 602: 97-116Crossref PubMed Scopus (111) Google Scholar, 2Costello B. Chadwick A. Siao A. Chu A. Maurer A. Fleischer S. J. Cell Biol. 1986; 103: 741-753Crossref PubMed Scopus (82) Google Scholar, 3Mitchell R. Saito A. Palade P. Fleischer S. J. Cell Biol. 1983; 96: 1017-1029Crossref PubMed Scopus (31) Google Scholar). There are three distinct mammalian isoforms of RyR identified and characterized, with RyR1 and RyR2 being the predominant forms found in skeletal and cardiac muscle, respectively (4Takeshima 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 (862) Google Scholar, 5Zorzato F. Fujii J. Otsu K. Phillips M. Green N. Lai F. Meissner G. Maclennan D. J. Biol. Chem. 1990; 265: 2244-2256Abstract Full Text PDF PubMed Google Scholar, 6Tunwell E. Wickenden C. Bertrand B. Shevchenko V. Walsh M. Allen P. Lai F. Biochem. J. 1996; 318: 477-487Crossref PubMed Scopus (127) Google Scholar, 7Otsu K. Willard H. Khanna V. Zorato F. Green N. J. Biol. Chem. 1990; 265: 13472-13483Abstract Full Text PDF PubMed Google Scholar, 8Nakai J. Imagawa T. Hakamata Y. Shigekawa M. Takeshima H. Numa S. FEBS Lett. 1990; 271: 169-177Crossref PubMed Scopus (287) Google Scholar, 9Hakamata Y. Nakai J. Takeshima H. Imoto K. FEBS Lett. 1992; 312: 229-235Crossref PubMed Scopus (351) Google Scholar). To constitute a functional Ca2+ release channel, four RyR subunits, each of ∼560 kDa, coalesce to form a stable homotetramer that surrounds the single ion channel pore (10Lai F. Misra M. Xu L. Smith A. Meissner G. J. Biol. Chem. 1989; 264: 16776-16785Abstract Full Text PDF PubMed Google Scholar). The unique size of this homotetrameric RyR complex, about 30 nm across (11Radermacher M. Rao V. Grassucci R. Frank J. Timerman A. Fleicher S. Wagenknecht T. J. Cell Biol. 1994; 127: 411-423Crossref PubMed Scopus (240) Google Scholar, 12Anderson K. Lai F. Liu Q.-Y. Rousseau E. Erickson H. Meissner G. J. Biol. Chem. 1989; 264: 1329-1335Abstract Full Text PDF PubMed Google Scholar, 13Jeyakumar L. Copello J. O'Malley A. Wu G.-M. Grassucci R. Wagenknetch T. Fleischer S. J. Biol. Chem. 1998; 273: 16011-16020Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), has enabled a detailed study of its morphology in the hydrated form using cryoelectron microscopy combined with image reconstruction analysis (14Samso M. Wagenknecht T. J. Struct. Biol. 1998; 121: 172-180Crossref PubMed Scopus (38) Google Scholar). A remarkable similarity is observed in topological models of three-dimensional reconstructions of RyR1, RyR2, and RyR3, each showing a structure with tetragonal symmetry (15Serysheva I.I. Orlova E.V. Chiu W. Sherman M.B. Hamilton S.L. van Heel M. Nat. Struct. Biol. 1995; 2: 18-24Crossref PubMed Scopus (169) Google Scholar, 16Wagenknecht T. Radermacher M. FEBS Lett. 1995; 369: 43-46Crossref PubMed Scopus (40) Google Scholar, 17Sharma M. Penczek P. Grassucci R. Xin H.-B. Fleischer S. Wagenknecht T. J. Biol. Chem. 1998; 273: 18429-18434Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 18Serysheva I.I. Schatz M. van Heel M. Chiu W. Hamilton S.L. Biophys. J. 1999; 77: 1936-1944Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Analysis of the intracellular organization of RyR particles using freeze-fractured, negatively stained muscle SR junctions, revealed that RyRs are packed into discrete, two-dimensional arrays termed couplons (19Franzini-Armstrong C. Kish J. J. Mus. Res. Cell Motil. 1995; 16: 319-324Crossref PubMed Scopus (67) Google Scholar, 20Rios E. Stern M.D. Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 47-82Crossref PubMed Scopus (115) Google Scholar, 21Stern M.D. Pizarro G. Rios E. J. Gen. Physiol. 1997; 110: 415-440Crossref PubMed Scopus (183) Google Scholar, 22Franzini-Armstrong C. Protasi F. Ramesh V. Biophys. J. 1999; 77: 1528-1539Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar). In recent studies using RyR1 purified from rabbit muscle SR, the assembly of RyRs into a regular, chessboard-like two-dimensional array was found to be an intrinsic property of RyR oligomers and not dependent upon insertion in the SR membrane or ancillary proteins (23Yin C.C. Lai F.A. Nat. Cell Biol. 2000; 2: 669-671Crossref PubMed Scopus (100) Google Scholar). The projection maps derived from these reconstituted RyR arrays resembled those described previously for junctional SR arrays observed in situ (23Yin C.C. Lai F.A. Nat. Cell Biol. 2000; 2: 669-671Crossref PubMed Scopus (100) Google Scholar). Thus, RyR molecules associate together to form an ordered array, enabling individual oligomers to interact allosterically, creating an integrated microdomain structure within the SR membrane. The RyR-RyR interaction site is found within the cytoplasmic domain (23Yin C.C. Lai F.A. Nat. Cell Biol. 2000; 2: 669-671Crossref PubMed Scopus (100) Google Scholar), which is highly conserved in mammalian and non-mammalian species (4Takeshima 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 (862) Google Scholar, 5Zorzato F. Fujii J. Otsu K. Phillips M. Green N. Lai F. Meissner G. Maclennan D. J. Biol. Chem. 1990; 265: 2244-2256Abstract Full Text PDF PubMed Google Scholar, 6Tunwell E. Wickenden C. Bertrand B. Shevchenko V. Walsh M. Allen P. Lai F. Biochem. J. 1996; 318: 477-487Crossref PubMed Scopus (127) Google Scholar, 7Otsu K. Willard H. Khanna V. Zorato F. Green N. J. Biol. Chem. 1990; 265: 13472-13483Abstract Full Text PDF PubMed Google Scholar, 8Nakai J. Imagawa T. Hakamata Y. Shigekawa M. Takeshima H. Numa S. FEBS Lett. 1990; 271: 169-177Crossref PubMed Scopus (287) Google Scholar, 9Hakamata Y. Nakai J. Takeshima H. Imoto K. FEBS Lett. 1992; 312: 229-235Crossref PubMed Scopus (351) Google Scholar) in regard to topology and size (14Samso M. Wagenknecht T. J. Struct. Biol. 1998; 121: 172-180Crossref PubMed Scopus (38) Google Scholar, 24Sharma M.R. Jeyakumar L.H. Fleischer S. Wagenknecht T. J. Biol. Chem. 2000; 275: 9485-9491Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), suggestive of an essential functional role (25Stern M.D. Song L.S. Cheng H. Sham J.S. Yang H.T. Boheler K.R. Rios E. J. Gen. Physiol. 1999; 113: 469-489Crossref PubMed Scopus (228) Google Scholar). RyR array formation is a shared feature of the distinct isoforms because both RyR1 and RyR3, when transfected and expressed independently in a dyspedic myotube cell line, also formed similar two-dimensional arrays in situ (26Protasi F. Takekura H. Wang Y. Chen S.R. Meissner G. Allen P.D. Franzini-Armstrong C. Biophys. J. 2000; 79: 2494-2508Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 27Fessenden J.D. Wang Y. Moore R.A. Chen S.R. Allen P.D. Pessah I.N. Biophys. J. 2000; 79: 2509-2525Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The concept of the couplon, comprising arrays of interacting RyRs (and ancillary proteins such as calmodulin, sorcin, and FK506-binding protein (FKBP12)) (20Rios E. Stern M.D. Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 47-82Crossref PubMed Scopus (115) Google Scholar, 21Stern M.D. Pizarro G. Rios E. J. Gen. Physiol. 1997; 110: 415-440Crossref PubMed Scopus (183) Google Scholar), provides a model to explore the underlying structure and function relationship. The lateral dimensions of individual couplons and their distribution within the myocytes vary with muscle type (22Franzini-Armstrong C. Protasi F. Ramesh V. Biophys. J. 1999; 77: 1528-1539Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar) and may influence the kinetics of local and global Ca2+ signals. Ca2+ sparks originating from the SR were first observed in cardiac myocytes using fluo-3 confocal microscopy (28Cheng H. Lederer W. Cannell M. Science. 1993; 262: 740-744Crossref PubMed Scopus (1607) Google Scholar). However, it remains unclear whether a spark emanates from a finite structural entity, i.e. it equates to activation of an entire couplon, or whether it involves a fraction of the RyRs. The extent of recruitment could depend upon both the mechanisms initiating the spark and the regulatory influences modulating RyR activity (29Rios E. Stern M. Gonzalwez A. Pizarro G. Shirokova N. J. Gen. Physiol. 1999; 114: 31-48Crossref PubMed Scopus (69) Google Scholar, 30Gonzalez A. Kirsch W.G. Shirokova N. Pizarro G. Brum G. Pessah I.N. Stern M.D. Cheng H. Rios E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4380-4385Crossref PubMed Scopus (100) Google Scholar), including allosteric interactions and the disposition of other proteins (31Rice J.J. Jafri M.S. Winslow R.L. Biophys. J. 1999; 77: 1871-1884Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). In cardiac muscle, the “local control” model of Ca2+ signaling (“cluster bomb” hypothesis) postulates that a small number of RyR channels interact spatially with each L-type voltage-operated Ca2+ channel (dihydropyridine receptor), and then calcium-induced calcium release operates to trigger the opening of other RyRs (20Rios E. Stern M.D. Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 47-82Crossref PubMed Scopus (115) Google Scholar, 32Stern M. Biophys. J. 1992; 63: 497-517Abstract Full Text PDF PubMed Scopus (529) Google Scholar, 33Klein M. Cheng H. Santana L. Jiang Y.-H. Lederer W. Schneider M. Nature. 1996; 379: 455-458Crossref PubMed Scopus (276) Google Scholar, 34Cannel M. Soeller C. Trends Pharmacol. Sci. 1998; 19: 16-20Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 35Williams A. Eur. Heart J. 1997; 18: A27-A35Crossref PubMed Google Scholar). Combined patch clamp and confocal measurement of Ca2+ in the subsarcolemmal space estimates that each SR Ca2+ release event (“spark”) requires the concerted opening of four to six RyR molecules, following Ca2+ influx via one dihydropyridine receptor in isolated cardiac myocytes (36Wang S.Q. Sang L.S. Lakatta E.G. Cheng H. Nature. 2001; 410: 592-596Crossref PubMed Scopus (334) Google Scholar). The mechanism of simultaneous activation of the RyR molecules within the cluster to produce a Ca2+ spark remains uncertain. Stochastic gating theory suggests that each RyR molecule activates and closes independently (20Rios E. Stern M.D. Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 47-82Crossref PubMed Scopus (115) Google Scholar, 33Klein M. Cheng H. Santana L. Jiang Y.-H. Lederer W. Schneider M. Nature. 1996; 379: 455-458Crossref PubMed Scopus (276) Google Scholar). An alternative hypothesis proposes coupled gating, involving the functional interaction of adjacent RyR molecules, mediated by FKBP12 or 12.6, as demonstrated for RyR1 and RyR2 channels recorded in planar bilayer experiments, respectively (37Marx S. Ondrias K. Marks A. Science. 1998; 281: 818-821Crossref PubMed Scopus (351) Google Scholar, 38Marx S.O. Gaburjakova J. Gaburjakova M. Henrikson C. Ondrias K. Marks A.R. Circ. Res. 2001; 88: 1151-1158Crossref PubMed Scopus (328) Google Scholar). Similar results with coupled gating of inositol trisphosphate receptors (39Dargan S.L. Lea E.J.A. Dawson A.P. Biochem. J. 2002; 361: 401-407Crossref PubMed Scopus (44) Google Scholar) suggest that this may be a common mechanism for related Ca2+ release channels. Thus, RyR-RyR oligomeric interaction may underlie a central mechanism for regulation of EC coupling in muscle cells. The aim of this study was to identify potential sites of interaction between RyR oligomers. The strategy used was to trypsinize RyR and detect liberated peptides that specifically bound to immobilized tetramers of RyR, using site-specific antibodies for different regions of the RyR. A putative binding region was identified using this approach. The corresponding RyR protein fragments were expressed in vitro and as GST fusion proteins and then tested for binding to RyR by immunoprecipitation, ELISA, and GST pull-down assays. The results describe a potential RyR binding region near the center of the linear RyR sequence, within which were a number of potential binding domains. Materials—Restriction endonucleases were purchased from Amersham Biosciences, and all biochemicals, unless indicated otherwise, were purchased from Sigma. Primary antibodies (Abs) to RyR were rabbit antisera raised against peptides derived from RyR sequences; Ab2142 was to RyR1 residues 830–845 (peptide sequence RREGPRGPHLVGPSRC), Ab34 to the RyR2 residues 2872–2893 (peptide sequence CTLTAKEKAKDREKAQDLKFL, conserved in RyR1), Ab2160 to the RyR2 C terminus residues 4951–4965 (peptide sequence FFPAGDCFRKQYEDQL, conserved in RyR1), and Ab1093 to the hRyR2 residues 4459–4478 (peptide sequence EDKGKQKLRQLHTHRYGEPEC). The GST polyclonal antibody was raised against purified, recombinant GST protein. Preparation of hRyR2 Expression Vector Constructs—The coordinates of the expression vector constructs, corresponding to the protein sequence of human RyR2 (hRyR2), are given in Figs. 2A and 3A (6Tunwell E. Wickenden C. Bertrand B. Shevchenko V. Walsh M. Allen P. Lai F. Biochem. J. 1996; 318: 477-487Crossref PubMed Scopus (127) Google Scholar). Forward and reverse oligonucleotide primers were designed to contain appropriate restriction sites in-frame with the vectors used and were synthesized by Sigma Genosys. RyR constructs 1, 2, and 3 were inserted into a pGBKT7 vector (Clontech) for in vitro expression. For RyR construct 1 the primers were 5′-AAGGATCCACGCTTCTCTCATTGAC-3′ (forward) and 5′-AGGACTCGAGTTCCAGGTCCTTAAA-3′ (reverse); for RyR construct 2, 5′-CCGGATCCATAATATATGGCCAAAG-3′ (forward) and 5′-CTGGCTCGAGTTTCTCCAAAG-3′ (reverse); and for RyR construct 3, 5′-AGGGATCCTTAACCCACAACCT-3′ (forward) and 5′-CCTGCTCGAGGTTTTCCATGGT-3′ (reverse). DNA constructs encoding GST fusion proteins were made using pGEX vectors (Amersham Biosciences). GST-RyR 4, 4A, 4B, and 5 were constructed in pGEX-5X-2, whereas GST-RyR 6 used pGEX-4T-3. For GST-RyR 4 the forward primer was that used for GST-RyR construct 1, and the reverse was 5′-CAGCGTCGACAGAAACCTGGCT-3′. For GST-RyR construct 4A the forward primer was that used for RyR construct 1, and the reverse primer was 5′-GAGGCTCGAGTGTTTTTCCATCATATCGACATA-3′. For GST-RyR construct 4B the forward primer was 5′-CGCGGGATCCAGTCATCAATGGATTCTGAA-3′, and the reverse primer was that used for GST-RyR construct 4. For GST-RyR 5 the primers were 5′-CCGGATCCTTGACATGAGCAATGTTA-3′ (forward) and 5′-ACATGTCGACTGAGTTGTTGGAGGAAAC-3′ (reverse). For GST-RyR construct 6 the forward primer was 5′-CTATGGATCCGTGATGAAGACTGGCCTGGAG-3′, and the reverse primer was that used for RyR construct 2. All constructs were generated by PCR using Pfu DNA polymerase (Promega) and full-length hRyR2 DNA as the template (6Tunwell E. Wickenden C. Bertrand B. Shevchenko V. Walsh M. Allen P. Lai F. Biochem. J. 1996; 318: 477-487Crossref PubMed Scopus (127) Google Scholar) and were confirmed by DNA sequencing (ABI PRISM Big Dye kit; Applied Biosystems).Fig. 3Sandwich ELISAs of GST-RyR constructs. A, hRyR2 coordinates of GST-RyR constructs 4, 4A, 4B, 5, and 6 expressed and purified for ELISA. B, concentration-dependent binding of GST-RyR constructs to ELISA plates coated with an enriched RyR1 preparation (closed circles) or BSA (open circles). The binding of the GST moiety alone to either RyR1 (closed triangles) or BSA (open triangles) on the same ELISA plates is shown for each construct. The asterisks denote significant binding to RyR1 compared with BSA, p > 0.05, n = 3, ±S.E. C, Western blot analysis of the purified GST-RyR constructs and GST-FKBP12 developed with an anti-GST antibody. The expected molecular mass of each GST fusion protein (in kDa) is given beneath each blot and corresponds to the uppermost band observed.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In Vitro Expression of RyR Fragments and Immunoprecipitation by RyR—A TNT T7 Quick-coupled transcription/translation system (Promega) was used to incorporate [35S]methionine and [35S]cysteine (Promix, Amersham Biosciences) into RyR constructs 1, 2, and 3. A 10-μl aliquot of TNT Quick Master Mix was incubated with 1 μg of DNA vector template and 1 μl of Promix for 90 min at 30 °C to synthesize proteins in vitro.5 μl of the reaction mix was then added to native RyR1 protein and incubated for 16 h at 4 °C before immunoprecipitation. The final immunoprecipitation reaction mix contained 250 μg of enriched RyR1 protein, 100 mm NaCl, 20 mm PIPES buffer, pH 7.1, 0.5% CHAPS, and 1% bovine serum albumin (BSA), 20 μl of Protein G PLUS-agarose beads (Santa Cruz Biotechnology), and 10 μl of Ab2142. The Protein G PLUS beads were then washed five times with the immunoprecipitation buffer and twice more with phosphate-buffered saline (PBS: 137 mm NaCl, 8 mm Na2HPO4, 1.5 mm KH2PO4, and 2.7 mm KCl, pH 7.4) and then boiled in SDS sample buffer (0.2 m Tris-HCl, pH 6.8, 0.3 m dithiothreitol (DTT), 15 mm EDTA, 6% SDS, 30% glycerol, and bromphenol blue) for 1 min. Samples were loaded and separated electrophoretically on 12% SDS-polyacrylamide gels. The gel was fixed for 30 min (40% methanol, 10% acetic acid in H2O) and then incubated with Amplify enhancer solution (Amersham Biosciences) for 30 min. The gel was dried and exposed to x-ray film at –80 °C. Ab2142 was omitted from the controls to estimate nonspecific binding to the Protein G PLUS-agarose beads and plasticware. Preparation of GST-RyR Fragments—The protocol was based on the manufacturer's instructions for pGEX vectors (Amersham Biosciences). GST-RyR fusion protein expression in bacterial cultures (1l, 25 °C, Escherichia coli BL21 Rosetta; Novagen) was induced by 0.1 mm isopropyl β-d-1-thiogalactopyranoside when the absorbance (A600) reached 0.5. Cells were harvested after 3 h and the pellets frozen at –80 °C until required for purification. The batch purification protocol with glutathione-Sepharose 4B beads and elution with reduced glutathione was used (Amersham Bioscience). Reduced glutathione was removed by overnight dialysis of the eluted recombinant protein against PBS, pH 7.4. Purified GST fusion proteins were stored at –80 °C until use. GST-RyR Fusion Protein Pull-down Assays, Silver Stain, and Immunoblot Analysis—Pull-down assays using GST-RyR fusion proteins were performed with native RyR1- or RyR2-enriched preparations. 20 μg of GST fusion protein was mixed gently overnight at 4 °C with 40 μl of glutathione-Sepharose 4B and 200 μg of RyR1 or RyR2 in binding buffer (20 mm PIPES, pH 7.1, 0.5% CHAPS, 1% BSA, 200 mm NaCl) in a total volume of 800 μl. The beads were washed four times with PBS, eluted with SDS sample buffer, and boiled for 1 min. The sample was separated by 5.5% SDS-PAGE and either silver stained (Bio-Rad silver stain kit) or transferred to polyvinylidene difluoride membrane for 4 h at 20 V using a semidry blotter (Bio-Rad) and the recommended transfer solution (39 mm glycine, 48 mm Tris, 0.0375% SDS, 20% methanol). The membrane was blocked overnight at 4 °C with blocking buffer (0.2% Tween 20, 5% milk proteins). The remaining blotting procedure was carried out at 22 °C, using primary antibodies Ab2142 (1:1,000) and Ab1093 (1:500) to detect RyR1 and RyR2 (1 h), respectively. Horseradish peroxidase-conjugated secondary Ab (1:10,000) was used; the blots were developed with Supersignal West Dura reagent (Perbio) and the images recorded on a ChemiDoc acquisition system (Bio-Rad). GST-RyR Fusion Protein Overlay Assays—To dissociate the RyR1 tetramers to monomers, enabling analysis on native polyacrylamide gels, the enriched RyR1 preparations were incubated for 1 h at 4 °C with zwittergent (0.9%) (10Lai F. Misra M. Xu L. Smith A. Meissner G. J. Biol. Chem. 1989; 264: 16776-16785Abstract Full Text PDF PubMed Google Scholar). Zwittergent-treated RyR1 preparations were run on native 5.5% SDS gels, as described above, except that SDS was omitted from the gel and sample and running buffers, and DTT from the sample buffer. Proteins were transferred to polyvinylidene difluoride membrane as above for 4 h (>200 kDa) or for 30 min (<200 kDa). A Bio-Rad antibody screening apparatus was used to create distinct wells covering the length of the membrane, which was incubated with 500 μl of 40 μg/ml GST-RyR fusion proteins in binding buffer (as in the above GST pull-down assays, except 5% milk protein replaced 1% BSA) with rocking at 4 °C overnight. The membrane was washed three times with binding buffer and developed with either anti-RyR Ab2142 or anti-GST as described above. Binding of RyR Tryptic Fragments to Immobilized RyR—Solubilized RyR from skeletal muscle microsomes was purified by sucrose gradient centrifugation (23Yin C.C. Lai F.A. Nat. Cell Biol. 2000; 2: 669-671Crossref PubMed Scopus (100) Google Scholar). To lower the NaCl concentration to 200 mm, the RyR peak fraction was diluted with 20 mm PIPES buffer, pH 7.1, 0.5% CHAPS, 100 μm EGTA, 150 mm CaCl2, 2 mm DTT, and protease inhibitors (0.2 mm AEBSF, 0.5 mm benzamidine, 1 mm iodoacetamide) and concentrated to ∼1.5 mg/ml protein using centrifugal concentrators (Amicon). Protease inhibitors were omitted from gradients when used for trypsinization experiments, and DTT was omitted from preparations used to couple RyR1 to agarose. Trypsin digestion was performed in 20 mm PIPES buffer, pH 7.1, 0.5% CHAPS, 100 μm EGTA, 150 mm CaCl2, 2 mm DTT, 200 mm NaCl, with 800 μg of RyR protein and 2 μg of trypsin (protein:trypsin ratio 400:1 w/w (40Meissner G. Rousseaux E. Lai F.A. J. Biol. Chem. 1989; 264: 1715-1722Abstract Full Text PDF PubMed Google Scholar)). At specific intervals up to 30 min, trypsin digests were stopped by the addition of 100 μg of trypsin inhibitor and AEBSF to 0.2 mm. RyR1 was coupled to an agarose support using Sulfolink coupling gel according to the manufacturer's protocol (Perbio). The coupling buffer used to equilibrate a 2-ml bed volume column was 50 mm Tris, pH 8.5, 5 mm EDTA, 1 m NaCl, 0.5% CHAPS, and 5% sucrose. 1 ml of this buffer was combined with 1 ml of RyR1 preparation (1.5 mg of protein/ml) and then mixed with coupling gel for 15 min and allowed to settle for 30 min at room temperature. The coupling gel was washed with 5 bed volumes of coupling buffer. To quench unbound coupling groups, 2 ml of 50 mm cysteine was added and mixed as before. A control column lacking RyR protein was quenched in a similar way. The coupling gel columns were washed four times with 2 bed volumes of 1 m NaCl in PBS to remove unbound protein and equilibrated with 5 bed volumes of 20 mm PIPES buffer, pH 7.1, 0.5% CHAPS, 100 μm EGTA, 150 mm CaCl2, 2 mm DTT, 200 mm NaCl, and 0.2 mm AEBSF. A 30-min tryptic digest of 1.2 ml of RyR1 was loaded into the RyR and control columns, which were capped and incubated at room temperature for 1 h and then washed with 5 bed volumes of equilibration buffer. Bound protein was eluted with 1 ml of the same buffer but containing 1 m NaCl. Eluates were concentrated using centrifugal concentrators, desalted using Microbiospin chromatography columns (Bio-Rad) to reduce the NaCl concentration, and the eluates were boiled with sample buffer and subjected to SDS-PAGE and Western blotting with Abs 2142, 34, and 2160. Preparation of Enriched RyR1—Native RyR1 was prepared from solubilized skeletal muscle microsomes (23Yin C.C. Lai F.A. Nat. Cell Biol. 2000; 2: 669-671Crossref PubMed Scopus (100) Google Scholar) using fast protein liquid chromatography anion exchange chromatography with a 20-ml Resource Q column (Bio-Rad) in place of sucrose gradient centrifugation. RyR1 was eluted with a 125–1,000 mm NaCl gradient in 20 mm PIPES buffer, pH 7.1, 0.5% CHAPS, 100 μm EGTA, 150 mm CaCl2, 2 mm DTT, and protease inhibitors 0.2 mm AEBSF, 0.5 mm benzamidine, and 1 mm iodoacetamide. The RyR protein peak eluting at ∼350 mm NaCl provided the RyR1 preparation used for all experiments other than those with trypsin digests. The protein concentration in this fraction was typically 1 mg/ml. In all of the experimental protocols described, the final NaCl concentration was calculated to allow for the 350 mm NaCl contribution of the buffer containing the enriched RyR preparation. Preparation of Enriched RyR2—Native RyR2 protein was prepared from pig heart microsomes, solubilized, and enriched using sucrose density gradient centrifugation with the same protocol as that for the RyR1 preparation used for the trypsin digest experiments (described above). RyR Interaction ELISAs—Either 10 μg of RyR1-enriched preparation or BSA was added to the wells of a 96-well ELISA plate (ICN) for 2hat22 °C in PBS and bicarbonate buffer (41Harlow E. Lane D. Using Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1999Google Scholar). The wells were washed three times with PBS. 100 μl of GST-RyR fusion protein (1,000 ng/well), made up in assay buf" @default.
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• W1970571883 date "2004-04-01" @default.
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• W1970571883 title "Ryanodine Receptor Oligomeric Interaction" @default.
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• W1970571883 doi "https://doi.org/10.1074/jbc.m308014200" @default.
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