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• W2013297848 abstract "Phosphorylation of the skeletal muscle (RyR1) and cardiac muscle (RyR2) ryanodine receptors has been reported to modulate channel activity. Abnormally high phosphorylation levels (hyperphosphorylation) at Ser-2843 in RyR1 and Ser-2809 in RyR2 and dissociation of FK506-binding proteins from the receptors have been implicated as one of the causes of altered calcium homeostasis observed during human heart failure. Using site-directed mutagenesis, we prepared recombinant RyR1 and RyR2 mutant receptors mimicking constitutively phosphorylated and dephosphorylated channels carrying a Ser/Asp (RyR1-S2843D and RyR2-S2809D) and Ser/Ala (RyR1-S2843A and RyR2-S2809A) substitution, respectively. Following transient expression in human embryonic kidney 293 cells, the effects of Ca2+, Mg2+, and ATP on channel function were determined using single channel and [3H]ryanodine binding measurements. In both assays, neither the skeletal nor cardiac mutants showed significant differences compared with wild type. Similarly essentially identical caffeine responses were observed in Ca2+ imaging measurements. Co-immunoprecipitation and Western blot analysis showed comparable binding of FK506-binding proteins to wild type and mutant receptors. Finally metabolic labeling experiments showed that the cardiac ryanodine receptor was phosphorylated at additional sites. Taken together, the results did not support the view that phosphorylation of a single site (RyR1-Ser-2843 and RyR2-Ser-2809) substantially changes RyR1 and RyR2 channel function. Phosphorylation of the skeletal muscle (RyR1) and cardiac muscle (RyR2) ryanodine receptors has been reported to modulate channel activity. Abnormally high phosphorylation levels (hyperphosphorylation) at Ser-2843 in RyR1 and Ser-2809 in RyR2 and dissociation of FK506-binding proteins from the receptors have been implicated as one of the causes of altered calcium homeostasis observed during human heart failure. Using site-directed mutagenesis, we prepared recombinant RyR1 and RyR2 mutant receptors mimicking constitutively phosphorylated and dephosphorylated channels carrying a Ser/Asp (RyR1-S2843D and RyR2-S2809D) and Ser/Ala (RyR1-S2843A and RyR2-S2809A) substitution, respectively. Following transient expression in human embryonic kidney 293 cells, the effects of Ca2+, Mg2+, and ATP on channel function were determined using single channel and [3H]ryanodine binding measurements. In both assays, neither the skeletal nor cardiac mutants showed significant differences compared with wild type. Similarly essentially identical caffeine responses were observed in Ca2+ imaging measurements. Co-immunoprecipitation and Western blot analysis showed comparable binding of FK506-binding proteins to wild type and mutant receptors. Finally metabolic labeling experiments showed that the cardiac ryanodine receptor was phosphorylated at additional sites. Taken together, the results did not support the view that phosphorylation of a single site (RyR1-Ser-2843 and RyR2-Ser-2809) substantially changes RyR1 and RyR2 channel function. Contraction of striated muscle is induced by the release of Ca2+ from the sarcoplasmic reticulum (SR) 1The abbreviations used are: SRsarcoplasmic reticulumRyRryanodine receptorRyR1skeletal muscle isoform of RyRRyR2cardiac muscle isoform of RyRFKBPFK506-binding proteinHEKhuman embryonic kidneyPKAcAMP-dependent protein kinaseCaMcalmodulinCaMKIICa2+/calmodulin-dependent kinase IIWTwild typePipes1,4-piperazinediethanesulfonic acidPBSphosphate-buffered salineKRHKrebs-Ringer-Henseleit solutionAMP-PCPadenosine 5′-(β,γ-methylenetriphosphate).1The abbreviations used are: SRsarcoplasmic reticulumRyRryanodine receptorRyR1skeletal muscle isoform of RyRRyR2cardiac muscle isoform of RyRFKBPFK506-binding proteinHEKhuman embryonic kidneyPKAcAMP-dependent protein kinaseCaMcalmodulinCaMKIICa2+/calmodulin-dependent kinase IIWTwild typePipes1,4-piperazinediethanesulfonic acidPBSphosphate-buffered salineKRHKrebs-Ringer-Henseleit solutionAMP-PCPadenosine 5′-(β,γ-methylenetriphosphate). in a series of signal transduction steps known as excitation-contraction coupling. SR Ca2+ release is initiated by an action potential on the surface membrane that activates SR calcium release channels also known as ryanodine receptors (RyRs). In mammalian skeletal muscle, this process is mediated by a direct physical interaction between voltage-gated, dihydropyridine-sensitive Ca2+ channels located in the surface membrane and transverse tubular infoldings (t-tubules) and the skeletal muscle RyR (RyR1) (1Rios E. Pizarro G. Physiol. Rev. 1991; 71: 849-908Crossref PubMed Scopus (495) Google Scholar). In mammalian myocardium, an action potential initiates cardiac ryanodine receptor (RyR2) activation and SR Ca2+ release by a mechanism referred to as calcium-induced calcium release (2Fabiato A. Am. J. Physiol. 1983; 245: C1-C14Crossref PubMed Google Scholar). During calcium-induced calcium release, the influx of Ca2+ ions through voltage-gated, dihydropyridine-sensitive Ca2+ channels triggers the massive release of Ca2+ from the SR by activating a group of closely opposed RyR2s (3Franzini-Armstrong C. Protasi F. Ramesh V. Biophys. J. 1999; 77: 1528-1539Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar). sarcoplasmic reticulum ryanodine receptor skeletal muscle isoform of RyR cardiac muscle isoform of RyR FK506-binding protein human embryonic kidney cAMP-dependent protein kinase calmodulin Ca2+/calmodulin-dependent kinase II wild type 1,4-piperazinediethanesulfonic acid phosphate-buffered saline Krebs-Ringer-Henseleit solution adenosine 5′-(β,γ-methylenetriphosphate). sarcoplasmic reticulum ryanodine receptor skeletal muscle isoform of RyR cardiac muscle isoform of RyR FK506-binding protein human embryonic kidney cAMP-dependent protein kinase calmodulin Ca2+/calmodulin-dependent kinase II wild type 1,4-piperazinediethanesulfonic acid phosphate-buffered saline Krebs-Ringer-Henseleit solution adenosine 5′-(β,γ-methylenetriphosphate). Modulation of skeletal and cardiac excitation-contraction coupling results, in part, from changes in the phosphorylation state of numerous SR proteins involved in Ca2+ homeostasis. One of these proteins has been suggested to be the ryanodine receptor. RyR1 and RyR2 belong to a family of ligand-gated ion channels composed of four large subunits with molecular masses of ∼565 kDa with each subunit being associated with one small 12- and 12.6-kDa FK506-binding protein (FKBP12 and -12.6), respectively (4Franzini-Armstrong C. Protasi F. Physiol. Rev. 1997; 77: 699-729Crossref PubMed Scopus (592) Google Scholar, 5Fill M. Copello J.A. Physiol. Rev. 2002; 82: 893-922Crossref PubMed Scopus (880) Google Scholar). The FKBPs belong to the family of immunophilins, exhibit cis/trans isomerase activity, and have been reported to stabilize channel protein conformation and synchronize the gating of neighboring RyRs (coupled gating) (6Brillantes A.B. Ondrias K. Scott A. Kobrinsky E. Ondriasova E. Moschella M.C. Jayaraman T. Landers M. Ehrlich B.E. Marks A.R. Cell. 1994; 77: 513-523Abstract Full Text PDF PubMed Scopus (702) Google Scholar, 7Marx S.O. Ondrias K. Marks A.R. Science. 1998; 281: 818-821Crossref PubMed Scopus (352) Google Scholar, 8Marx S.O. Gaburjakova J. Gaburjakova M. Henrikson C. Ondrias K. Marks A.R. Circ. Res. 2001; 88: 1151-1158Crossref PubMed Scopus (330) Google Scholar). RyR1 and RyR2 are part of a multiprotein complex that includes cAMP-dependent protein kinase (PKA), protein phosphatase 1, and muscle protein kinase A-anchoring protein (9Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1673) Google Scholar). The RyR2 complex also contains and is regulated by protein phosphatase 2A (9Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1673) Google Scholar, 10Bandyopadhyay A. Shin D.W. Ahn J.O. Kim D.H. Biochem. J. 2000; 352: 61-70Crossref PubMed Scopus (75) Google Scholar). Although isoform-specific differences are observed, the skeletal and cardiac ryanodine receptors are regulated by a common group of endogenous ligands including Ca2+, Mg2+, ATP, and calmodulin (5Fill M. Copello J.A. Physiol. Rev. 2002; 82: 893-922Crossref PubMed Scopus (880) Google Scholar, 11Meissner G. Front. Biosci. 2002; 7: d2072-d2080Crossref PubMed Google Scholar). Initially RyR2 was proposed to have a unique phosphorylation site (RyR2-Ser-2809) for Ca2+/calmodulin-dependent kinase II (CaMKII). The site was also phosphorylated by PKA but to a lesser extent (12Witcher D.R. Kovacs R.J. Schulman H. Cefali D.C. Jones L.R. J. Biol. Chem. 1991; 266: 11144-11152Abstract Full Text PDF PubMed Google Scholar). Although initial studies found that the homologous site in skeletal RyR (RyR1-Ser-2843) was not phosphorylated by CaMKII (12Witcher D.R. Kovacs R.J. Schulman H. Cefali D.C. Jones L.R. J. Biol. Chem. 1991; 266: 11144-11152Abstract Full Text PDF PubMed Google Scholar), other studies have observed in vitro CaMKII phosphorylation of this site and additional threonine residue(s) (13Chu A. Sumbilla C. Inesi G. Jay S.D. Campbell K.P. Biochemistry. 1990; 29: 5899-5905Crossref PubMed Scopus (58) Google Scholar, 14Suko J. Maurer F.I. Plank B. Bertel O. Wyskovsky W. Hohenegger M. Hellmann G. Biochim. Biophys. Acta. 1993; 1175: 193-206Crossref PubMed Scopus (102) Google Scholar). Different functional effects following CaMKII and PKA phosphorylation also indicated the presence of additional phosphorylation sites (15Hain J. Onoue H. Mayrleitner M. Fleischer S. Schindler H. J. Biol. Chem. 1995; 270: 2074-2081Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Metabolic labeling of RyR2 provided direct evidence for additional phosphorylation sites by showing that CaMKII phosphorylates at least four additional sites in RyR (16Rodriguez P. Bhogal M.S. Colyer J. J. Biol. Chem. 2003; 278: 38593-38600Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). In support of a physiological role, CaMKII co-purified with cardiac muscle RyR (17Hohenegger M. Suko J. Biochem. J. 1993; 296: 303-308Crossref PubMed Scopus (71) Google Scholar) and, in skeletal SR vesicles, remained anchored to RyR in lipid bilayer measurements, altering the regulation of channel activity by ATP and Ca2+ (18Dulhunty A.F. Laver D. Curtis S.M. Pace S. Haarmann C. Gallant E.M. Biophys. J. 2001; 81: 3240-3252Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In skeletal muscle, αKAP, a non-kinase protein, has been identified as one of the anchoring proteins necessary for targeting the CaMKII holoenzyme to the SR membrane (19Bayer K.U. Harbers K. Schulman H. EMBO J. 1998; 17: 5598-5605Crossref PubMed Scopus (109) Google Scholar). In single channel measurements, PKA-mediated phosphorylation of RyR decreased ATP and Mg2+ sensitivity, and the phosphorylated RyR channels exhibited an increase in the number of channel events and mean open time (20Uehara A. Yasukochi M. Mejia-Alvarez R. Fill M. Imanaga I. Pflueg. Arch. Eur. J. Physiol. 2002; 444: 202-212Crossref PubMed Scopus (32) Google Scholar) and altered the Ca2+ sensitivity under non-steady-state conditions (21Valdivia H.H. Kaplan J.H. Ellis-Davies G.C. Lederer W.J. Science. 1995; 267: 1997-2000Crossref PubMed Scopus (316) Google Scholar). In failing hearts, release of protein phosphatases from the RyR2 protein complex has been suggested to increase the endogenous PKA-mediated phosphorylation levels of RyR2 (“hyperphosphorylation”) (9Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1673) Google Scholar). Recently hyperphosphorylation and FKBP12 depletion of the skeletal ryanodine receptor during heart disease have been described and linked to the severely decreased exercise capability observed in these patients (22Ward C.W. Reiken S. Marks A.R. Marty I. Vassort G. Lacampagne A. FASEB J. 2003; 17: 1517-1519Crossref PubMed Google Scholar). Failing to observe dissociation of protein phosphatase 1, the only phosphatase known to associate with RyR1, during heart failure indicates a novel mechanism for PKA-mediated hyperphosphorylation of RyR1 in skeletal muscle (23Reiken S. Lacampagne A. Zhou H. Kherani A. Lehnart S.E. Ward C. Huang F. Gaburjakova M. Gaburjakova J. Rosemblit N. Warren M.S. He K.L. Yi G.H. Wang J. Burkhoff D. Vassort G. Marks A.R. J. Cell Biol. 2003; 160: 919-928Crossref PubMed Scopus (189) Google Scholar). In both RyR1 and RyR2, hyperphosphorylation of the receptor was accompanied by an almost complete elimination of FKBP12 and FKBP12.6 binding to the receptor (9Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1673) Google Scholar, 23Reiken S. Lacampagne A. Zhou H. Kherani A. Lehnart S.E. Ward C. Huang F. Gaburjakova M. Gaburjakova J. Rosemblit N. Warren M.S. He K.L. Yi G.H. Wang J. Burkhoff D. Vassort G. Marks A.R. J. Cell Biol. 2003; 160: 919-928Crossref PubMed Scopus (189) Google Scholar). Loss of FKBP was proposed to increase RyR sensitivity to the agonist Ca2+ and thereby increase SR Ca2+ “leakiness” at diastolic Ca2+ levels (9Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1673) Google Scholar, 23Reiken S. Lacampagne A. Zhou H. Kherani A. Lehnart S.E. Ward C. Huang F. Gaburjakova M. Gaburjakova J. Rosemblit N. Warren M.S. He K.L. Yi G.H. Wang J. Burkhoff D. Vassort G. Marks A.R. J. Cell Biol. 2003; 160: 919-928Crossref PubMed Scopus (189) Google Scholar, 24Yano M. Ono K. Ohkusa T. Suetsugu M. Kohno M. Hisaoka T. Kobayashi S. Hisamatsu Y. Yamamoto T. Noguchi N. Takasawa S. Okamoto H. Matsuzaki M. Circulation. 2000; 102: 2131-2136Crossref PubMed Scopus (197) Google Scholar). Other studies failed to establish a role for PKA-mediated phosphorylation on RyR activity (25Jiang M.T. Lokuta A.J. Farrell E.F. Wolff M.R. Haworth R.A. Valdivia H.H. Circ. Res. 2002; 91: 1015-1022Crossref PubMed Scopus (210) Google Scholar, 26Li Y. Kranias E.G. Mignery G.A. Bers D.M. Circ. Res. 2002; 90: 309-316Crossref PubMed Scopus (231) Google Scholar). To investigate the functional effects of RyR phosphorylation in more detail, we prepared recombinant RyR1 and RyR2 phosphorylation mutants that cannot be phosphorylated at their known phosphorylation site, RyR1-Ser-2843 and RyR2-Ser-2809, due to an alanine or aspartic acid substitution. These amino acid substitutions mimic a constitutively dephosphorylated and phosphorylated receptor state, respectively. Functional and biochemical effects of these mutations were assessed using [3H]ryanodine binding, single channel, and calcium imaging measurements as well as co-immunoprecipitation. The results presented here suggest that ryanodine receptors may be phosphorylated at additional site(s) in vivo and that phosphorylation of RyR1-Ser-2843 and RyR2-Ser-2809, per se, does not cause major functional changes. Materials—Phospholipids were obtained from Avanti Polar Lipids (Birmingham, AL). [3H]Ryanodine and [32P]PO43- were purchased from PerkinElmer Life Sciences and ICN Biomedicals Inc. (Costa Mesa, CA), respectively. FK506 was generously provided by Dr. Bekershy, Fujisawa Healthcare Inc., Deerfield, IL. All chemicals used were of analytical grade. Site-directed Mutagenesis and Cell Culture—The full-length rabbit RyR1 cDNA was constructed as described previously (27Gao L. Tripathy A. Lu X. Meissner G. FEBS Lett. 1997; 412: 223-226Crossref PubMed Scopus (56) Google Scholar). Full-length rabbit RyR2 was kindly provided by Dr. Junichi Nakai, National Institute of Physiological Science, Okazaki, Japan. cDNAs encoding RyR1-S2843A and -S2843D and RyR2-S2809A and -S2809D mutants were prepared using Pfu polymerase-based chain reaction and the QuikChange site-directed mutagenesis kit according to the gmanufacturer's instructions (Stratagene, La Jolla, CA). The sequences of the primers used for mutagenesis were as follows: RyR1-S2843A, g aca cgg aag att Gcc cag act gcc cag; RyR1-S2843D, g aaa aag aca cgg aag att GAc cag act gcc cag acc tac; RyR2-S2809A, ga act cgt cgt att Gct cag aca agc cag g; RyR2-S2809D, cc ctt tat aac cga act cgt cgt att GAt cag aca agc c. The PvuI/NdeI (positions 8600–11304) fragment of RyR1 cDNA and the MunI/XbaI (positions 7736–10066) fragment of RyR2 subcloned into vector served as the template for mutagenesis. The complete mutated sequences were confirmed by DNA sequencing. The RyR1 mutated sequence was subcloned back into the original position of RyR1 in two steps: to a vector containing PvuI/XbaI (positions 8600–15276) fragment and preparation of mutated RyR1 full-length plasmids by ligation of two fragments (ClaI/PvuI and PvuI/XbaI containing the mutated sequence) and pCMV5 (ClaI/XbaI). The RyR2 fragment with the mutation was subcloned back into the original position of RyR2 in two steps: to a vector containing BbrPI/SacII (positions 5038–11203) fragment and to full-length RyR2 in pCIneo. Nucleotide numbering is as described previously (27Gao L. Tripathy A. Lu X. Meissner G. FEBS Lett. 1997; 412: 223-226Crossref PubMed Scopus (56) Google Scholar, 28Nakai J. Imagawa T. Hakamata Y. Shigekawa M. Takeshima H. Numa S. FEBS Lett. 1990; 271: 169-177Crossref PubMed Scopus (287) Google Scholar). WT and mutant RyRs were transiently expressed, in the presence of FKBP12.6 where indicated, in HEK293 cells using FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions. Cells were maintained at 37 °C and 5% CO2 in high glucose Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and plated the day before transfection. For each 10-cm culture dish, 3.5 μg of cDNA was used. Cells were harvested 48 h after transfection. Crude membrane fractions were prepared as described previously (27Gao L. Tripathy A. Lu X. Meissner G. FEBS Lett. 1997; 412: 223-226Crossref PubMed Scopus (56) Google Scholar) and stored under liquid nitrogen until use. Preparation of SR Vesicles—SR vesicle fractions enriched in [3H]ryanodine binding were prepared from rabbit skeletal and canine cardiac muscle in the presence of protease inhibitors (100 nm aprotinin, 1 μm leupeptin, 1 μm pepstatin, 1 mm benzamidine, 0.2 mm phenylmethylsulfonyl fluoride) (29Meissner G. J. Biol. Chem. 1984; 259: 2365-2374Abstract Full Text PDF PubMed Google Scholar, 30Meissner G. Henderson J.S. J. Biol. Chem. 1987; 262: 3065-3073Abstract Full Text PDF PubMed Google Scholar). Single Channel Measurements—Single channel measurements were performed in symmetric KCl solutions (250 mm, 20 mm KHepes, pH 7.4) containing additions as indicated (31Stange M. Tripathy A. Meissner G. Biophys. J. 2001; 81: 1419-1429Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Membrane fractions containing the WT and mutant receptors were added to the cis chamber of a bilayer apparatus and fused in the presence of an osmotic gradient with Mueller-Rudin type planar bilayers containing a 5:3:2 mixture of bovine brain phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine (35 mg of total phospholipid/ml n-decane). The gradient was formed across the bilayer membrane with 250 mmcis KCl and 20 mmtrans KCl solutions. After appearance of single channel activity, an increase in trans KCl concentration to 250 mm prevented further incorporation of RyRs. The trans side of the bilayer was defined as ground. Channel activities were recorded at –20 mV and with 10 mm Ca2+ in the trans chamber at 0-mV holding potential using a commercially available patch clamp amplifier with a bilayer headstage (Axopatch 1D, Axon Instruments, Burlingame, CA). Measurement of the sensitivity of the channels to cytosolic Ca2+ indicated that in a majority of recordings (>98%) the cytosolic side of RyRs faced the cis side and the luminal side faced the trans side of the bilayer. Electrical signals were filtered at 2 kHz or 300 Hz with K+ and Ca2+ as current carrier, respectively, and digitized at 10 kHz. Data acquisition and analysis were performed with the software package pClamp 9.0.2. (Axon Instruments, Union City, CA). Data files were directly acquired using the continuous gap-free mode. Channel parameters were calculated from current recordings of 2-min duration using a threshold setting of 50% of the current amplitude between the closed and open states. Channel open probability (Po) in multichannel recordings was calculated using the formula Σ iPo,i/N where N is the total number of channels, and Po,i is channel open probability of the ith channel. The presence of possible substates relative to the full-open amplitude was probed using current amplitude histograms obtained from 2-min single channel recordings. [3H]Ryanodine Binding—Ryanodine binds with high specificity to the RyRs and is widely used as a probe of channel activity because of its preferential binding to the open channel state (4Franzini-Armstrong C. Protasi F. Physiol. Rev. 1997; 77: 699-729Crossref PubMed Scopus (592) Google Scholar, 32Sutko J.L. Airey J.A. Welch W. Ruest L. Pharmacol. Rev. 1997; 49: 53-98PubMed Google Scholar). Unless otherwise indicated, experiments were performed as described previously (33Balshaw D.M. Xu L. Yamaguchi N. Pasek D.A. Meissner G. J. Biol. Chem. 2001; 276: 20144-20153Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Membrane fractions (25–75 μg of protein in 120 μl of total sample volume) were incubated with 2.5 nm [3H]ryanodine in 10 mm imidazole, pH 7.0 buffer containing 150 mm KCl, 0.3 m sucrose, under oxidizing (1 mm oxidized glutathione, GSSG) or reducing (5 mm reduced glutathione, GSH) conditions, and the indicated additions of channel agonists and/or antagonists. After 24 h at room temperature, samples were diluted with 8.5 volumes of ice-cold water and placed on Whatman GF/B filters preincubated with 2% polyethylenimine in water. Radioactivity remaining on the filters was determined by liquid scintillation counting to obtain bound [3H]ryanodine. Nonspecific binding was determined using a 1000-fold excess of unlabeled ryanodine in the presence of 1 mm EGTA. The total number of ryanodine binding sites (Bmax) was determined in 10 mm imidazole, pH 7.0 buffer containing 800 mm KCl, 200 μm free Ca2+ by Scatchard plot analysis or at 30 nm [3H]ryanodine, a condition that caused maximal binding of [3H]ryanodine to the receptors. If not stated differently, data were normalized using Bmax values. In experiments using protein phosphatase-treated SR, acid phosphatase (Sigma) was dialyzed for 5 h at 4 °C against sample buffer, and membranes were subsequently incubated with phosphatase at 60 μg of protein/unit of protein phosphatase for 1 h at 37 °C. RyR2-Ser-2809 phosphorylation levels of treated and untreated samples were determined by immunoblot analysis using a phospho-RyR2-Ser-2809- and dephospho-RyR2-Ser-2809-specific antibody (Badrilla, Leeds, UK). Development and analysis of Western blots were performed as described under “Co-immunoprecipitation of RyRs and FKBPs” except that 5% bovine serum albumin in Tris-buffered saline was used for blocking and primary antibody incubation medium, and Tris-buffered saline with 0.05% Tween 20 was used as washing buffer. Co-immunoprecipitation of RyRs and FKBPs—For immunoprecipitation experiments, membranes (1–2 mg of protein) were homogenized in ice-cold 20 mm NaPipes, pH 7.2 buffer containing 0.3 m sucrose, 1 m NaCl, 2.5% Triton X-100, and protease inhibitors (Complete choice protease inhibitor tablets, Roche Applied Science) using a Teflon homogenizer. Following a 10-min incubation on ice, samples were diluted with ice-cold water to 1.25% Triton X-100 and incubated on ice for an additional 20 min. Solubilized receptors were separated by centrifugation at 100,000 × g for 40 min, diluted with 20 mm NaPipes, pH 7.0 buffer containing 0.3 m sucrose, 5 mm dithiothreitol, 1.2 mm CaCl2, and protease inhibitors to 0.3% Triton X-100 and 125 mm NaCl, and incubated with mouse monoclonal antibody RyRD110 (1:10) raised against rabbit skeletal muscle RyR and mouse monoclonal antibody RyRC3-33 (1:10) raised against cardiac muscle RyR at 4 °C. After 12 h, anti-mouse IgG-agarose beads (Sigma) were added, and the sample was incubated for an additional 2 h. Subsequently beads were washed three times with 20 mm NaPipes, pH 7.0 buffer containing 0.3% Triton X-100 and 125 mm NaCl and once with phosphate-buffered saline (PBS) to remove residual Triton X-100. Bound proteins were solubilized in SDS buffer at 37 °C for 15 min and separated on 3–15% gradient SDS-polyacrylamide gels. Proteins in the top half of the gel (>60 kDa) were transferred in a cooled wet chamber for 1 h at 400 mA and 12 h at 1000 mA onto a polyvinylidene difluoride membrane (Millipore) and blocked with 5% milk and 0.05% Tween 20 in PBS. RyRs were detected using monoclonal antibody RyRD110 and monoclonal antibody RyRC3-33, both diluted to 1:10 with 5% milk and 0.05% Tween 20 in PBS. The bottom half of the gel was transferred in a semidry chamber overnight at 50 mA onto polyvinylidene difluoride, blocked with 5% milk and 0.05% Tween 20 in PBS, and blotted with anti-FKBP antibody (Alexis) diluted to 1:2000 in 5% milk and 0.05% Tween 20 in PBS. RyR and co-immunoprecipitated FKBP were detected using enhanced chemiluminescence (ECL) (Amersham Biosciences), and proteins were quantified using Kodak Scientific Imaging Systems 1D version 3.6 software. Calcium Imaging—Cellular Ca2+ release in response to caffeine was determined by intracellular fluo-4 fluorescence. HEK293 cells transfected with cDNA encoding WT and mutant RyRs were grown on glass coverslips. RyR2 cDNAs were co-transfected with FKBP12.6 cDNA. For control experiments, non-transfected cells or cells transfected with vector-only plasmids were used. Calcium imaging experiments were performed similarly to those described in Ref. 34Fessenden 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. Approximately half-confluent cells were loaded with 5 μm fluo-4 ester, 0.1% dispersion agent (F-127) at 37 °C in Krebs-Ringer-Henseleit solution (KRH) containing 125 mm NaCl, 5 mm KCl, 1.2 mm KH2PO4, 6 mm glucose, 1.2 mm MgCl2, 2 mm CaCl2, and 25 mm Hepes, pH 7.4. After 1 h, cells were rinsed with KRH to remove non-hydrolyzed fluorophore and transferred to a chamber. The fluorescence response was measured using a Nikon Eclipse TE300 microscope and Photon Technology International Deltascan system. Fluo-4 was excited at 490 nm, and emitted fluorescence was recorded at 526 nm. Base-line fluorescence was adjusted to 30–40 response units and recorded for 30 s, and caffeine responsiveness of cells was measured by rapid (∼1-s) addition of freshly made caffeine solution to a final concentration of 10 mm. Preliminary experiments indicated that this concentration was sufficient to result in maximal caffeine response. To allow normalization, cells were subsequently treated with ionomycin to measure fluorescence in the presence of 2 mm Ca2+. For analysis, individual cells were defined as regions of interest, and average fluorescence was quantified using the program ImageMaster (Photon Technology International). Background levels were subtracted, and responsiveness was normalized using the analysis program Felix (Photon Technology International). Metabolic Labeling of RyR2—In metabolic labeling experiments, ∼2–3 × 107 HEK293 cells were used. To increase the extent of metabolic 32Pi labeling, cells were incubated for 1 h in phosphate-free Dulbecco's modified Eagle's medium prior to labeling for 12 h in fresh phosphatefree medium supplemented with 100 μCi/ml 32Pi (ICN Biomedicals Inc.). PBS and all other buffers used for harvesting and processing the cells contained Complete choice protease inhibitor tablets (Roche Applied Science) and phosphatase inhibitor mixture I (Sigma), 1 mm Na3VO4, and 75 mm Na3PO4. Crude membrane fractions from metabolically labeled HEK293 cells were solubilized in 20 mm NaPipes, pH 7.4 buffer containing 0.3 m sucrose, 2.5% Triton X-100, and 1 m NaCl. Extended solubilization times of 4 h were required before affinity purification to eliminate a 32P-labeled high molecular weight band traveling closely above the RyR on the gels. Following dilution to 0.3% Triton X-100 and 125 mm NaCl, RyR2 was purified by binding to CaM-agarose beads in the presence of 900 μm free Ca2+ and 3 mm dithiothreitol. Following washing of the beads, proteins were separated by 3–10% SDS-PAGE. Gels were silver-stained and dried using a gel drying solution (Invitrogen), and the incorporation of 32Pi was visualized using phosphorimaging (Storm PhosphorImager, ImageQuant analysis software, and general purpose screens, all obtained from Amersham Biosciences). In parallel experiments, unlabeled cells were processed identically. The position of RyR2 as determined by Western blot analysis was used to identify the silver-stained RyR2 bands in the 32Pi-labeled gels and PhosphorImager scans. To quantify the amount of 32Pi incorporation, identically sized gel pieces containing the RyR2 band were incubated with scintillation fluid for 24 h and counted using a scintillation counter. To account for potential differences in initial 32Pi uptake by the cells, 32P content of a second gel piece at 200-kDa molecular mass was determined and used to normalize the RyR2 signal. Background signal intensities of non- and vector-only-transfected cells were determined and subtracted from all samples, and final results were adjusted for RyR2 protein content based on signal intensity in silver-stai" @default.
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• W2013297848 date "2003-12-01" @default.
• W2013297848 modified "2023-09-26" @default.
• W2013297848 title "Characterization of Recombinant Skeletal Muscle (Ser-2843) and Cardiac Muscle (Ser-2809) Ryanodine Receptor Phosphorylation Mutants" @default.
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