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• W2067686089 abstract "Metabolically 35S-labeled calmodulin (CaM) was used to determine the CaM binding properties of the cardiac ryanodine receptor (RyR2) and to identify potential channel domains for CaM binding. In addition, regulation of RyR2 by CaM was assessed in [3H]ryanodine binding and single-channel measurements. Cardiac sarcoplasmic reticulum vesicles bound approximately four CaM molecules per RyR2 tetramer in the absence of Ca2+; in the presence of 100 μmCa2+, the vesicles bound 7.5 CaM molecules per tetramer. Purified RyR2 bound approximately four [35S]CaM molecules per RyR tetramer, both in the presence and absence of Ca2+. At least four CaM binding domains were identified in [35S]CaM overlays of fusion proteins spanning the full-length RyR2. The affinity (but not the stoichiometry) of CaM binding was altered by redox state as controlled by the presence of either GSH or GSSG. Inhibition of RyR2 activity by CaM was influenced by Ca2+ concentration, redox state, and other channel modulators. Parallel experiments with the skeletal muscle isoform showed major differences in the CaM binding properties and regulation by CaM of the skeletal and cardiac ryanodine receptors. Metabolically 35S-labeled calmodulin (CaM) was used to determine the CaM binding properties of the cardiac ryanodine receptor (RyR2) and to identify potential channel domains for CaM binding. In addition, regulation of RyR2 by CaM was assessed in [3H]ryanodine binding and single-channel measurements. Cardiac sarcoplasmic reticulum vesicles bound approximately four CaM molecules per RyR2 tetramer in the absence of Ca2+; in the presence of 100 μmCa2+, the vesicles bound 7.5 CaM molecules per tetramer. Purified RyR2 bound approximately four [35S]CaM molecules per RyR tetramer, both in the presence and absence of Ca2+. At least four CaM binding domains were identified in [35S]CaM overlays of fusion proteins spanning the full-length RyR2. The affinity (but not the stoichiometry) of CaM binding was altered by redox state as controlled by the presence of either GSH or GSSG. Inhibition of RyR2 activity by CaM was influenced by Ca2+ concentration, redox state, and other channel modulators. Parallel experiments with the skeletal muscle isoform showed major differences in the CaM binding properties and regulation by CaM of the skeletal and cardiac ryanodine receptors. ryanodine receptor skeletal muscle RyR cardiac muscle RyR sarcoplasmic reticulum calmodulin reduced glutathione oxidized glutathione myosin light chain kinase-derived calmodulin binding peptide bovine serum albumin adenosine 5′-(β,γ-methylene)triphosphate 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid 4-morpholinepropanesulfonic acid 1,4-piperazinediethanesulfonic acid glutathione S-transferase amino acids The ryanodine receptors (RyRs)1 are large, high conductance Ca2+ release channels found in a specialized subcompartment of the endoplasmic reticulum of many tissues (1Sorrentino V. Volpe P. Trends Pharmacol. Sci. 1993; 14: 98-103Abstract Full Text PDF PubMed Scopus (276) Google Scholar). In muscle cells, this subcompartment is referred to as the sarcoplasmic reticulum (SR). There are three known mammalian RyR isoforms: RyR1, which is the dominant isoform in skeletal muscle; RyR2, which is found in cardiac muscle; and RyR3, which is expressed in many tissues at low levels but is mostly associated with diaphragm and brain. In both skeletal and cardiac muscle, Ca2+ release through the RyR in response to a signal received from the T-tubule membrane via the dihydropyridine receptor is a crucial step in excitation-contraction coupling (2Tanabe T. Mikami A. Numa S. Beam K.G. Nature. 1990; 344: 451-453Crossref PubMed Scopus (194) Google Scholar). This event is highly regulated by small molecules such as Ca2+, Mg2+ and adenine nucleotides (3Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (843) Google Scholar, 4Franzini-Armstrong C. Protasi F. Physiol. Rev. 1997; 77: 699-729Crossref PubMed Scopus (596) Google Scholar), and through protein-protein interactions such as with triadin and calmodulin (CaM) (5Zhang L. Kelley J. Schmeisser G. Kobayashi Y.M. Jones L.R. J. Biol. Chem. 1997; 272: 23389-23397Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar, 6Ohkura M. Furukawa K. Fujimori H. Kuruma A. Kawano S. Hiraoka M. Kuniyasu A. Nakayama H. Ohizumi Y. Biochemistry. 1998; 37: 12987-12993Crossref PubMed Scopus (84) Google Scholar, 7Tripathy A. Xu L. Mann G. Meissner G. Biophys. J. 1995; 69: 106-119Abstract Full Text PDF PubMed Scopus (261) Google Scholar, 8Moore C.P. Rodney G. Zhang J.Z. Santacruz-Toloza L. Strasburg G. Hamilton S.L. Biochemistry. 1999; 38: 8532-8537Crossref PubMed Scopus (120) Google Scholar). CaM is a small (16.7 kDa) cytosolic protein, the structure of which has been determined by both x-ray crystallography and NMR (9Babu Y.S. Sack J.S. Greenhough T.J. Bugg C.E. Means A.R. Cook W.J. Nature. 1985; 315: 37-40Crossref PubMed Scopus (807) Google Scholar, 10Zhang M. Tanaka T. Ikura M. Nat. Struct. Biol. 1995; 2: 758-767Crossref PubMed Scopus (655) Google Scholar). The protein resembles a dumbbell, with two globular heads linked by a solvent-exposed α-helical stalk. Each of the N- and C-terminal domains contains two EF-hand Ca2+ binding motifs. Ca2+ binding domains I and II in the N domain have a lower Ca2+ affinity (10−5m) and are less Ca2+-selective than the corresponding domains III and IV (10−6m) in the C domain. CaM binds to and regulates a myriad of target proteins involved in almost every biological function in three distinct manners: 1) as CaCaM 2) as apoCaM (without Ca2+ bound), and 3) Ca2+ independent or constituently bound (11Celio M.R. Pauls T. Schwaller B. Guidebook to the Calcium-binding Proteins. Oxford University Press, New York1995: 34-40Google Scholar, 12Rhoads A.R. Friedberg F. FASEB J. 1997; 11: 331-340Crossref PubMed Scopus (749) Google Scholar). Each of these binding events occurs through one of several poorly defined CaM binding motifs, the most common of which are composed of an amphipathic helix of ∼20 amino acid residues that bind CaCaM or an IQ sequence motif that preferentially binds apoCaM. CaM shows a biphasic regulation of RyR1, activating the channel at submicromolar cytosolic Ca2+ while inhibiting the channel at higher Ca2+ concentrations (7Tripathy A. Xu L. Mann G. Meissner G. Biophys. J. 1995; 69: 106-119Abstract Full Text PDF PubMed Scopus (261) Google Scholar). RyR2, on the other hand, does not show activation by apoCaM but is inhibited by CaCaM in a manner similar to RyR1 (13Meissner G. Henderson J.S. J. Biol. Chem. 1987; 262: 3065-3073Abstract Full Text PDF PubMed Google Scholar, 14Fruen B.R. Bardy J.M. Byrem T.M. Strasburg G.M. Louis C.F. Am. J. Physiol. 2000; 279: C724-C733Crossref PubMed Google Scholar). Several studies have reported the stoichiometry of CaM binding to the RyR1. Early studies using125I-CaM (7Tripathy A. Xu L. Mann G. Meissner G. Biophys. J. 1995; 69: 106-119Abstract Full Text PDF PubMed Scopus (261) Google Scholar) or fluorescently labeled (15Yang H.C. Reedy M.M. Burke C.L. Strasburg G.M. Biochemistry. 1994; 33: 518-525Crossref PubMed Scopus (80) Google Scholar) CaM indicated a stoichiometry of 1 molecule of CaCaM and 4–6 molecules of apoCaM bound per subunit. Binding site localization studies with fusion proteins and synthetic peptides indicated three to six potential binding sites per subunit with a variable Ca2+ dependence (16Chen S.R. MacLennan D.H. J. Biol. Chem. 1994; 269: 22698-22704Abstract Full Text PDF PubMed Google Scholar, 17Menegazzi P. Larini F. Treves S. Guerrini R. Quadroni M. Zorzato F. Biochemistry. 1994; 33: 9078-9084Crossref PubMed Scopus (71) Google Scholar). More recent studies using metabolically 35S-labeled CaM indicate a stoichiometry of one binding site per RyR1 subunit for both apo- and CaCaM (8Moore C.P. Rodney G. Zhang J.Z. Santacruz-Toloza L. Strasburg G. Hamilton S.L. Biochemistry. 1999; 38: 8532-8537Crossref PubMed Scopus (120) Google Scholar). A recent report suggests that cardiac SR vesicles bind five CaCaM molecules but only 1 apoCaM molecule per RyR2 tetramer (14Fruen B.R. Bardy J.M. Byrem T.M. Strasburg G.M. Louis C.F. Am. J. Physiol. 2000; 279: C724-C733Crossref PubMed Google Scholar). This study presents a systematic analysis of the CaM binding properties of RyR2 and compares them to RyR1 assayed under identical conditions. Our data indicate that the purified RyR2 binds approximately one [35S]CaM molecule per subunit in both 100 μm Ca2+ and 5 mm EGTA. In native cardiac SR vesicles, [35S]CaM binds approximately two sites per RyR2 subunit in the presence of Ca2+ and a single site per subunit in the absence of Ca2+. Two possible explanations for this discrepancy are that a second CaCaM-binding protein in SR vesicles is lost on purification or that purification induces a conformational change masking the second site. We have also analyzed the effects of redox state on CaM binding to both RyR1 and RyR2. RyR1 and RyR2 are sensitive to redox regulation, showing a 2–3-fold reduction in CaCaM affinity in the presence of GSSG, which is accentuated in the absence of Ca2+ to a 4–9-fold reduction in affinity. CaCaM inhibition of native RyR2, unlike that of RyR1, is greatly diminished by the presence of two allosteric regulators of the ryanodine receptor, caffeine and AMPPCP. Unlike RyR1, apoCaM inhibited RyR2, as determined in [3H]ryanodine binding and single-channel measurements. Heavy SR vesicles were isolated from rabbit hind limb and back muscle and canine cardiac muscle as previously described (18Xu L. Tripathy A. Pasek D.A. Meissner G. J. Biol. Chem. 1999; 274: 32680-32691Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). In selected experiments, endogenous CaM was removed by incubating SR vesicles for 30 min at 24 °C with 1 μm myosin light chain kinase-derived calmodulin binding peptide (CaMBP) in the presence of 100 μm Ca2+ followed by centrifugation through a layer of 15% sucrose to remove complexed CaM and CaMBP. Where indicated, the centrifugation step was omitted. For purification, SR vesicles were solubilized in CHAPS, purified by sucrose density gradient centrifugation, and reconstituted into phosphatidylcholine liposomes (19Lee H.B. Xu L. Meissner G. J. Biol. Chem. 1994; 269: 13305-13312Abstract Full Text PDF PubMed Google Scholar). The endogenous SR-associated concentration of CaM was determined by the ability of CaM to stimulate phosphodiesterase hydrolysis of cAMP as described previously (20Eu J.P. Sun J. Xu L. Stamler J.S. Meissner G. Cell. 2000; 102: 499-509Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). Calmodulin was metabolically labeled with35S according to a protocol generously provided by Drs. Gerald Carlson and Kenneth Traxler (University of Missouri at Kansas City). The cDNA encoding CaM was the generous gift of Dr. Claude Klee (National Institutes of Health). Escherichia colitransformed with the plasmid DNA were grown in M63 minimal media, and expression was induced by heat shock at 42 °C followed by the addition of 1 mCi/100 ml Tran35S-label (ICN Radiochemicals, Costa Mesa, CA). Expression was allowed to continue for 3 h before the bacteria were pelleted and resuspended in lysis buffer (50 mm MOPS, pH 7.5, 100 mm KCl, 1 mmEDTA, 1 mm dithiothreitol, 100 μg/ml lysozyme) and allowed to lyse overnight at 4 °C. After centrifugation at 30,000 × g for 30 min, CaM was purified from the cleared lysate on phenyl-Sepharose in the presence of 4 mmCa2+ and eluted with 1 mm EDTA. Peak elution fractions were dialyzed versus two changes of 0.15m KCl, 20 mm K+-Pipes, 100 μm CaCl2, pH 7.0. CaM protein concentration was determined by absorption spectroscopy by the equation [CaM] = (A277 − A320)/ε. For expressed CaM, ε was assumed to be 0.20 ml/(mg cm) (21Richman P.G. Klee C.B. J. Biol. Chem. 1979; 254: 5372-5376Abstract Full Text PDF PubMed Google Scholar). Unless otherwise indicated, SR vesicles or purified RyR preparations were incubated with 1–300 nm [35S]CaM in 150 mm KCl, 20 mm K-Pipes, pH 7.0, 0.1 mg/ml bovine serum albumin (BSA, Sigma A-0281), 0.2 mm Pefabloc, 20 μmleupeptin with either 5 mm GSH or GSSG and 100 μm (200 μm CaCl2, 100 μm EGTA) or <10 nm (5 mm EGTA, no added CaCl2) free Ca2+. Equilibrium [35S]CaM binding was assayed after incubation at room temperature for 2 h by centrifugation in a Beckman Airfuge for 30 min at 90,000 × g (SR vesicles) or for 180 min at 225,000 × g in a Beckman Type 75 Ti rotor (soluble and reconstituted, purified RyR preparations). Centrifugation-based binding assays are ideal in situations of rapid ligand dissociation and low affinity since the receptor and ligand remain in equilibrium throughout the separation period. Nonspecific binding, including the trapped volume of [35S]CaM, was determined using a 100–1000-fold excess of unlabeled calmodulin (SR vesicles) or by determining [35S]CaM binding to CHAPS-solubilized phospholipid or liposomes that lacked RyR2. Bound [35S]CaM was determined by scintillation counting after solubilization of pellets in Tris-HCl buffer, pH 8.5, containing 2% sodium dodecylsulfate. The time course of [35S]CaM dissociation was determined at 23 °C with the use of a filter assay. To minimize nonspecific binding of [35S]CaM, Whatman GF/B filters were blocked for 1 h in 0.15 m KCl, 10 mm K-Pipes, pH 7.0, buffer containing 10 mg/ml BSA. Vesicles on the filters were washed with 3 × 5 ml of 0.15 KCl, 10 mm K-Pipes, pH 7.0, buffer containing 0.1 mg/ml BSA. Specific [3H]ryanodine binding was determined in the buffer system used for [35S]CaM binding after incubation with 1–2 nm [3H]ryanodine for 20 h at 23 °C as previously described (18Xu L. Tripathy A. Pasek D.A. Meissner G. J. Biol. Chem. 1999; 274: 32680-32691Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Bmax values of [3H]ryanodine binding were determined by Scatchard analysis or with 50 nm [3H]ryanodine in 0.6m KCl buffer. Fusion proteins spanning the full-length coding sequences of rabbit RyR1 (fused to TrpE or glutathione S-transferase) and RyR2 (fused to glutathioneS-transferase) were generated by using polymerase chain reaction to add unique restriction sites to the 5′ and 3′ ends of the region of interest (RyR2) or using existing restriction sites (RyR1) followed by cloning in-frame into pATH or pGEX-5X (RyR1) or pGEX-5X (Amersham Pharmacia Biotech, RyR2). The sequences expressed for each fusion protein are as follows: for RyR2, FP1 (1), FP2 (263), FP3 (561), FP4 (872–1207), FP5 (1157–1509), FP6 (1487–1817), FP7 (1791–2112), FP8 (2084–2401), FP9 (2385–2754), FP10 (2724–3016), FP11 (3003–3182), FP12 (3160–3352), FP13 (3298–3595), FP13short (3298–3577), FP14 (3543–3961), FP15 (3931–4229), FP16 (4205–4478), FP17 (4404–4563), FP18 (4548–4748), FP19 (4726–4968); for RyR1, FPA (1), FPB (282), FPC (799–1209), FPD (1209–1632), FPE (1632–2157), FPF (2156–2592), FPG (2502–2874), FPH (2804–3224), FPI (3225–3662), FPJ (3622–3880), FPK (3879–4222), FPL (4223–4302), FPM (4302–4430), FPN (4431–4771), FPO (4771–5037). Fusion proteins were expressed in the BL21 Gold strain of E. coli (Stratagene) by induction (with 1 mmisopropyl-1-thio-β-d-galactopyranoside for GST and 10 μg/ml indoacrylic acid for TrpE fusion proteins) and a 2-h incubation at 37 °C. Whole cell pellets were collected by centrifugation at 1500 × g for 15 min followed by resuspension in phosphate-buffered saline containing Complete protease inhibitors (Roche Molecular Biochemicals) and lysis by sonication. Equivalent amounts of each fusion protein (as judged by Coomassie Brilliant Blue stain and Western analysis) were loaded onto 10% SDS-polyacrylamide electrophoresis gels. After electrophoresis, the proteins were transferred to nitrocellulose (0.45-μm pore, Schleicher & Schuell) by semidry techniques. The membranes were blocked (2 × 30 min) in 150 mm KCl, 20 mm K-Pipes, pH 7.0, 1 mg/ml BSA with either 100 μm CaCl2 or 5 mmEGTA. The membranes were then exposed to 100 nm[35S]CaM in 150 mm KCl, 20 mmK-Pipes, pH 7.0, 0.04% Tween 20 with the appropriate Ca2+concentration for 1 h at room temperature followed by four 10-min washes in blocking buffer at 4 °C. The membranes were dried overnight then exposed to Biomax MR x-ray film (Eastman Kodak Co.) for 1–10 days. Single-channel measurements using purified RyR2 were carried out as previously described (18Xu L. Tripathy A. Pasek D.A. Meissner G. J. Biol. Chem. 1999; 274: 32680-32691Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) in planar lipid bilayers containing phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine in the ratio of 5:3:2 (25 mg of total phospholipid/ml of n-decane). The side of the bilayer to which the proteoliposomes containing the purified RyR2 were added was defined as the cis (cytoplasmic) side. The trans (SR lumenal) side of the bilayer was defined as ground. Measurements were made with symmetrical 0.25 m KCl, 20 mm K-Pipes, pH 7.0, with 50 μm free Ca2+ in the trans (lumenal) chamber. The cis (cytosolic) solution was varied according to experimental conditions. Data were acquired using test potentials of ±35 mV and were sampled at 10 kHz and filtered at 2 kHz. Channel open probabilities (Po) were determined from at least 2 min of recordings for each condition. Free Ca2+ concentrations were determined as previously described (18Xu L. Tripathy A. Pasek D.A. Meissner G. J. Biol. Chem. 1999; 274: 32680-32691Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). All data analyses were done using SigmaPlot version 5. Fig.1 A shows the results of Scatchard analysis of CaM binding to cardiac SR vesicles in reducing (5 mm reduced glutathione, GSH, circles) and oxidizing (5 mm oxidized glutathione, GSSG,triangles) conditions with 100 μm(filled symbols) and <10 nm free (open symbols) Ca2+, respectively. The data are also summarized in Table I. In the presence of 100 μm Ca2+, cardiac SR vesicles bound 7.5 mol of [35S]CaM/mol of bound [3H]ryanodine. Since there is only one high affinity [3H]ryanodine binding site per tetramer (3Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (843) Google Scholar, 4Franzini-Armstrong C. Protasi F. Physiol. Rev. 1997; 77: 699-729Crossref PubMed Scopus (596) Google Scholar), this suggests that in the presence of Ca2+, RyR2 binds 2 CaM molecules per subunit or that other CaM-binding proteins are present in cardiac SR vesicles. One of the binding sites appeared to be CaCaM-specific since, in the absence of free Ca2+, the stoichiometry approximates one CaM molecule per RyR2 subunit. Skeletal SR vesicles bound ∼1 molecule of [35S]CaM per RyR1 subunit both in the presence and absence of Ca2+, as has been previously reported (8Moore C.P. Rodney G. Zhang J.Z. Santacruz-Toloza L. Strasburg G. Hamilton S.L. Biochemistry. 1999; 38: 8532-8537Crossref PubMed Scopus (120) Google Scholar). The results from these experiments have been corrected for the presence of endogenous CaM in the vesicle preparations (1.0 ± 0.6 (n = 4) and 0.13 ± 0.05 (n = 5) CaM per subunit for cardiac and skeletal SR vesicles, respectively), as determined by phosphodiesterase activation assay.Table I[35S]Calmodulin binding to cardiac and skeletal SR vesicles and purified RyR2 and RyR1with 100 μmCa2+with <10 nm Ca2+[35S]CaM/[3H]RyanodineKD[35S]CaM/[3H]RyanodineKDnmnmCardiac SR+GSH7.4 ± 1.5 (12)3.5 ± 2.0 (12)3.8 ± 0.8 (5)72 ± 30 (5)+GSSG7.6 ± 1.3 (5)6.8 ± 2.3 (5) 1-aP < 0.01 when compared to KD in presence of GSH by unpaired Student'st test.2.6 ± 0.8 (5)261 ± 103 (5) 1-aP < 0.01 when compared to KD in presence of GSH by unpaired Student'st test.Purified RyR2+GSH3.9 ± 1.0 (5)5.0 ± 2.2 (5)3.8 ± 1.5 (6)54 ± 34 (6)Skeletal SR+GSH4.9 ± 0.7 (16)4.7 ± 1.6 (16)4.2 ± 0.8 (8)12.9 ± 3.4 (8)+GSSG5.3 ± 0.9 (5)12.1 ± 1.8 (5) 1-aP < 0.01 when compared to KD in presence of GSH by unpaired Student'st test.4.4 ± 0.7 (5)108 ± 41 (5) 1-aP < 0.01 when compared to KD in presence of GSH by unpaired Student'st test.Purified RyR1+GSH4.6 ± 0.9 (6)5.5 ± 2.6 (6)3.3 ± 1.3 (6)5.4 ± 2.5 (6)Values are the mean ± S.D. with the number of experiments in parenthesis. [35S]CaM/[3H]ryanodine is the ratio ofBmax values in pmol/mg for [35S]CaM and [3H]ryanodine as determined by either Scatchard analysis or using saturating concentrations.1-a P < 0.01 when compared to KD in presence of GSH by unpaired Student'st test. Open table in a new tab Values are the mean ± S.D. with the number of experiments in parenthesis. [35S]CaM/[3H]ryanodine is the ratio ofBmax values in pmol/mg for [35S]CaM and [3H]ryanodine as determined by either Scatchard analysis or using saturating concentrations. After purification of RyR2 from cardiac SR vesicles (shown in Fig.1 B) in the presence of 5 mm GSH and after reconstitution into proteoliposomes, the CaCaM:[3H]ryanodine binding stoichiometry dropped from ∼2 to 1 molecule per subunit in the presence of 100 μmCa2+, suggesting that other CaM-binding proteins have been removed. Alternatively, one of the two CaCaM binding sites in RyR2 may have been conformationally destroyed or buried during purification. We also considered the possibility that endogenous CaM remains associated with the purified RyR. However, if there is such a population of CaM, it would have to be very tightly bound to the receptor and, therefore, without effect on CaCaM binding measured in this study, which has a high rate of dissociation (see Fig. 2). Purification did not significantly alter the CaM:[3H]ryanodine binding stoichiometry for RyR2 in the absence of Ca2+ or for RyR1 both in the presence and absence of Ca2+ (Table I). Decrease in free Ca2+ concentration from 100 μm to <10 nm lowered the affinity for CaM in cardiac SR vesicles 20-fold in the presence of reduced glutathione (GSH) (Fig. 1, A and B, circles) and ∼40-fold in the presence of oxidized glutathione (GSSG) (triangles). In either the presence or absence of Ca2+, oxidized glutathione decreased the affinity for CaM relative to reduced glutathione without changingBmax. Likewise, the CaM binding affinity of RyR1 was reduced 2.5-fold by GSSG in the presence of Ca2+ and 9-fold in the absence, again without changes inBmax (Table I). The Ca2+-dependent changes in CaM binding to cardiac SR vesicles are shown in Fig. 1 C. Increase in free Ca2+ from <10 to 100 nm was without significant effect on CaM binding affinity or CaM:[3H]ryanodine binding stoichiometry, implying that there is apoCaM binding at resting cytosolic Ca2+ levels. The binding stoichiometry nearly doubled as Ca2+concentration was raised to 600 nm, to a value close to the one at 100 μm Ca2+. Conversely, the increase in CaM binding affinity occurred over a broad Ca2+concentration range, requiring Ca2+ concentrations between 1 and 10 μm for maximal affinity. Dissociation experiments were performed to determine the effects of redox state on the stability of the apoCaM and CaCaM RyR complexes. As shown in Fig. 2, the rate of dissociation from RyR2 is largely independent of whether CaM is bound in the presence of 5 mmGSH or 5 mm GSSG. The rate of dissociation in EGTA containing media occurred with a τ1/2 of ∼40 s. The rate of dissociation was decreased by more than 10-fold in the presence of Ca2+, occurring with τ1/2 of ∼9 min. The results suggest that the rate of dissociation of CaM from RyR2 is largely independent of redox state but rather on whether Ca2+ is present in the dissociation buffer, with apoCaM dissociating at a significantly greater rate than CaCaM. Similar results were obtained for RyR1 (see the legend of Fig. 2), with less pronounced differences between the rates of dissociation of CaCaM and apoCaM. To identify potential calmodulin binding sites within the linear sequence of RyR1 and RyR2, we have generated fusion proteins spanning the full coding sequence of the subunit. The design for the fusion proteins is indicated under “Experimental Procedures.” RyR1 sequences were fused to TrpE (with the exception of RyR1 FP E, L, M, and N, which are fused to GST to improve expression), whereas the RyR2 fusion proteins were fused to GST. Since the vast majority of the fusion proteins were insoluble, [35S]CaM overlays were performed with whole cell fractions. Fig. 3 A shows that, in the presence of 100 μm CaCl2 and 100 nm [35S]CaM, five of the RyR2 fusion proteins showed pronounced [35S]CaM binding. CaM binding was detected for RyR2 fusion proteins 2 (aa 263–615), 10 (2724–3016), 13 (3298–3595), 14 (3543–3961), and 18 (4548–4748), whereas inconsistent binding was observed for fusion proteins 1 (1), 3 (561), 6 (1487–1817), and 16 (4205–4478). Fig. 3 Bshows that in 5 mm EGTA, the binding to FP10 is only slightly decreased, whereas the binding to the remaining fusion proteins is greatly reduced, indicative of a Ca2+dependence of binding to FPs 2, 13, 14, and 18. The CaM binding to FP13 is localized to the C-terminal portion of the fusion protein since a truncated form, FP13short (3298–3577), does not bind either CaCaM or apoCaM in overlay experiments (not shown). Hence, it is likely that FPs 13 and 14 contain the same CaM binding domain. RyR1 fusion proteins I (aa 3225–3662) and M (4302–4430) bound CaCaM; in the presence of 5 mm EGTA, binding to FPM was not significantly altered, and binding to FPI was abolished (not shown). These results suggest that there are multiple potential CaM binding sites within the linear RyR sequences, particularly RyR2. Most appear to be buried in the large intact RyR2 channel protein because their number exceeds the number of CaM binding sites in the intact receptor (Table I). It has been previously reported that CaCaM inhibits Ca2+ efflux from both skeletal and cardiac SR vesicles (13Meissner G. Henderson J.S. J. Biol. Chem. 1987; 262: 3065-3073Abstract Full Text PDF PubMed Google Scholar, 22Meissner G. Biochemistry. 1986; 25: 244-251Crossref PubMed Scopus (112) Google Scholar). In addition, CaCaM inhibits [3H]ryanodine binding to RyR1 in skeletal SR vesicles (7Tripathy A. Xu L. Mann G. Meissner G. Biophys. J. 1995; 69: 106-119Abstract Full Text PDF PubMed Scopus (261) Google Scholar, 14Fruen B.R. Bardy J.M. Byrem T.M. Strasburg G.M. Louis C.F. Am. J. Physiol. 2000; 279: C724-C733Crossref PubMed Google Scholar, 23Zhang J.Z. Wu Y. Williams B.Y. Rodney G. Mandel F. Strasburg G.M. Hamilton S.L. Am. J. Physiol. 1999; 276: C46-C53Crossref PubMed Google Scholar) with little effect on [3H]ryanodine binding to RyR2 in cardiac SR vesicles (14Fruen B.R. Bardy J.M. Byrem T.M. Strasburg G.M. Louis C.F. Am. J. Physiol. 2000; 279: C724-C733Crossref PubMed Google Scholar). We have used a CaM binding peptide derived from the myosin light chain kinase (CaMBP) to determine and correct for the presence of endogenous calmodulin (see above) in studies of [3H]ryanodine binding, which has not been done in previous studies. Relatively low CaMBP concentrations (0.1 μm) were sufficient to reduce [35S]CaM binding in the presence of 100 μmfree Ca2+ to skeletal SR vesicles to 4% of the control value (Fig. 4 A). For cardiac SR vesicles, a higher CaMBP concentration (1 μm) was required to reduce [35S]CaM binding to comparable, low levels. It is crucial that relatively low concentrations of CaMBP be used if the experiments are done in the presence of the peptide since, as shown in Fig. 4 B, vesicles assayed in the presence of high concentrations of CaMBP show a marked stimulation of [3H]ryanodine binding to skeletal SR in either GSH or GSSG by CaMBP at concentrations greater than 1 μm. A lower degree of stimulation was observed with cardiac SR. CaMBP was less effective in reducing [35S]CaM binding to SR vesicles at [Ca2+] < 1 μm. Where indicated, experiments were therefore done with SR vesicles pretreated with CaMBP, as described under “Experimental Procedures.” To correlate the binding of CaCaM to the inhibition of [3H]ryanodine binding, we measured [3H]ryanodine binding at increasing concentrations of CaM both in the presence of GSH and GSSG for cardiac (Fig.5) and skeletal (not shown) SR vesicles. For cardiac SR, a higher extent of inhibition was observed in GSH than GSSG. The KHi for inhibition of ryanodine binding (0.6 ± 0.2 and 1.7 ± 0.7 nm for RyR2 in GSH and GSSG, respectively) was considerably lower than theKD for [35S]CaM binding (see Table I). The Hill coefficients for CaM inhibition of ryanodine binding to RyR2 were 1.0 ± 0.3 both in GSH and GSSG. Skeletal SR also hadKHi values (legend of Fig. 5) that were lower than the KD values and had Hill coefficients near unity. The results suggest that inhibition of ryanodine binding perhaps requires only a single CaCaM bound/tetramer. CaM inhibition of [3H]ryanodine binding is also modulated by various regulators of the RyRs as indicated in Fig.6 and TableII. At [Ca2+] > 1 μm, CaM (1 μm) inhibition of both cardiac and skeletal RyR is observed in both oxidizing and reducing conditions in the absence of MgAMPPCP (AMPPCP is a nonhydrolyzable ATP analogue). CaM inhibits [3H]ryanodine binding to RyR2 by both rendering the receptor less sensitive to activation by Ca2+ and more sensitive to inhibition at high Ca2+ as well as by lowering the maximal level of [3H]ryanodine binding (Fig. 6, upper left panel). In the presence of MgAMPPCP and at [Ca2+] < 10 μm, CaM inhibits cardiac SR ryanodine binding in both reducing and oxidizing conditions (Fig. 6, upper right panel). However, at [Ca2+] > 10 μm, CaM inhibits RyR2 ryanodine binding only in reducing conditions. Thelower left panel of Fig. 6 indicates that in agreement with a previous report (24Zable A.C. Favero T.G. Abramson J.J. J. Biol. Chem. 1997; 272: 7069-7077Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), skeletal SR [3H]ryanodine binding is markedly inhibited in the presence of GSH. [3H]Ryanodine binding to skeletal SR is activated by CaM at low Ca2+ concentrations both" @default.
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• W2067686089 date "2001-01-01" @default.
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• W2067686089 title "Calmodulin Binding and Inhibition of Cardiac Muscle Calcium Release Channel (Ryanodine Receptor)" @default.
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• W2067686089 doi "https://doi.org/10.1074/jbc.m010771200" @default.
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