Title: Molecular Basis of Calmodulin Binding to Cardiac Muscle Ca2+ Release Channel (Ryanodine Receptor)
Abstract: Calmodulin (CaM) is a ubiquitous Ca2+-binding protein that regulates the ryanodine receptors (RyRs) by direct binding. CaM inhibits the skeletal muscle ryanodine receptor (RyR1) and cardiac muscle receptor (RyR2) at >1 μm Ca2+ but activates RyR1 and inhibits RyR2 at <1 μm Ca2+. Here we tested whether CaM regulates RyR2 by binding to a highly conserved site identified previously in RyR1. Deletion of RyR2 amino acid residues 3583–3603 resulted in background [35S]CaM binding levels. In single channel measurements, deletion of the putative CaM binding site eliminated CaM inhibition of RyR2 at Ca2+ concentrations below and above 1 μm. Five RyR2 single or double mutants in the CaM binding region (W3587A, L3591D, F3603A, W3587A/L3591D, L3591D/F3603A) eliminated or greatly reduced [35S]CaM binding and inhibition of single channel activities by CaM depending on the Ca2+ concentration. An RyR2 mutant, which assessed the effects of 4 amino acid residues that differ between RyR1 and RyR2 in or flanking the CaM binding domain, bound [35S]CaM and was inhibited by CaM, essentially identical to wild type (WT)-RyR2. Three RyR1 mutants (W3620A, L3624D, F3636A) showed responses to CaM that differed from corresponding mutations in RyR2. The results indicate that CaM regulates RyR1 and RyR2 by binding to a single, highly conserved CaM binding site and that other RyR type-specific sites are likely responsible for the differential functional regulation of RyR1 and RyR2 by CaM. Calmodulin (CaM) is a ubiquitous Ca2+-binding protein that regulates the ryanodine receptors (RyRs) by direct binding. CaM inhibits the skeletal muscle ryanodine receptor (RyR1) and cardiac muscle receptor (RyR2) at >1 μm Ca2+ but activates RyR1 and inhibits RyR2 at <1 μm Ca2+. Here we tested whether CaM regulates RyR2 by binding to a highly conserved site identified previously in RyR1. Deletion of RyR2 amino acid residues 3583–3603 resulted in background [35S]CaM binding levels. In single channel measurements, deletion of the putative CaM binding site eliminated CaM inhibition of RyR2 at Ca2+ concentrations below and above 1 μm. Five RyR2 single or double mutants in the CaM binding region (W3587A, L3591D, F3603A, W3587A/L3591D, L3591D/F3603A) eliminated or greatly reduced [35S]CaM binding and inhibition of single channel activities by CaM depending on the Ca2+ concentration. An RyR2 mutant, which assessed the effects of 4 amino acid residues that differ between RyR1 and RyR2 in or flanking the CaM binding domain, bound [35S]CaM and was inhibited by CaM, essentially identical to wild type (WT)-RyR2. Three RyR1 mutants (W3620A, L3624D, F3636A) showed responses to CaM that differed from corresponding mutations in RyR2. The results indicate that CaM regulates RyR1 and RyR2 by binding to a single, highly conserved CaM binding site and that other RyR type-specific sites are likely responsible for the differential functional regulation of RyR1 and RyR2 by CaM. The ryanodine receptors (RyRs) 1The abbreviations used are: RyR, ryanodine receptor; RyR1, skeletal muscle RyR; RyR2, cardiac muscle RyR; CaM, calmodulin; apoCaM, Ca2+-free CaM; CaCaM, Ca2+-bound CaM; HEK, human embryonic kidney; WT, wild type; Pipes, 1,4-piperazinediethanesulfonic acid. 1The abbreviations used are: RyR, ryanodine receptor; RyR1, skeletal muscle RyR; RyR2, cardiac muscle RyR; CaM, calmodulin; apoCaM, Ca2+-free CaM; CaCaM, Ca2+-bound CaM; HEK, human embryonic kidney; WT, wild type; Pipes, 1,4-piperazinediethanesulfonic acid. are Ca2+ channels that release Ca2+ from an intracellular Ca2+ storing, membrane-bound compartment, the endo/sarcoplasmic reticulum (1Franzini-Armstrong C. Protasi F. Physiol. Rev. 1997; 77: 699-729Scopus (590) Google Scholar, 2Fill M. Copello J.A. Physiol. Rev. 2002; 82: 893-922Google Scholar, 3Meissner G. Front. Biosci. 2002; 7: d2072-d2080Google Scholar). In mammalian cells, three structurally and functionally related RyR isoforms include RyR1, predominant in skeletal muscle, RyR2, predominant in cardiac muscle, and RyR3, which was initially isolated from brain but is found in many tissues. The three isoforms are comprised of four 560-kDa RyR subunits and four 12-kDa FK506-binding protein subunits. Multiple endogenous effectors regulate the RyRs, including Ca2+, Mg2+, ATP, and calmodulin (CaM) (1Franzini-Armstrong C. Protasi F. Physiol. Rev. 1997; 77: 699-729Scopus (590) Google Scholar, 2Fill M. Copello J.A. Physiol. Rev. 2002; 82: 893-922Google Scholar, 3Meissner G. Front. Biosci. 2002; 7: d2072-d2080Google Scholar, 4Balshaw D.M. Yamaguchi N. Meissner G. J. Membr. Biol. 2002; 185: 1-8Google Scholar).CaM is a ubiquitous cytosolic Ca2+-binding protein that modulates proteins through CaM-dependent protein kinases or by direct binding (5Rhoads A.R. Friedberg F. FASEB J. 1997; 11: 331-340Google Scholar). CaM modulates the RyRs by direct binding since CaM affects channel function in the absence of ATP (6Meissner G. Biochemistry. 1986; 25: 244-251Google Scholar, 7Meissner G. Henderson J.S. J. Biol. Chem. 1987; 262: 3065-3073Google Scholar). CaM inhibits all three RyRs at Ca2+ concentrations above 1 μm; however, differences in the regulation of the RyRs at submicromolar Ca2+ concentrations have been described. At free Ca2+ concentrations below 1 μm, CaM has a stimulatory effect on RyR1 and RyR3 channel activities (8Buratti R. Prestipino G. Menegazzi P. Treves S. Zorzato F. Biochem. Biophys. Res. Comm. 1995; 213: 1082-1090Google Scholar, 9Tripathy A. Xu L. Mann G. Meissner G. Biophys. J. 1995; 69: 106-119Google Scholar, 10Chen S.R.W. Li X. Ebisawa K. Zhang L. J. Biol. Chem. 1997; 272: 24234-24246Google Scholar), whereas RyR2 is unaffected (11Fruen B.R. Bardy J.M Byrem T.M. Strasburg G.M. Louis C.F. Am. J. Physiol. 2000; 279: C724-C733Google Scholar) or inhibited (12Balshaw D.M. Xu L. Yamaguchi N. Pasek D.A. Meissner G. J. Biol. Chem. 2001; 276: 20144-20153Google Scholar) by CaM.Studies investigating the CaM binding properties of the RyRs have focused on RyR1. Trypsin digestion and peptide binding studies indicate that Ca2+-free CaM (apoCaM) and Ca2+-bound CaM (CaCaM) bind RyR1 amino acid residues 3614–3643 (13Rodney G.G. Moore C.P. Williams B.Y. Zhang J.Z. Krol J. Pedersen S.E. Hamilton S.L. J. Biol. Chem. 2001; 276: 2069-2074Google Scholar, 14Moore C.P. Rodney G. Zhang J.Z. Santacruz-Toloza L. Strasburg G. Hamilton S.L. Biochemistry. 1999; 38: 8532-8537Google Scholar). Mutations in this region resulted in loss of high affinity CaCaM and apoCaM binding and modulation of RyR1 channel activity (15Yamaguchi N. Xin C. Meissner G. J. Biol. Chem. 2001; 276: 22579-22585Google Scholar).The present study was undertaken to identify the CaM binding sites in RyR2. We generated eight RyR2 mutants focusing on the domain corresponding to the apo- and CaCaM regulatory domain that is highly conserved between RyR1 and RyR2. One mutant assessed the significance of a 1,5,10 CaM recognition motif (5Rhoads A.R. Friedberg F. FASEB J. 1997; 11: 331-340Google Scholar) by substituting a corresponding amino acid residue in RyR1 and RyR2. The results of the study show that (i) like RyR1, RyR2 has a high affinity CaM binding domain that is shared by apoCaM and CaCaM; (ii) deletion of the CaM binding site eliminates inhibition of RyR2 by CaM at submicromolar and micromolar Ca2+; and (iii) corresponding mutations in the CaM binding site differentially alter the CaM binding properties and regulation by CaM of the skeletal and cardiac RyRs.EXPERIMENTAL PROCEDURESMaterials—[3H]Ryanodine was obtained from Perkin Elmer Life Sciences, Tran35S label was obtained from ICN Radiochemicals (Costa Mesa, CA), unlabeled ryanodine was obtained from Calbiochem, unlabeled CaM was obtained from Sigma, Complete protease inhibitors were obtained from Roche Applied Science, and human embryonic kidney (HEK) 293 cells were obtained from ATCC. Full-length RyR2 cDNA was kindly provided by Dr. Junichi Nakai at National Institute of Physiological Sciences, Okazaki, Japan.Construction of Mutant cDNAs—The full-length rabbit RyR2 cDNA (16Nakai J. Imagawa T. Hakamata Y. Shigekawa M. Takeshima H. Numa S. FEBS Lett. 1990; 271: 169-177Google Scholar) was subcloned into pCIneo. Single and multiple base changes and deletions were introduced by Pfu-turbo polymerase-based chain reaction by using mutagenic oligonucleotides and the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The complete mutated sequences were confirmed by DNA sequencing. A 721-bp fragment (ClaI/SacII, 10483–11203) subcloned into pBluescript vector (Stratagene) was used as template for mutagenesis of RyR2. The fragment with the mutation was subcloned back into the original position of RyR2 in two steps: to a vector containing a BbrPI/SacII (residues 5038–11203) fragment and to full-length RyR2 in pCIneo.The full-length rabbit RyR1 cDNA (ClaI/XbaI) was subcloned into pCMV5 (17Gao L. Tripathy A. Lu X. Meissner G. FEBS Lett. 1997; 412: 223-226Google Scholar). For construction of RyR1-F3636A, a 243-bp RyR1 cDNA fragment (EclXI/BamHI, 10872–11114) subcloned into pBluescript vector was used as template for mutagenesis. The mutated sequence was confirmed by DNA sequencing and subcloned back into the original position of RyR1 in three steps: the sequence was subcloned back to a vector containing a PvuI/NdeI (residues 8600–11304) fragment and then back to a vector containing a PvuI/XbaI (residues 8600–15276) fragment, and finally, mutated RyR1 full-length plasmids were prepared by ligation of two fragments (ClaI/PvuI, PvuI/XbaI containing the mutated sequence) and pCMV5 (ClaI/XbaI). Nucleotide numbering is as described (16Nakai J. Imagawa T. Hakamata Y. Shigekawa M. Takeshima H. Numa S. FEBS Lett. 1990; 271: 169-177Google Scholar, 17Gao L. Tripathy A. Lu X. Meissner G. FEBS Lett. 1997; 412: 223-226Google Scholar).Expression of Full-length RyRs in HEK293 Cells—RyR cDNAs were transiently expressed in HEK293 cells transfected with 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 medium containing 10% fetal bovine serum and plated the day before transfection. For each 10-cm tissue culture dish, 3.5 μg of cDNA was used. Cells were harvested about 48 h after transfection as described (15Yamaguchi N. Xin C. Meissner G. J. Biol. Chem. 2001; 276: 22579-22585Google Scholar).[3H]Ryanodine Binding—[3H]Ryanodine binding experiments were performed with crude membrane fractions prepared from HEK293 cells as described (15Yamaguchi N. Xin C. Meissner G. J. Biol. Chem. 2001; 276: 22579-22585Google Scholar). Unless otherwise indicated, membranes were incubated at room temperature with 2.5 nm [3H]ryanodine in 20 mm imidazole, pH 7.0, 250 mm KCl, 5 mm glutathione (oxidized), 20 μm leupeptin, and 200 μm Pefabloc and the indicated free Ca2+ concentrations. Nonspecific binding was determined using 1000-fold excess of unlabeled ryanodine. After 20 h, aliquots of the samples were diluted with 8.5 volumes of ice-cold water and placed on Whatman GF/B filters preincubated with 2% polyethyleneimine in water. Filters were washed with three 5 ml of ice-cold 100 mm KCl, 1 mm KPipes, pH 7.0. Radioactivity remaining on the filters was determined by liquid scintillation counting to obtain bound [3H]ryanodine.[35S]Calmodulin Binding—[35S]CaM was metabolically labeled using Tran35S label and purified as described (12Balshaw D.M. Xu L. Yamaguchi N. Pasek D.A. Meissner G. J. Biol. Chem. 2001; 276: 20144-20153Google Scholar). Crude membrane fractions prepared from HEK293 cells (15Yamaguchi N. Xin C. Meissner G. J. Biol. Chem. 2001; 276: 22579-22585Google Scholar) were incubated for 2 h at room temperature with 15–200 nm [35S]CaM in 10 mm KPipes, 20 mm imidazole, pH 7.0, 0.15 m sucrose, 150 mm KCl, 100 μg/ml bovine serum albumin, 5 mm glutathione (reduced), 20 μm leupeptin, 200 μm Pefabloc, and 1 mm EGTA plus Ca2+ concentrations to yield <10 nm, 0.4 μm, or 100 μm free Ca2+. Samples were centrifuged for 30 min at 30 p.s.i. in a Beckman Airfuge after aliquots were taken for determination of total radioactivity. Radioactivity in the pellet fractions was determined by scintillation counting to obtain bound [35S]CaM. Nonspecific binding of [35S]CaM was determined by incubating equal protein amounts of membranes obtained from vector-transfected HEK293 cells. In parallel experiments, Bmax values of [3H]ryanodine binding were determined by incubating membranes for 4 h at room temperature with a saturating concentration of [3H]ryanodine (40 nm) in 20 mm imidazole, pH 7.0, 0.6 m KCl, 0.15 m sucrose, 1 mm glutathione (oxidized), 20 μm leupeptin, 200 μm Pefabloc, and 200 μm Ca2+. Specific [3H]ryanodine binding was determined as described above.Single Channel Recordings—Single channel measurements were performed using the planar lipid bilayer method (18Gao L. Balshaw D. Xu L. Tripathy A. Xin C. Meissner G. Biophys. J. 2000; 79: 828-840Google Scholar). Planar lipid bilayers contained phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine in the ratio of 5:3:2 (25 mg of total phospholipid/ml of n-decane). Membrane fractions of HEK293 cells expressing wild type (WT) or mutant RyRs were pretreated for 30 min with 1 μm myosin light chain kinase-derived calmodulin binding peptide to remove endogenous CaM (12Balshaw D.M. Xu L. Yamaguchi N. Pasek D.A. Meissner G. J. Biol. Chem. 2001; 276: 20144-20153Google Scholar). Final peptide concentration was 10 nm following the addition of membranes to the cis (cytosolic) chamber of the bilayer apparatus. A strong dependence of single channel activities on cis Ca2+ concentration indicated that the large cytosolic "foot" region faced the cis chamber of the bilayers. The trans (lumenal) side of the bilayer was defined as ground. Measurements were made with symmetrical 0.25 m KCl, 20 mm KHepes, pH 7.4, with the indicated concentration of Ca2+. Exogenous CaM was added to the cis solution. Electrical signals were filtered at 2 kHz, digitized at 10 kHz, and analyzed as described (18Gao L. Balshaw D. Xu L. Tripathy A. Xin C. Meissner G. Biophys. J. 2000; 79: 828-840Google Scholar). Po values in multichannel recordings were calculated using the equation Po = Σ iPo,i/N, where N is the total number of channels, and Po,i is channel open probability of the ith channel.Biochemical Assays and Data Analysis—Free Ca2+ concentrations were obtained by including in the solutions the appropriate amounts of Ca2+ and EGTA as determined using the stability constants and computer program published by Schoenmakers et al. (19Schoenmakers T.J. Visser G.J. Flik G. Theuvenet A.P. BioTechniques. 1992; 12: 870-879Google Scholar). Free Ca2+ concentrations of ≥1 μm were verified with the use of a Ca2+ selective electrode.Results are given as means ± S.E. Significances of differences in the data (p < 0.05) were determined using Student's t test.RESULTSIdentification of the CaM Binding Site in RyR2—Previous mutagenesis studies identified 2 residues in the RyR1 CaM binding domain that were required for high affinity CaCaM binding and inhibition of RyR1 channel activity. One mutation also resulted in loss of apoCaM binding and activation of RyR1 (15Yamaguchi N. Xin C. Meissner G. J. Biol. Chem. 2001; 276: 22579-22585Google Scholar). Because the region of the RyR1 apoCaM and CaCaM binding site is highly conserved among the RyRs (Fig. 1), we introduced the corresponding mutations into RyR2. Membrane fractions prepared from HEK293 cells transiently expressing WT or mutant RyR2s were incubated with increasing [35S]CaM concentrations in the presence of <10 nm Ca2+ to study apoCaM binding, 0.4 μm Ca2+ (a Ca2+ concentration that results in activation of RyR1 but inhibition of RyR2 by CaM (12Balshaw D.M. Xu L. Yamaguchi N. Pasek D.A. Meissner G. J. Biol. Chem. 2001; 276: 20144-20153Google Scholar)), and 100 μm Ca2+ to study CaCaM binding. Bound [35S]CaM activities were measured using a centrifugation assay. The Bmax values of [3H]ryanodine binding were determined in parallel experiments.The tetrameric WT-RyR2 bound [35S]CaM in a concentration-dependent manner with 1.9 ± 0.2 [35S]CaM/high affinity [3H]ryanodine binding site at 200 nm CaM and <10 nm Ca2+, which corresponds to 0.5 CaM/RyR2 subunit (Fig. 2), as there is only one high affinity [3H]ryanodine binding site/RyR2 tetramer. The use of 200 nm CaM likely did not result in saturation binding. However, higher CaM concentrations could not be used because these resulted in high background binding levels. The mean numbers of bound [35S]CaM/RyR2 subunit were 0.65 at 200 nm CaM and 0.4 μm Ca2+ and 0.73 at 75 nm CaM and 100 μm Ca2+ (Fig. 2.). By comparison, WT-RyR1 bound/subunit ∼1 apoCaM at <10 nm Ca2+ and ∼1 CaCaM at 100 μm Ca2+ (15Yamaguchi N. Xin C. Meissner G. J. Biol. Chem. 2001; 276: 22579-22585Google Scholar). Fig. 2 also shows the [35S]CaM binding properties of RyR2 mutants RyR2-W3587A and RyR2-L3591D that correspond to RyR1-W3620A and RyR1-L3624D. Each RyR1 mutation eliminated CaCaM binding at 100 μm Ca2+ with one of the mutations (RyR1-L3624D) resulting in loss of apoCaM binding at <10 nm Ca2+ (15Yamaguchi N. Xin C. Meissner G. J. Biol. Chem. 2001; 276: 22579-22585Google Scholar). RyR2-W3587A retained and RyR2-L3591D lost CaM binding at <10 nm Ca2+ (Fig. 2), as observed previously for the two corresponding RyR1 mutants (15Yamaguchi N. Xin C. Meissner G. J. Biol. Chem. 2001; 276: 22579-22585Google Scholar). However, CaM binding to the RyR2 mutants was not eliminated at 100 μm Ca2+ or 0.4 μm Ca2+. Thus, the corresponding RyR1 and RyR2 mutants have similar apoCaM but different CaCaM binding properties.Fig. 2[35S]CaM binding to WT- and mutant RyR2s. Membrane fractions prepared from HEK293 cells expressing WT or mutant RyR2s were incubated for 2 h at room temperature with indicated concentrations of [35S]CaM in the presence of <10 nm Ca2+ (apoCaM) (top), 0.4 μm Ca2+ (middle), and 100 μm Ca2+ (CaCaM) (bottom). The ratios of [35S]CaM binding values to maximal binding values of [3H]ryanodine were obtained, taking into account that there is one high affinity [3H]ryanodine binding site/RyR2 tetramer. Maximal values of [3H]ryanodine binding (pmol/mg of protein), determined as detailed under "Experimental Procedures," ranged from 0.4 to 1.2 for WT- and mutant RyR2s. RyR2–4M is RyR2-Q3580Y/R3581K/K3596R/A3606T. Data are the mean ± S.E. of 4–15 experiments.View Large Image Figure ViewerDownload (PPT)Preliminary experiments indicated that CaM did not inhibit [3H]ryanodine binding to WT-RyR2 expressed in HEK293 cells (not shown) but was inhibitory in single channel measurements. Membrane fractions prepared from HEK293 cells transiently expressed with WT- and mutant RyR2 cDNAs were incorporated into planar lipid bilayers. Single WT- and mutant RyR2 channel activities were recorded with K+ as current carrier in the absence and presence of exogenously added CaM. The use of K+ rather than Ca2+ as current carrier improved control of the cis Ca2+ concentration (20Xu L. Meissner G. Biophys. J. 1998; 75: 2302-2312Google Scholar). The functional effects of 50 nm and 1 μm CaM were examined with 0.4 and 2 μm free Ca2+ in the cis (cytosolic) chamber, i.e. at two Ca2+ concentrations where CaM inhibits the native RyR2. Under these conditions, RyR1 is activated by CaM at 0.4 μm and inhibited at 2 μm free Ca2+ (see Fig. 6). A low micromolar Ca2+ concentration of 2 μm was used because CaM is less effective in inhibiting the RyR2 ion channel at elevated Ca2+ concentrations (12Balshaw D.M. Xu L. Yamaguchi N. Pasek D.A. Meissner G. J. Biol. Chem. 2001; 276: 20144-20153Google Scholar).Fig. 6Effects of CaM on single WT-RyR1 and RyR1-F3636A ion channels. Single channel currents were recorded as described in the legend Fig. 3 at –20 mV (downward deflections from closed level, c) in symmetric 0.25 m KCl, 20 mm KHepes, pH 7.4, media with 0.3 μm Ca2+ and 1 mm ATP (left panels) or 2 μm Ca2+ (right panels) in the cis chamber before (top traces) and after the addition of 50 nm CaM (middle traces) and 1 μm CaM (bottom traces). Data of 4–6 single channel recordings are summarized in Fig. 4.View Large Image Figure ViewerDownload (PPT)Figs. 3, A–C, and 4 compare the effects of CaM on single WT-RyR2, RyR2-W3587A, and RyR2-L3591D channels. The averaged channel open probability (Po) of WT-RyR2 in the presence of 0.4 μm free Ca2+ was reduced to 35% of the control activity with 50 nm CaM and to 21% with 1 μm CaM in the cis chamber (Fig. 4). In the presence of 2 μm free Ca2+, 50 nm and 1 μm CaM were less effective in inhibiting WT-RyR2, reducing Po to 73 and 54% of the control, respectively.Fig. 3Effects of CaM on single WT- and mutant RyR2 ion channels. Membrane fractions prepared from HEK293 cells expressing WT-RyR2 (A), RyR2-W3587A (B), RyR2-L3591D (C), or RyR2-Δ3583–3603 (D) were fused with a lipid bilayer. Single channel currents were recorded at –20 mV (downward deflections from closed level, c) in symmetric 0.25 m KCl, 20 mm KHepes, pH 7.4, media with 0.4 μm Ca2+ (left panels) or 2 μm Ca2+ (right panels) before (top traces) and after the addition of 50 nm CaM (middle traces) and 1 μm CaM (bottom traces). Data of 4–8 single channel recordings are summarized in Fig. 4.View Large Image Figure ViewerDownload (PPT)Fig. 4Channel open probabilities of WT- and mutant RyR2s and RyR1s. Data were obtained as described in legend for Fig. 3. RyR2–4M is RyR2-Q3580Y/R3581K/K3596R/A3606T. Data show the relative mean channel open probability (Po,–CaM = 100%) ± S.E. at 0.4 μm Ca2+ (top) and 2 μm Ca2+ (bottom) of 4–8 single channel recordings for RyR2s, 4–6 single channel recordings for RyR1s.View Large Image Figure ViewerDownload (PPT)Single channel recordings showed that at 2 μm Ca2+, CaM inhibited RyR2-W3587A (Figs. 3B and 4) and RyR2-L3591D (Figs. 3C and 4) to an extent comparable with WT-RyR2. These results are in agreement with a similar extent of [35S]CaM binding to wild type and the two mutant RyR2s in the presence of 100 μm Ca2+. In contrast, CaM failed to inhibit RyR2-W3587A and RyR2-L3591D when the Ca2+ concentration was lowered from 2 to 0.4 μm Ca2+ despite the fact that both mutants bound CaM at 0.4 μm Ca2+.Both apoCaM and CaCaM binding to RyR2 was eliminated by deleting 21 amino acid residues (amino acids 3583–3603) (Fig. 2) corresponding to the CaM binding domain of RyR1 (Fig. 1). Loss of CaM binding resulted in loss of inhibition of RyR2-Δ3583–3603 activity by CaM in single channel measurements at 0.4 and 2 μm Ca2+ (Figs. 3D and 4). The deletion of amino acid residues 3583–3603 did not introduce major global protein conformational changes because the mutant displayed a single channel open probability (Po = 0.33 ± 0.12 versus 0.39 ± 0.05 for WT-RyR2 at 2 μm Ca2+), single channel conductance (Fig. 3D), and Ca2+ activation/inactivation profile (Fig. 5) not significantly different from RyR2, as determined in single channel and [3H]ryanodine binding measurements, respectively. The results indicate that like RyR1, RyR2 has a single functional CaM binding site.Fig. 5Ca2+ dependence of [3H]ryanodine binding to WT- and mutant RyR2s. Specific binding was determined as described under "Experimental Procedures" in 0.15 m KCl, 20 mm imidazole, pH 7.0, media containing 5 mm glutathione (reduced), 2.5 nm [3H]ryanodine, and the indicated Ca2+ concentrations. Normalized [3H]ryanodine binding data are the average of 4–5 experiments. Standard errors were 20% or less.View Large Image Figure ViewerDownload (PPT)Role of a 1,5,10 CaM Recognition Motif—RyR2 has a 1,5,10 CaM recognition motif (Val-3599, Phe-3603, Leu-3608) (5Rhoads A.R. Friedberg F. FASEB J. 1997; 11: 331-340Google Scholar) that is conserved in RyR1 (Fig. 1). We assessed the significance of this motif in the regulation of the RyRs by CaM by preparing RyR2-F3603A and corresponding RyR1-F3636A. For RyR2-F3603A, CaM binding was at background levels at <10 nm Ca2+ but was present at 0.4 and 100 μm Ca2+ (Fig. 2). The results suggest that RyR2-Phe-3603 is required for apoCaM but not for CaCaM binding. The expression level of RyR1-F3636A was too low to determine its CaM binding levels.Single channel recordings showed that CaM inhibited RyR2-F3603A activity at 2 μm Ca2+ but was without a significant effect at 0.4 μm Ca2+ (Fig. 4). CaM inhibited WT-RyR1 and RyR1-F3636A single channel activities at >1 μm Ca2+ (Fig. 6). A decrease in [3H]ryanodine binding by 1 μm CaM also indicated a decrease in WT and mutant RyR1 activities at >1 μm Ca2+ (Fig. 7). At 0.3 μm Ca2+ in the presence of 1 mm ATP to increase the otherwise very low channel activities, addition of CaM yielded the expected increase in activity of WT-RyR1 in both assays. In contrast, in the presence of 0.3 μm Ca2+, a significant decrease in RyR1-F3636A [3H]ryanodine binding and single channel activities was observed after the addition of 1 μm CaM (although in single channel measurements not at 50 nm CaM). Thus, the loss of CaM modulation of RyR2-F3603A evident at <1 μm Ca 2+ is no longer present when the corresponding Phe in RyR1 is substituted with Ala. Remarkably, the RyR1 mutation led to inhibition by 1 μm CaM at submicromolar Ca2+, as compared with activation of WT-RyR1.Fig. 7CaM inhibition and activation of [3H]ryanodine binding to WT-RyR1 and RyR1-F3636A. Specific [3H]ryanodine binding to WT-RyR1 and RyR1-F3636A were determined as described under "Experimental Procedures" in presence of 0.3 μm Ca2+ and 1 mm AMP-PCP (a nonhydrolyzable ATP analog) (top)or25 μm Ca2+ (bottom)inthe absence (open bars) or presence (filled bars) of 1 μm CaM. Normalized [3H]ryanodine binding data are the means ± S.E. of 4–5 experiments. *, p < 0.05, as compared with control (–CaM).View Large Image Figure ViewerDownload (PPT)Effects of CaM on Two RyR2 Double Mutations—None of the single site RyR2 mutants described above abolished CaM inhibition of RyR2 at 2 μm Ca2+. The effects of two double mutations (W3587A/L3591D, L3591D/F3603A) were therefore determined. Both mutants had low [35S]CaM binding activities at <10 nm and 100 μm Ca2+ (Fig. 2). Single channel recordings showed that, consistent with the binding data, addition of 50 nm or 1 μm CaM did not inhibit the single channel activity of either mutant at 0.4 μm Ca2+ (Fig. 4). CaM also failed to inhibit RyR2-L3591D/F3603A at 2 μm Ca2+ (Fig. 4). In contrast, at 2 μm Ca2+, CaM inhibited W3587A/L3591D, notwithstanding that the mutant had low CaM binding levels ([35S]CaM/[3H]ryanodine = 0.25 ± 0.15 at 50 nm CaM, as compared with 1.6 ± 0.2 for WT-RyR2, n = 3).Role of RyR2-specific 12-amino-acid Residues—RyR2 has a 12-amino-acid insert near the CaM binding site that is absent from RyR1 (Fig. 1). An RyR2 mutant with a deletion of this region (RyR2-Δ3564–3575) was tested as a possible explanation for the differential regulation of RyR2 and RyR1 by CaM at 0.4 μm Ca2+. The deletion did not alter apoCaM and CaCaM binding (Fig. 2) or CaM inhibition at 0.4 and 2 μm Ca2+ (Fig. 4). Furthermore, the deletion mutant displayed a single channel conductance (not shown) and Ca2+ activation/inactivation profile (Fig. 5) essentially identical to WT-RyR2. The results suggest that the RyR2-specific 12-amino-acid sequence does not directly contribute to modulation by CaM.Role of Nonidentical Amino Acid Residues in or Flanking RyR1 and RyR2 CaM Binding Domains—The CaM binding region identified in RyR2 (amino acids 3583–3603) is highly conserved in RyR1 with a single conserved charge difference where Arg-3629 in RyR1 corresponds to Lys-3596 in RyR2 (Fig. 1). To assess the effects of the nonidentical amino acid residue, as well as three additional amino acids in or flanking the CaM binding domain, we prepared an RyR2 quadruple mutant (RyR2–4M) by substituting 4 amino acids in RyR2 with the corresponding amino acids in RyR1 (in Fig. 1, substituted amino acids are indicated by the asterisk). RyR2–4M exhibited [35S]CaM binding (Fig. 2) and effects of CaM on single channel activities (Fig. 4) essentially identical to WT-RyR2. The results indicate that receptor sites other than the CaM binding domain are responsible for the differential regulation of the skeletal and cardiac RyRs by CaM.DISCUSSIONTwo experimental strategies were taken to identify the CaM binding site in RyR2, [35S]CaM binding measurements and single channel recordings using the planar lipid bilayer method. Deletion of amino acid residues 3583–3603 was sufficient to eliminate CaM binding and inhibition of RyR2 channel activity by CaM at submicromolar and micromolar Ca2+ concentrations. Mutagenesis generated four RyR2 single or double mutants in this region that eliminated or greatly reduced apoCaM binding with the double mutants also resulting in loss of or reduced CaCaM binding levels. Single channel recordings showed that at 0.4 μm Ca2+, RyR2-W3587A, -L3591D, and -F3603A bound CaM but were not inhibited by CaM concentrations as high as 1 μm. On the other hand, RyR2-W3587A/L3591D was inhibited by 50 nm CaM at 2 μm Ca2+ despite a low CaM binding level. Furthermore, an unexpected finding was that corresponding mutations in the CaM binding site affected the CaM binding properties and regulation by CaM of the skeletal and cardiac RyRs differently (Table I).Table I[35S]CaM binding and CaM regulation of WT and mutant RyR1s and RyR2sCaM BindingCaM Regulation<10 nM Ca2+0.4 μM Ca2+>1 μM Ca2+0.4 μM Ca2+>1 μM Ca2+WT-RyR1+++ND+++++++++RyR1-W3620A+++aData are from Ref. 15.N