Molecular Identification of the Ryanodine Receptor Ca2+ Sensor

We have investigated the molecular basis for ryanodine receptor (RyR) activation by Ca2+ by using site-directed mutagenesis together with functional assays consisting of Ca2+ release measurements and single channel recordings in planar lipid bilayers. We report here that a single substitution of alanine for glutamate at position 3885 (located in the putative transmembrane sequence M2 of the type 3 RyR) reduces the Ca2+ sensitivity, as measured by single channel activation, by more than 10,000-fold, without apparent changes in channel conductance and in modulation by other ligands (e.g. ATP and ryanodine). Co-expression of the wild type and mutant RyR proteins results in the synthesis of single channels that have intermediate Ca2+ sensitivities. These results suggest that the glutamates at position 3885 of each monomer may act in a coordinated way to form the Ca2+ sensor in the tetrameric structure corresponding to RyR. We have investigated the molecular basis for ryanodine receptor (RyR) activation by Ca2+ by using site-directed mutagenesis together with functional assays consisting of Ca2+ release measurements and single channel recordings in planar lipid bilayers. We report here that a single substitution of alanine for glutamate at position 3885 (located in the putative transmembrane sequence M2 of the type 3 RyR) reduces the Ca2+ sensitivity, as measured by single channel activation, by more than 10,000-fold, without apparent changes in channel conductance and in modulation by other ligands (e.g. ATP and ryanodine). Co-expression of the wild type and mutant RyR proteins results in the synthesis of single channels that have intermediate Ca2+ sensitivities. These results suggest that the glutamates at position 3885 of each monomer may act in a coordinated way to form the Ca2+ sensor in the tetrameric structure corresponding to RyR. Ryanodine receptors (RyRs) 1The abbreviations used are: RyR, ryanodine receptor; PCR, polymerase chain reaction; CHAPS , 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid. are a family of Ca2+ channels which mediate intracellular Ca2+release that is essential for a variety of cellular functions including muscle contraction, egg fertilization, and synaptic transmission (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6174) Google Scholar,2Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2268) Google Scholar). Three RyR isoforms (RyR1, RyR2, and RyR3) have been identified in mammalian tissues; all three are activated by Ca2+ (3Coronado R. Morrissette J. Sukhareva M. Vaughan D.M. Am. J. Physiol. 1994; 266: C1485-C1504Crossref PubMed Google Scholar, 4Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (842) Google Scholar, 5Ogawa Y. Crit. Rev. Biochem. Mol. 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Thus, understanding the molecular mechanism that controls the Ca2+ sensitivity is fundamental to the understanding of RyR regulation and intracellular Ca2+ signaling. RyR activation by Ca2+ is thought to be mediated by high affinity Ca2+ binding sites in the protein (14Meissner G. Darling E. Eveleth J. Biochemistry. 1985; 25: 236-244Crossref Scopus (348) Google Scholar), but the molecular identity of these Ca2+ activation sites, the Ca2+ sensor, has yet to be defined. It has been shown that negatively charged residues within a transmembrane sequence are often involved in binding and translocation of cations across the membrane (15Clarke D.M. Loo T.W. Inesi G. MacLennan D.H. Nature. 1989; 339: 476-478Crossref PubMed Scopus (470) Google Scholar, 16Heinemann S.H. Terlau H. Stuhmer W. Imoto K. Numa S. Nature. 1992; 356: 441-443Crossref PubMed Scopus (630) Google Scholar, 17Yang J. Ellinor P.T. Sather W.A. Zhang J-F. Tsien R.W. Nature. 1993; 366: 158-161Crossref PubMed Scopus (529) Google Scholar). Analysis of the amino acid sequences of RyRs reveals that of the 12 predicted transmembrane sequences of RyR (18Zorzato F. Fujii J. Otsu K. Phillips M. Green N.M. Lai F.A. Meissner G. MacLennan D.H. J. Biol. Chem. 1990; 265: 2244-2256Abstract Full Text PDF PubMed Google Scholar), four (M1, M2, M7, and M10) contain negatively charged amino acid residues that are conserved in all known RyR isoforms (Fig. 1 A ) (19Takeshima 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 (867) Google Scholar, 20Otsu K. Willard H.F. Khanna V.K. Zorzato F. Green N.M. MacLennan D.H. J. Biol. Chem. 1990; 265: 13472-13483Abstract Full Text PDF PubMed Google Scholar, 21Nakai J. Imagawa T. Hakamata Y. Shigekawa M. Takeshima H. Numa S. FEBS Lett. 1990; 271: 169-177Crossref PubMed Scopus (288) Google Scholar, 22Hakamata Y. Nakai J. Takeshima H. Imoto K. FEBS Lett. 1992; 312: 229-235Crossref PubMed Scopus (351) Google Scholar, 23Takeshima H. Nishi M. Iwabe N. Miyata T. Hosoya T. Masai I. Hotta Y. FEBS Lett. 1994; 337: 81-87Crossref PubMed Scopus (138) Google Scholar, 24Oyamada H. Murayama T. Takagi T. Iino M. Iwabe N. Miyata T. Ogawa Y. Endo M. J. Biol. Chem. 1994; 269: 17206-17214Abstract Full Text PDF PubMed Google Scholar, 25Ottini L. Marziali G. Conti A. Charlesworth A. Sorrentino V. Biochem. J. 1996; 315: 207-216Crossref PubMed Scopus (95) Google Scholar, 26Sakube Y. Ando H. Kagawa H. J. Mol. Biol. 1997; 267: 849-864Crossref PubMed Scopus (41) Google Scholar). To investigate their roles in RyR function, we have mutated these negatively charged residues in the rabbit type 3 RyR. The functional consequence of one of these point mutations, a glutamate-to-alanine mutation at position 3885 (E3885A) located in the M2 transmembrane sequence (Fig. 1 B ), was assessed. Our results demonstrate that glutamate 3885 plays an essential role in determining the Ca2+ sensitivity and provide important new insights into the Ca2+-sensing mechanism of RyR. Cloning and sequencing of the rabbit uterus RyR3 cDNA have been described previously (27Chen S.R.W. Li X. Ebisawa K. Zhang L. J. Biol. Chem. 1997; 272: 24234-24246Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Substitution of alanine for glutamate 3885 (E3885A) was carried out by the overlap extension method (28Ho S.N. Hunt H.D. Morton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6832) Google Scholar) using polymerase chain reaction (PCR). The “outer” two oligonucleotides used were: forward, 5′-TACTCCAGAATGATGAG-3′; and reverse, 5′-TCCATGGCCTTCTGGAATTC-3′. The oligonucleotides for the E3885A mutation were: forward, 5′-CCCTCCTGGCAGGGAATGT-3′; and reverse, 5′-ACATTCCCTGCCAGGAGGG-3′. The sequences of the PCR products were confirmed by DNA sequencing. TheApa I (11,469)–Rco RI (11,833) fragment was removed from the PCR product and subcloned into the Spe I (10,590)–Spe I (12,795) subfragment and subsequently into the full-length RyR3 cDNA. Transfection of HEK293 cells were carried out using Ca2+ phosphate precipitation. Free cytosolic Ca2+ concentration in HEK293 cells was measured with the fluorescence Ca2+ indicator dye fluo-3 (27Chen S.R.W. Li X. Ebisawa K. Zhang L. J. Biol. Chem. 1997; 272: 24234-24246Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Cells grown for 18 h after transfection were washed three times with KRH buffer without MgCl2 and CaCl2 (KRH buffer: 125 mm NaCl, 5 mm KCl, 1.2 mmKH2PO4, 6 mm glucose, 1.2 mm MgCl2, 2 mm CaCl2, and 25 mm Hepes, pH 7.4) and incubated in the same buffer at room temperature for 30 min and at 37 °C for 30 min. After being detached from culture dishes by pipetting, cells from two 100-mm tissue culture dishes were collected by centrifugation at 2,500 rpm for 2 min in a Beckman TH-4 rotor. Cell pellets were suspended and loaded with 5 μm fluo-3 AM in Dulbecco's modified Eagle's medium at room temperature for 30 min followed by washing with KRH buffer three times and resuspended in 150 μl of KRH buffer plus 0.1 mg/ml bovine serum albumin and 250 μm sulfinpyrazone. The fluo-3-loaded cells (150 μl) were added to 2 ml (final volume) of KRH buffer in a cuvette. Fluorescence intensity of fluo-3 at 530 nm was measured in an SLM-Aminco series 2 luminescence spectrometer with 480 nm excitation at 25 °C (SLM Instruments, Urbana, IL). Preparation of microsomal membranes from transfected HEK293 cells and purification of the expressed RyR were carried out as described previously (27Chen S.R.W. Li X. Ebisawa K. Zhang L. J. Biol. Chem. 1997; 272: 24234-24246Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Single channel recordings were carried out using CHAPS-solubilized and sucrose density gradient-purified wild type and mutant recombinant RyRs as reported previously (27Chen S.R.W. Li X. Ebisawa K. Zhang L. J. Biol. Chem. 1997; 272: 24234-24246Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Free Ca2+ concentrations were calculated using the computer program of Fabiato and Fabiato (29Fabiato A. Fabiato F. J. Physiol. (Paris). 1979; 75: 463-505PubMed Google Scholar). Fig. 1 C shows that addition of 2 mm caffeine to HEK293 cells transfected with the wild type cDNA caused an increase in the fluo-3 fluorescence (n = 5), similar to that observed previously (27Chen S.R.W. Li X. Ebisawa K. Zhang L. J. Biol. Chem. 1997; 272: 24234-24246Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). In contrast, the fluo-3 fluorescence in HEK293 cells transfected with mutant E3885A cDNA failed to increase in response to 2 mm caffeine but did respond to 100 nmCa2+ ionophore A23187 (n = 7). Caffeine is known to sensitize RyR to Ca2+ (12Meissner G. Rios E. Tripathy A. Pasek D.A. J. Biol. Chem. 1997; 272: 1628-1638Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The lack of caffeine response suggests that the E3885A mutation may interfere with the Ca2+ activation pathway. To examine directly whether the E3885A mutation alters Ca2+ activation, we incorporated the recombinant E3885A mutant proteins into planar lipid bilayers and determined the response of single mutant channels to a wide range of Ca2+ concentrations. As shown in Fig.2 A , the E3885A mutant channel was essentially closed at micromolar Ca2+ and remained closed until the Ca2+ concentration was raised to about 500 μm. The mutant channel was further activated by increasing the Ca2+ concentration, but the maximal open probability was low. On the other hand, the wild type channel was activated at about 100 nm Ca2+ and reached full activation at micromolar Ca2+ (Fig. 2, B andC ) (27Chen S.R.W. Li X. Ebisawa K. Zhang L. J. Biol. Chem. 1997; 272: 24234-24246Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Open times of the mutant channels were short and exponentially distributed with a time constant of 0.296 ± 0.095 ms (mean ± S.D.) (n = 10) (Fig. 2 D ), as compared with the open time constant of 1.16 ms of the wild type channels (27Chen S.R.W. Li X. Ebisawa K. Zhang L. J. Biol. Chem. 1997; 272: 24234-24246Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Thus the E3885A mutant channel, although still regulated by Ca2+, differs markedly from the wild type channel in the sensitivity to Ca2+ activation, the extent of maximal Ca2+ activation, and the gating kinetics. To examine whether the mutant channel is still sensitive to other modulators of RyR, we assessed the effect of ATP, caffeine, and ryanodine on the single channel activity of the mutant channel (Fig. 2,E and F ). In the presence of 1 mmcytoplasmic Ca2+, the mutant channel was activated by the addition of 2 mm ATP (n = 3). The ATP-activated channel was inhibited by subsequent addition of EGTA. The mutant channel was also activated by caffeine and was sensitive to ryanodine (n = 3). The mutant channel exhibited a linear I-V relationship similar to that of the wild type (Fig.2 G ). The unitary conductance of the mutant channel, determined in the presence of both ATP and caffeine, was 806 ± 16 pS (n = 4), compared with 777 pS of the wild type (27Chen S.R.W. Li X. Ebisawa K. Zhang L. J. Biol. Chem. 1997; 272: 24234-24246Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). These data suggest that the E3885A mutation does not cause gross alterations in channel function. The E3885A mutation thus appears to alter specifically the Ca2+ activation of the channel. Quantification of the extent of alteration in Ca2+ activation of the mutant channel was difficult because of its low open probability. To overcome this problem, we assessed the relative sensitivity to activation by Ca2+ of the mutant channel in the presence of 2 mm ATP and 4 mm caffeine. Under these conditions, the mutant channel was activated at micromolar Ca2+, and it was fully activated at about 150–200 μm Ca2+ (Fig.3 A ). In comparison, under the same conditions, the wild type channel was activated at less than 1 nm Ca2+, and it was fully activated at about 15–20 nm Ca2+ (Fig. 3 B ). The Hill equation was used to fit the data obtained from two wild type and nine mutant single channels (Fig. 3 C ). This analysis revealed that half-maximal activation was achieved at a Ca2+concentration of 61.4 μm for the mutant channels and 4.7 nm for the wild type channels. Thus the sensitivity to Ca2+ activation of the mutant channel is more than 10,000-fold lower than that of the wild type. Based on the 10,000-fold difference in Ca2+ sensitivity and the threshold of the wild type channel (about 100 nm) (27Chen S.R.W. Li X. Ebisawa K. Zhang L. J. Biol. Chem. 1997; 272: 24234-24246Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), the threshold for Ca2+ activation of the E3885A mutant in the absence of ATP and caffeine would be about 1 mm. The high threshold for Ca2+ activation of the mutant channel is close to the threshold for Ca2+ inactivation (about 1 mm) (27Chen S.R.W. Li X. Ebisawa K. Zhang L. J. Biol. Chem. 1997; 272: 24234-24246Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). The very low activation by Ca2+ of the mutant channel (Fig. 2, A and C ) is, therefore, most likely because of simultaneous inactivation by Ca2+. This high threshold may also explain why we could not detect caffeine-induced Ca2+ release in E3885A-transfected HEK293 cells where the resting cytoplasmic Ca2+concentration is about 100 nm, although mutant channels in the lipid bilayers proved to be sensitive to caffeine. Taken together, these results indicate that glutamate 3885 is essential in determining the sensitivity of RyR to activation by Ca2+. The mechanism by which substitution of a single amino acid residue results in a more than 10,000-fold change in the Ca2+sensitivity of RyR is not clear. One possibility is that the four glutamates at position 3885 of each monomer may be located in close proximity to each other and act in concert to form the Ca2+sensor in the homotetrameric RyR. To examine this possibility, we co-expressed the wild type and mutant cDNA in HEK293 cells and determined caffeine-induced Ca2+ release. We reasoned that co-expression of the wild type and mutant E3885A would result in the formation of a Ca2+ sensor with a mixture of glutamates and alanines. This hybrid Ca2+ sensor would be expected to have reduced caffeine response and Ca2+ sensitivity. Fig.4 A shows that the caffeine-induced Ca2+ release in these co-transfected HEK293 cells was reduced as a result of the decreasing ratio of wild type over mutant cDNA (n = 4). Single-channel analysis revealed that co-transfection of HEK293 cells with a 1:1 ratio of wild type and mutant cDNA produced channels that have intermediate Ca2+ sensitivities (Fig. 4 B ). Half-maximal activation of these hybrid single channels was achieved at Ca2+ concentrations ranging from 36.6 nm to 7.5 μm. Most of the hybrid channels displayed half-maximal activation at about 238 nm Ca2+. These different Ca2+ responses most likely resulted from hybrid channels with different ratios of wild type and mutant subunits. Thus, these findings are consistent with the possibility that the glutamates at position 3885 of the monomers act coordinately to form the Ca2+ sensor. The putative transmembrane sequence M2 is highly conserved in all RyR isoforms sequenced to date (Fig. 1 B ). It is most likely that the corresponding glutamate in other RyR isoforms also plays a fundamental role in determining the Ca2+ sensitivity. Further studies of the Ca2+ sensor will provide clues to how the Ca2+ sensitivity is modulated by various modulators and by disease states and how binding of Ca2+ to the Ca2+ sensor couples to channel opening. The oligomeric coordination of the Ca2+ sensor may provide the basis for controlling and manipulating the Ca2+ sensitivity of RyR. We thank Drs. Henry J. Duff, Michael Fill, Wayne R. Giles, and Henk E. D. J. ter Keurs for critical reviews of this manuscript, and Paul M. Schnetkamp for the use of his luminescence spectrometer.