COMMUNICATION
Molecular Identification of the Ryanodine Receptor Ca2+ Sensor*

S. R. Wayne ChenDagger , Katsuto Ebisawa, Xiaoli Li, and Lin Zhang

From the Cardiovascular Research Group, Department of Medical Biochemistry, University of Calgary, Calgary, Alberta T2N 4N1, Canada

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

Ryanodine receptors (RyRs)1 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 (1, 2). Three RyR isoforms (RyR1, RyR2, and RyR3) have been identified in mammalian tissues; all three are activated by Ca2+ (3-8). Activation of RyR by Ca2+ is the mechanism underlying Ca2+-induced Ca2+ release from the sarco(endo)plasmic reticulum (9-11).

Of the many ligands known to modulate the activity of RyR, Ca2+ is the essential regulator. Most other ligands exert their effect on RyR activity by influencing the Ca2+ sensitivity of RyR (3-8, 12). Alterations in the Ca2+ sensitivity of RyR have been implicated in at least one disease, malignant hyperthermia (13). 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 (14), 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 (15-17). Analysis of the amino acid sequences of RyRs reveals that of the 12 predicted transmembrane sequences of RyR (18), four (M1, M2, M7, and M10) contain negatively charged amino acid residues that are conserved in all known RyR isoforms (Fig. 1A) (19-26). 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. 1B), 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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References

Site-directed Mutagenesis-- Cloning and sequencing of the rabbit uterus RyR3 cDNA have been described previously (27). Substitution of alanine for glutamate 3885 (E3885A) was carried out by the overlap extension method (28) 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. The ApaI (11,469)-RcoRI (11,833) fragment was removed from the PCR product and subcloned into the SpeI (10,590)-SpeI (12,795) subfragment and subsequently into the full-length RyR3 cDNA. Transfection of HEK293 cells were carried out using Ca2+ phosphate precipitation.

Ca2+ Release Measurements-- Free cytosolic Ca2+ concentration in HEK293 cells was measured with the fluorescence Ca2+ indicator dye fluo-3 (27). 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 mM KH2PO4, 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).

RyR Purification and Single Channel Recordings-- Preparation of microsomal membranes from transfected HEK293 cells and purification of the expressed RyR were carried out as described previously (27). Single channel recordings were carried out using CHAPS-solubilized and sucrose density gradient-purified wild type and mutant recombinant RyRs as reported previously (27). Free Ca2+ concentrations were calculated using the computer program of Fabiato and Fabiato (29).

    RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References

Fig. 1C 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 (27). 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 nM Ca2+ ionophore A23187 (n = 7).


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Fig. 1.   A schematic diagram of the proposed transmembrane topology of RyR (A), amino acid sequences of the M2 segment of different RyR isoforms (B), and effect of caffeine on intracellular Ca2+ release in transfected HEK293 cells (C). A, up to 12 transmembrane sequences have been predicted (M', M", M1-M10) (18). The four negatively charged amino acid residues located in the transmembrane sequences M1, M2, M7, and M10 that are conserved in all known RyRs are depicted by single-letter amino acid codes. The remainder of the molecule has been predicted to form the cytoplasmic domain known as the "foot structure." B, sequences shown are from Caenorhabditis elegans (CelRyR), Drosophila melanogaster (DroRyR), bullfrog (BfRyRalpha and BfRyRbeta ), chicken (ChRyR3), and rabbit (RaRyR1, RaRyR2, and RaRyR3). Amino acid numbers for each RyR sequence are given at right. The conserved glutamates (E) in M2 are boxed. C, HEK293 cells were transfected with 10 µg of wild type (WT) and 10 µg of mutant E3885A cDNA, respectively. Fluorescence intensity of fluo-3-loaded cells was measured before and after sequential addition of 2 mM caffeine (solid arrows) and 100 nM Ca2+ ionophore A23187 (open arrows).

Caffeine is known to sensitize RyR to Ca2+ (12). 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. 2A, 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 and C) (27). 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. 2D), as compared with the open time constant of 1.16 ms of the wild type channels (27). 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.


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Fig. 2.   Single channel properties of the wild type and mutant E3885A. A, single channel activities of mutant E3885A, recorded in a symmetrical recording solution containing 250 mM KCl, 200 µM CaCl2 and 25 mM Hepes, pH 7.4, were inhibited by the addition of 200 µM EGTA (trans), indicating that the channel was incorporated into the bilayer with its cytoplasmic side facing the trans chamber. Single channel current fluctuations in the presence of 4 µM, 500 µM, 2 mM, and 5 mM CaCl2 (cytoplasmic) (trans) are shown. The open probability (Po), arithmetic mean open time (To), and arithmetic mean closed time (Tc) at each Ca2+ level are indicated on the top of each panel. Base lines are indicated by a short line to the right of each current trace. The holding potential was -20 mV. B, single channel current fluctuations of the wild type channel in the presence of 2.5 µM CaCl2 (cytoplasmic) were recorded in a symmetrical recording solution containing 250 mM KCl and 25 mM Hepes, pH 7.4. The holding potential was +20 mV. C, the Po values (open circles) of single mutant channels at various Ca2+ concentrations are shown. A total of 56 measurements was made from 10 separate experiments. The relationship between Po and Ca2+ concentrations of single wild type (WT) channels is depicted by a dashed curve (taken from Ref. 27). D, the histogram of open time of the mutant channel in the presence of 1 mM CaCl2 (cytoplasmic). E, ligand gating properties of the mutant channel. A single mutant channel was activated by 1 mM CaCl2 (cis), indicating that the channel was incorporated into the bilayer with its cytoplasmic side facing the cis chamber. All subsequent additions were then made to the cis (cytoplasmic) chamber. Single channel current recordings in the presence of 1 mM CaCl2 and after sequential additions (cis) of 2 mM ATP, 1 mM EGTA, and 4 mM caffeine were made from the same channel at +20 mV. Current recordings in the presence of 10 µM ryanodine were obtained from a different channel. F, a diary plot of open probability. Each point represents the average Po of a 20-s recording. G, current-voltage (I-V) relationships of the mutant (open circles) and the wild type (dashed line) channel in symmetrical 250 mM KCl. The I-V curve of the wild type channel was taken from Ref. 27.

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 mM cytoplasmic 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. 2G). 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 (27). 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. 3A). 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. 3B). The Hill equation was used to fit the data obtained from two wild type and nine mutant single channels (Fig. 3C). 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.


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Fig. 3.   Assessment of the relative Ca2+ sensitivity of the mutant channel. A, Ca2+ response of the mutant channel in the presence of ATP and caffeine. Single channel activities were first inhibited by cis addition of EGTA and reactivated by cis addition of 2 mM ATP and 4 mM caffeine. An aliquot of 100 mM EGTA or CaCl2 solution was added to the cis chamber (cytoplasmic) to obtain various free Ca2+ concentrations. Single-channel current fluctuations of the ATP and caffeine-activated mutant channel in the presence of 16, 45, 77, and 120 µM free cytoplasmic Ca2+ are shown. The holding potential was +20 mV. B, Ca2+ response of the wild type (WT) channel in the presence of ATP and caffeine. Single-channel recordings of the ATP and caffeine-activated wild type channel were made in the presence of 1, 6, and 13 nM free cytoplasmic Ca2+ at +20 mV. C, the relationship between the open probability (Po) and Ca2+ concentrations (pCa) of the wild type and mutant channels in the presence of 2 mM ATP and 4 mM caffeine. Data shown are obtained from two wild type channels (solid circles) and nine mutant channels (open circles). Curve fit was done using the Hill equation. The Po values of the mutant channels at Ca2+ concentrations greater than 1 mM where Ca2+ inactivation was apparent were not included in the fitting.

Based on the 10,000-fold difference in Ca2+ sensitivity and the threshold of the wild type channel (about 100 nM) (27), 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) (27). 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. 4A 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. 4B). 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.


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Fig. 4.   Co-expression of mutant E3885A results in decreases in both the caffeine response and the sensitivity to activation by Ca2+. A, caffeine-induced Ca2+ release in HEK293 cells transfected with 2 µg of wild type (WT), 2 µg of wild type plus 2 µg of mutant E3885A (WT:E3885A (1:1)), or 2 µg of wild type plus 6 µg of mutant E3885A (WT:E3885A (1:3)) cDNA. B, HEK293 cells were transfected with 5 µg of wild type plus 5 µg of mutant E3885A cDNA (WT:Mutant). The Ca2+ responses of single hybrid channels were determined in the presence of 2 mM ATP and 4 mM caffeine. Solid circles indicate data obtained from a channel with relatively high Ca2+ sensitivity. Open squares display data from a channel with relatively low Ca2+ sensitivity. Open circles show data obtained from four channels with intermediate Ca2+ sensitivity. The curve fit of each group of data was performed using the Hill equation. The dashed curves depict the Po-pCa relationship of the wild type and mutant channel in the presence of 2 mM ATP and 4 mM caffeine as shown in Fig. 3C.

The putative transmembrane sequence M2 is highly conserved in all RyR isoforms sequenced to date (Fig. 1B). 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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* This work was supported by research grants from the Medical Research Council of Canada and the Alberta Heritage Foundation for Medical Research (to S. R. W. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Scholar of the Alberta Heritage Foundation for Medical Research. To whom correspondence should be addressed. Tel.: 403-220-4235; Fax: 403-283-4841; E-mail: swchen{at}acs.ucalgary.ca.

1 The abbreviations used are: RyR, ryanodine receptor; PCR, polymerase chain reaction; CHAPS , 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

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Abstract
Introduction
Procedures
Results & Discussion
References

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