Correspondence to: S.R. Wayne Chen, Department of Physiology and Biophysics, University of Calgary, 330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada. Fax:(403) 283-4841 E-mail:swchen{at}ucalgary.ca.
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Abstract |
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Activation of the cardiac ryanodine receptor (RyR2) by Ca2+ is an essential step in excitation-contraction coupling in heart muscle. However, little is known about the molecular basis of activation of RyR2 by Ca2+. In this study, we investigated the role in Ca2+ sensing of the conserved glutamate 3987 located in the predicted transmembrane segment M2 of the mouse RyR2. Single point mutation of this conserved glutamate to alanine (E3987A) reduced markedly the sensitivity of the channel to activation by Ca2+, as measured by using single-channel recordings in planar lipid bilayers and by [3H]ryanodine binding assay. However, this mutation did not alter the affinity of [3H]ryanodine binding and the single-channel conductance. In addition, the E3987A mutant channel was activated by caffeine and ATP, was inhibited by Mg2+, and was modified by ryanodine in a fashion similar to that of the wild-type channel. Coexpression of the wild-type and mutant E3987A RyR2 proteins in HEK293 cells produced individual single channels with intermediate sensitivities to activating Ca2+. These results are consistent with the view that glutamate 3987 is a major determinant of Ca2+ sensitivity to activation of the mouse RyR2 channel, and that Ca2+ sensing by RyR2 involves the cooperative action between ryanodine receptor monomers. The results of this study also provide initial insights into the structural and functional properties of the mouse RyR2, which should be useful for studying RyR2 function and regulation in genetically modified mouse models.
Key Words: excitation-contraction coupling, Ca2+-induced Ca2+ release, Ca2+ sensing, sarcoplasmic reticulum, planar lipid bilayers
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INTRODUCTION |
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Excitation-contraction (E-C)* coupling in heart muscle is believed to occur via a mechanism known as Ca2+-induced Ca2+ release (CICR;
Despite the central role of Ca2+ activation of RyR in CICR and in cardiac E-C coupling, the molecular mechanism of Ca2+ sensing by RyR has been elusive. Three RyR isoforms (RyR1, RyR2, and RyR3) have been identified and cloned, and their responses to Ca2+ have been investigated (
Activation of RyR by Ca2+ is believed to result from binding of Ca2+ ions to the high affinity Ca2+ binding sites in the channel protein (1,000 amino acid residues, rather than in the NH2-terminal region of RyR, since a truncated RyR1 lacking
4,000 residues from the NH2 terminus retains Ca2+ activation (
1,000 residues of RyR.
Site-specific mutational studies have provided new insights into the molecular determinant of Ca2+ activation (10,000-fold. It should be noted that the corresponding M2 sequence (
In line with the importance of this conserved glutamate in channel function and regulation, mutation of the corresponding glutamate 4032 to alanine (E4032A) in RyR1 abolished caffeine response and [3H]ryanodine binding (
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MATERIALS AND METHODS |
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Materials
Anti-RyR mAb 34C was obtained from Affinity BioReagents Inc. Brain phosphatidylserine was obtained from Avanti Polar Lipid. Egg phosphatidylcholine was purchased from Sigma-Aldrich. Synthetic 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were from Northern Lipids.
Cloning and Expression of the Mouse RyR2 cDNA and Construction of the Mutant Construct
Cloning of the mouse RyR2 cDNA and expression of RyR in HEK293 cells have been described previously (
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Preparation of Cell Lysate from Transfected HEK293 Cells and Heavy Sarcoplasmic Reticulum from Canine Heart Muscle
HEK293 cells grown for 2426 h after transfection using Ca2+ phosphate precipitation were washed three times with PBS (137 mM NaCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, and 2.7 mM KCl) plus 2.5 mM EDTA, and were harvested in the same solution by centrifugation. Cells from 15 tissue culture dishes (100 mm in diameter) were solubilized in 2.5 ml lysis buffer containing 25 mM Tris, 50 mM HEPES, pH 7.4, 137 mM NaCl, 1% CHAPS, 0.6% egg phosphatidylcholine, 2.5 mM DTT, and a protease inhibitor mix (1 mM benzamidine, 2 µg/ml leupeptin, 2 µg /ml pepstatin A, 2 µg /ml aprotinin, and 0.5 mM PMSF) on ice for 1 h. Cell lysate was obtained after removing the unsolubilized materials by centrifugation in microcentrifuge at 4°C for 30 min. Heavy SR was isolated from canine cardiac muscle according to the method described previously (
Sucrose Density Gradient Purification of Recombinant RyR2 and Canine RyR2 Proteins
2.5 ml of cell lysate or solubilized canine cardiac SR membranes (2 mg in 2.5 ml) was layered on top of a 10.5-ml (7.525%, wt/wt) linear sucrose gradient containing 25 mM Tris, 50 mM HEPES, pH 7.4, 0.3 M NaCl, 0.1 mM CaCl2, 0.3 mM EGTA, 0.25 mM PMSF, 4 µg/ml leupeptin, 5 mM DTT, 0.3% CHAPS, and 0.16% synthetic phosphatidylcholine. The gradient was centrifuged at 29,000 rpm in Beckman SW-41 rotor at 4°C for 17 h. Fractions of 0.7 ml each were collected. Peak fractions containing RyR proteins, as determined by immunoblotting, were pooled, aliquoted, and stored at -80°C.
Single-channel Recordings
Recombinant wt and E3987A mutant RyR2 and canine RyR2 proteins solubilized and purified by sucrose density gradient centrifugation were used for single-channel recordings as described previously (
[3H]Ryanodine Binding
Equilibrium [3H]ryanodine binding to cell lysate was performed as described previously (
Ca2+ Release Measurements
Free cytosolic Ca2+ concentration in transfected HEK293 cells was measured using the fluorescence Ca2+ indicator dye fluo-3-AM as described previously (
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RESULTS |
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Cloning and Functional Expression of the Mouse Cardiac Ryanodine Receptor cDNA
Fig 1 A outlines the strategy for the cloning of the mouse cardiac ryanodine receptor (RyR2) cDNA. The mouse RyR2 cDNA encodes a 565-kD protein composed of 4,967 amino acids. The deduced amino acid sequence of the mouse RyR2 shares 97% identity with that of the rabbit (
Mutation E3987A Altered the Ca2+ Response of Single Mouse RyR2 Channels
To further understand the defect in mutant E3987A, we incorporated the wt and E3987A mutant proteins into planar lipid bilayers and determined the open probability (Po) of single channels at a wide range of Ca2+ concentrations. As shown in Fig 2 A, a single wt channel was activated by Ca2+ at 100 nM and was inactivated at
10 mM. At Ca2+ concentrations between
1 and 2,000 µM, the channel was maximally activated, resulting in a bell-shaped Ca2+ response curve (Fig 2 C). Curve fitting of the wt Ca2+ response using the Hill equation yielded an EC50 of 0.26 µM and a Hill coefficient of 3.0 for activation by Ca2+ (n = 22) and an IC50 of 2.1 mM and a Hill coefficient of 1.3 for inactivation by Ca2+ (n = 19). The extent of inactivation of single wt channels by high concentrations of Ca2+ was found to vary from channel to channel. Some channels were inactivated, whereas the others remained highly active at high Ca2+ concentrations (Fig 2 C). In contrast, the E3987A mutant channels required several hundreds of micromolar Ca2+ for activation and hardly responded to increasing Ca2+ concentrations (Fig 2 B). The maximum Po of most E3987A mutant channels activated by a wide range of Ca2+ concentrations was <0.05 (Fig 2 C). In addition, opening events of the E3987A mutant channels were extremely brief. The mean open time of single E3987A mutant channels is
10-fold shorter than that of the wt channels (Fig 2A and Fig B). It appears that some opening events were too brief to be resolved completely under our recording conditions (Fig 2 B). Thus, the Po of the E3987A mutant channels activated by Ca2+ alone may have been under estimated. Nevertheless, these data indicate that mutation E3987A severely impairs the threshold, maximum extent, and kinetics of Ca2+ activation of the mouse RyR2.
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Ligand Gating Properties of Single wt and E3987A Mutant RyR2 Channels
We next examined the response of wt and E3987A mutant RyR2 channels to various modulators. Consistent with the results of [3H]ryanodine binding studies reported previously (200 nM) and mutant (
2 mM) channels differ considerably because of their differences in Ca2+ activation (Fig 2). Hence, the extent of activation or inhibition by a ligand of the wt and mutant channels may not be compared quantitatively. It should also be noted that Mg2+ could inhibit Ca2+-activated wt or mutant E3987A channels in the absence of ATP and caffeine (not shown). Both the wt and E3987A mutant channels were modified in a similar fashion by ryanodine, which shifted the channel into a state with high Po and reduced conductance (Fig 3 A, panel e, and Fig 3 B, panel f). The single-channel conductance of the E3987A mutant channel is 793 ± 2.8 pS (n = 4), similar to that of the wt channels (
800 pS). Thus, the E3987A mutation does not change the single-channel conductance and does not cause gross alterations in channel function.
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Mutant E3987A Retained High Affinity [3H]Ryanodine Binding
Different from mutant E3987A in RyR2, the corresponding mutant E4032A in RyR1 showed no response to either caffeine or ryanodine and lacked high affinity [3H]ryanodine binding. It has been suggested that the E4032A mutation may affect ryanodine binding directly (
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Mutation E3987A Markedly Reduced the Sensitivity of Mouse RyR2 Channels to Activating Ca2+
As seen in Fig 3 B, single E3987A mutant channels could be fully activated by Ca2+ in the presence of ATP and caffeine. This property allows us to quantify the relative Ca2+ sensitivity of the E3987A mutant channels at the single-channel level. As shown in Fig 5 A, in the presence of 2 mM ATP and 4 mM caffeine, a single wt channel was activated by 50 nM Ca2+ and reached maximum activation at
300 nM Ca2+. The Ca2+ response of single wt channels under these conditions could be described by an EC50 of 93 nM and a Hill coefficient of 3.1 (n = 5; Fig 5 D). On the other hand, under the same conditions, single mutant E3987A channels were activated by submicromolar Ca2+ and reached maximum activation at
500 µM (Fig 5B and Fig C). The difference in the extent of maximum activation was observed among single E3987A mutant channels. Some mutant channels could be fully activated, whereas others showed maximum activation of
50% (Fig 5, BD). This discrepancy is most likely attributable to different extents of Ca2+ inactivation, as seen among single wt channels (Fig 2 C). The Ca2+ responses of the high and low Po E3987A mutant channels were analyzed by using the Hill equation. These analyses yielded an EC50 of 109 µM and a Hill coefficient of 1.1 for Ca2+ activation of the high Po E3987A mutant channels (n = 5), and an EC50 of 246 µM and a Hill coefficient of 1.63 for Ca2+ activation of the low Po mutant channels (n = 5). Hence, single E3987A mutant channels exhibited
1,0003,000-fold reduction in Ca2+ sensitivity to activation as compared with single wt RyR2 channels. We have previously shown that the corresponding mutation E3885A in the RyR3 isoform reduced the Ca2+ sensitivity of single RyR3 channels to activation in lipid bilayers by >10,000-fold (
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Marked reduction in the sensitivity of the E3987A mutant channel to Ca2+ activation could be also demonstrated in the absence of ATP and caffeine by using [3H]ryanodine binding assay. Although mutant E3987A exhibited a binding affinity and maximum binding capacity similar to those of the wt (Fig 4), they differ considerably in the Ca2+ dependence of [3H]ryanodine binding. [3H]ryanodine binding to wt proteins was activated by Ca2+ with an EC50 of 0.22 ± 0.03 µM and a Hill coefficient of 2.6 ± 0.34 (n = 6), whereas activation by Ca2+ of [3H]ryanodine binding to mutant E3987A proteins could be described by an EC50 of 59 ± 14 µM and a Hill coefficient of 1.3 ± 0.3 (n = 6; Fig 6). Thus, the E3987A mutation resulted in 270-fold reduction in Ca2+ sensitivity to activation of [3H]ryanodine binding. This estimated reduction in Ca2+ sensitivity differs by
410-fold from that estimated by single-channel measurements. The Ca2+ sensitivity of the wt channels estimated by single-channel measurements (0.26 µM) is similar to that (0.22 µM) estimated by [3H]ryanodine binding analysis (Fig 2 and Fig 6). On the other hand, the relative Ca2+ sensitivity to activation of single E3987A mutant channels measured in lipid bilayers may have been underestimated due to the influence of Ca2+-dependent inhibition (Fig 5 D). As a result, the differences in Ca2+ sensitivity between single wt and E3987A mutant channels measured in lipid bilayers may have been overestimated. It is of interest to note that significant Ca2+-dependent inhibition of [3H]ryanodine binding was not detected at Ca2+ concentrations as high as 1 M (Fig 6). The lack of Ca2+-dependent inhibition of [3H]ryanodine binding to rabbit RyR2 proteins also has been reported previously (
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Coexpression of wt and E3987A Mutant RyR2 Proteins Produced Single Channels with Intermediate Ca2+ Sensitivities
To investigate the role of subunit interaction in Ca2+ activation of the tetrameric RyR channel, we coexpressed the wt and E3987A mutant proteins in HEK293 cells and determined the Ca2+ response of each single channel detected in lipid bilayers (Fig 7). A total of 19 single channels were observed and characterized. Based on their responses to Ca2+, these single channels could be divided into five groups. Group I (6/19) exhibited a Ca2+ response similar to that of the wt, with an EC50 of 0.40 µM and a Hill coefficient of 2.8 for activation by Ca2+ (Fig 7 D, solid circles). This group of single channels most probably corresponds to the wt homotetramer. On the other hand, single channels in group II (2/19) were hardly activated by Ca2+, resembling the E3987A mutant homotetramer (Fig 7 D, solid triangles). Group III (medium Po; 4/19) displayed a Ca2+ response clearly different from that of the wt homotetramer and E3987A mutant homotetramer, exhibiting an EC50 of 85 µM and a Hill coefficient of 2.0 for activation by Ca2+ (Fig 7B and Fig D, solid squares). Since the E3987A mutant protein forms a functional channel, presumably a homotetramer, it is most likely that the E3987A mutant protein is capable of forming a heterotetrameric channel with the wt protein. If so, group III single channels most probably represents hybrid channels formed by the wt and E3987A mutant subunits. The Ca2+ response of group IV (high Po) single channels (5/19; open circles) was found to be in between or similar to those of group I (wt homotetramer) and group III (presumably wt/mutant hybrid; Fig 7A and Fig D). Group V (low Po; 2/19) showed Ca2+ response in between or similar to those of the group III (presumably wt/mutant hybrid) and group II (E3987A mutant homotetramer) (Fig 7C and Fig D, open squares). Because of the overlap in Ca2+ response with the wt homotetramer or with the mutant homotetramer, we are not certain that single channels in groups IV and V are all wt/mutant hybrid channels. Nevertheless, it is clear from Fig 7 D that coexpression of the wt and E3987A mutant proteins produced single channels displaying sensitivities to activating Ca2+ in between those of the wt and E3987A mutant RyR2 channels. These results also indicate that the sensitivity to activation of the RyR2 Ca2+ sensor may depend on the cooperative interaction among RyR monomers.
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Single Mouse RyR2 Channels Exhibited Ca2+ Response Similar to that of Single Canine RyR2 Channels
The mouse heart is quite different from that of other mammalian species. The most distinctive feature of the mouse heart is its fast heart rate (500600 beats per minute;
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DISCUSSION |
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The Role of the Conserved Glutamate in Ca2+ Activation of Different RyR Isoforms
In earlier studies, we have provided evidence that glutamate 3885 is a major determinant of the sensitivity of RyR3 to activation by Ca2+ (
In view of the essential role of Ca2+ activation of RyR2 in CICR and in E-C coupling, we set out to investigate the molecular basis of activation of RyR2 by Ca2+ and to determine whether the Ca2+-sensing role of this conserved glutamate is maintained in the RyR2 isoform. To this end, we have cloned the cDNA encoding the mouse RyR2 and made the corresponding mutation E3987A. Single-channel properties of the wt and E3987A mutant RyR2 were assessed and compared. Our results demonstrate that the E3987A mutant RyR2 channels exhibit marked reduction in the sensitivity to activating Ca2+, while retaining similar single-channel conductance, high affinity [3H]ryanodine binding, and responses to ATP, caffeine, Mg2+, and ryanodine. These properties of the E3987A mutant RyR2 are very similar to those of the E3885A mutant RyR3, indicating that the specific role of glutamate 3987 in Ca2+ sensing is conserved in the RyR2 isoform.
The reasons for the lack of activity of the E4032A mutant RyR1 are not known. One possible explanation for the observed discrepancy between the E4032A mutant RyR1 and E3987A mutant RyR2 or E3885A mutant RyR3 may lie, in part, in different sensitivities of these mutant channels to inactivation by high Ca2+ concentrations. RyR1 is known to be 1020 times more sensitive to inactivation by Ca2+ than RyR2 or RyR3 (
As to the E3987A mutant RyR2, Ca2+ activation is partially overlapped with Ca2+ inactivation due to the much lower sensitivity of RyR2 to inactivating Ca2+. Residual activity of the E3987A mutant channel could be detected at submillimolar Ca2+, although its Ca2+ response was significantly suppressed at higher Ca2+ concentrations (Fig 2B and Fig C). This suppression could be alleviated by the addition of ATP and caffeine (Fig 5). ATP and caffeine are known to be able to increase the sensitivity to Ca2+ activation and decrease the sensitivity to Ca2+ inactivation of the channel (
The sensitivity to Ca2+ inactivation differs also among single recombinant mouse RyR2 channels (Fig 2 C). Some single recombinant mouse RyR2 channels displayed no significant level of Ca2+ inactivation. Heterogeneity in Ca2+ inactivation of single native rabbit RyR2 channels has also been observed (
Possible Mechanisms of Ca2+ Sensing by RyR
It is clear from the results of our present and previous studies that the absolutely conserved glutamate located in the putative transmembrane sequence M2 is a key residue in determining the sensitivity of RyR to activation by Ca2+. However, the molecular basis of how this glutamate is involved in Ca2+ sensing remains to be understood. One possibility is that each RyR subunit has one Ca2+ sensor and that the conserved glutamate contributes to the formation of the Ca2+ sensor in each subunit. Mutation of this glutamate would decrease the sensitivity of each sensor to Ca2+ activation and probably alter the cooperativity between sensors, thus, reducing the overall sensitivity and cooperativity of the tetrameric RyR channel to Ca2+ activation. This multi-sensor model is analogous to that proposed for Ca2+ activation of Ca2+-activated potassium channels in which activation of all four Ca2+ sensors is necessary for channel opening and the steeply cooperative channel gating would arise from the cooperative interaction between sensors (
Alternatively, each subunit may contribute partially to the formation of a single Ca2+ sensor in the tetrameric RyR channel, and the conserved glutamates of each subunit may be located in close proximity and act cooperatively to form a major part of the Ca2+ sensor. The sensor could be composed of two or more cooperative Ca2+ binding sites. Mutation of this glutamate would affect both the sensitivity and cooperativity of the sensor to activating Ca2+. This single-sensor model is reminiscent of the Ca2+ binding sites in the SR Ca2+ pump in which amino acid residues from four transmembrane segments contribute to the formation of two Ca2+ binding sites (
Channel Properties of the Mouse RyR2
Most information on RyR2 channel properties has primarily come from studies using sheep, canine, or rabbit hearts. Little is known about the channel properties of RyR2 from the mouse heart. This is due, in part, to the limited amount of mouse RyR2 proteins that could be isolated and used for either biochemical or electrophysiological analyses. In the present study, we were able to express the mouse RyR2 cDNA in HEK293 cells and functionally characterize the recombinant mouse RyR2 protein at the single-channel level. We show that recombinant mouse RyR2 channels can be activated by ATP and caffeine, inhibited by Mg2+, and modified by ryanodine. The Ca2+ response of the mouse RyR2 channels is biphasic, being activated by Ca2+ at low concentrations and inhibited by Ca2+ at high concentrations, similar to that of the canine RyR2 channels. Further mutational studies should lead to a better understanding of the structure and function relationships of the mouse RyR2 channel. This kind of knowledge should be useful for manipulating specific properties of the mouse RyR2 channel such as Ca2+ sensing and ion conduction and assessing the physiological significance of these properties in cardiac function via genetic engineering of the mouse RyR2 gene.
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Footnotes |
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* Abbreviations used in this paper: CICR, Ca2+-induced Ca2+ release; DHPR, dihydropyridine receptor; E-C coupling, excitation-contraction coupling; Po, open probability; RyR, ryanodine receptor; SR, sarcoplasmic reticulum; wt, wild-type.
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Acknowledgements |
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We would like to thank Lin Zhang and Xiaoli Li for invaluable technical assistance, Dr. Wayne R. Giles and the CIHR Group on Ion Channels and Transporters for continuous support, and Dr. Paul M. Schnetkamp for the use of his luminescence spectrometer.
This work was supported by research grants from the Canadian Institutes of Health Research and from the Heart and Stroke Foundation of Alberta to S.R.W. Chen. S.R.W. Chen is a Senior Scholar of the Alberta Heritage Foundation for Medical Research.
Submitted: 23 March 2001
Revised: 21 May 2001
Accepted: 22 May 2001
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References |
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