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Molecular basis of Ca(2)+ activation of the mouse cardiac Ca(2)+ release channel (ryanodine receptor).

Li P, Chen SR - J. Gen. Physiol. (2001)

Bottom Line: Single point mutation of this conserved glutamate to alanine (E3987A) reduced markedly the sensitivity of the channel to activation by Ca(2)+, as measured by using single-channel recordings in planar lipid bilayers and by [(3)H]ryanodine binding assay.However, this mutation did not alter the affinity of [(3)H]ryanodine binding and the single-channel conductance.Coexpression of the wild-type and mutant E3987A RyR2 proteins in HEK293 cells produced individual single channels with intermediate sensitivities to activating Ca(2)+.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Research Group, University of Calgary, Calgary, Alberta, Canada T2N 4N1.

ABSTRACT
Activation of the cardiac ryanodine receptor (RyR2) by Ca(2)+ is an essential step in excitation-contraction coupling in heart muscle. However, little is known about the molecular basis of activation of RyR2 by Ca(2)+. In this study, we investigated the role in Ca(2)+ 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 Ca(2)+, as measured by using single-channel recordings in planar lipid bilayers and by [(3)H]ryanodine binding assay. However, this mutation did not alter the affinity of [(3)H]ryanodine binding and the single-channel conductance. In addition, the E3987A mutant channel was activated by caffeine and ATP, was inhibited by Mg(2)+, 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 Ca(2)+. These results are consistent with the view that glutamate 3987 is a major determinant of Ca(2)+ sensitivity to activation of the mouse RyR2 channel, and that Ca(2)+ 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.

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Ca2+ responses of single channels produced by coexpression of the wt and E3987A mutant RyR2 proteins in HEK293 cells. HEK293 cells were transfected with an equal amount (6 μg) of wt and E3987A mutant RyR2 cDNA. The Ca2+ responses of single hybrid channels with high Po (A), medium Po (B), and low Po (C) were determined as described in the legend to Fig. 2. Single-channel activities shown in A and B were inhibited by addition of 0.1 mM EGTA to the cis chamber, indicating that the cytoplasmic side of the incorporated channel was facing the cis chamber. On the other hand, single-channel activities shown in C were inhibited by addition of 0.1 mM EGTA to the trans chamber, indicating that the cytoplasmic side of the incorporated channel was facing the trans chamber. To measure currents in the same direction, from the luminal to the cytoplasmic side of the channel, a +20-mV holding potential was applied in A and B and −20 mV in C. The Po-pCa relationships of single hybrid channels are shown in D. Data points represent individual measurements. According to their Ca2+ sensitivities to activation, single hybrid channels could be divided into at least five groups (I–V). Group I (solid circles) and group II (solid triangles) displayed Ca2+ sensitivity to activation similar to that of the wt and E3987A mutant channel, respectively. Group III (solid squares), group IV (open circles), and group V (open squares) showed sensitivities to Ca2+ activation in between those of the wt and E3987A mutant channels. These different Ca2+ sensitivities most probably resulted from hybrid channels with different compositions of wt and E3987A mutant subunits. Note that two single channels are present in recordings shown in C and Po indicates the average open probability.
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Figure 7: Ca2+ responses of single channels produced by coexpression of the wt and E3987A mutant RyR2 proteins in HEK293 cells. HEK293 cells were transfected with an equal amount (6 μg) of wt and E3987A mutant RyR2 cDNA. The Ca2+ responses of single hybrid channels with high Po (A), medium Po (B), and low Po (C) were determined as described in the legend to Fig. 2. Single-channel activities shown in A and B were inhibited by addition of 0.1 mM EGTA to the cis chamber, indicating that the cytoplasmic side of the incorporated channel was facing the cis chamber. On the other hand, single-channel activities shown in C were inhibited by addition of 0.1 mM EGTA to the trans chamber, indicating that the cytoplasmic side of the incorporated channel was facing the trans chamber. To measure currents in the same direction, from the luminal to the cytoplasmic side of the channel, a +20-mV holding potential was applied in A and B and −20 mV in C. The Po-pCa relationships of single hybrid channels are shown in D. Data points represent individual measurements. According to their Ca2+ sensitivities to activation, single hybrid channels could be divided into at least five groups (I–V). Group I (solid circles) and group II (solid triangles) displayed Ca2+ sensitivity to activation similar to that of the wt and E3987A mutant channel, respectively. Group III (solid squares), group IV (open circles), and group V (open squares) showed sensitivities to Ca2+ activation in between those of the wt and E3987A mutant channels. These different Ca2+ sensitivities most probably resulted from hybrid channels with different compositions of wt and E3987A mutant subunits. Note that two single channels are present in recordings shown in C and Po indicates the average open probability.

Mentions: 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.


Molecular basis of Ca(2)+ activation of the mouse cardiac Ca(2)+ release channel (ryanodine receptor).

Li P, Chen SR - J. Gen. Physiol. (2001)

Ca2+ responses of single channels produced by coexpression of the wt and E3987A mutant RyR2 proteins in HEK293 cells. HEK293 cells were transfected with an equal amount (6 μg) of wt and E3987A mutant RyR2 cDNA. The Ca2+ responses of single hybrid channels with high Po (A), medium Po (B), and low Po (C) were determined as described in the legend to Fig. 2. Single-channel activities shown in A and B were inhibited by addition of 0.1 mM EGTA to the cis chamber, indicating that the cytoplasmic side of the incorporated channel was facing the cis chamber. On the other hand, single-channel activities shown in C were inhibited by addition of 0.1 mM EGTA to the trans chamber, indicating that the cytoplasmic side of the incorporated channel was facing the trans chamber. To measure currents in the same direction, from the luminal to the cytoplasmic side of the channel, a +20-mV holding potential was applied in A and B and −20 mV in C. The Po-pCa relationships of single hybrid channels are shown in D. Data points represent individual measurements. According to their Ca2+ sensitivities to activation, single hybrid channels could be divided into at least five groups (I–V). Group I (solid circles) and group II (solid triangles) displayed Ca2+ sensitivity to activation similar to that of the wt and E3987A mutant channel, respectively. Group III (solid squares), group IV (open circles), and group V (open squares) showed sensitivities to Ca2+ activation in between those of the wt and E3987A mutant channels. These different Ca2+ sensitivities most probably resulted from hybrid channels with different compositions of wt and E3987A mutant subunits. Note that two single channels are present in recordings shown in C and Po indicates the average open probability.
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Figure 7: Ca2+ responses of single channels produced by coexpression of the wt and E3987A mutant RyR2 proteins in HEK293 cells. HEK293 cells were transfected with an equal amount (6 μg) of wt and E3987A mutant RyR2 cDNA. The Ca2+ responses of single hybrid channels with high Po (A), medium Po (B), and low Po (C) were determined as described in the legend to Fig. 2. Single-channel activities shown in A and B were inhibited by addition of 0.1 mM EGTA to the cis chamber, indicating that the cytoplasmic side of the incorporated channel was facing the cis chamber. On the other hand, single-channel activities shown in C were inhibited by addition of 0.1 mM EGTA to the trans chamber, indicating that the cytoplasmic side of the incorporated channel was facing the trans chamber. To measure currents in the same direction, from the luminal to the cytoplasmic side of the channel, a +20-mV holding potential was applied in A and B and −20 mV in C. The Po-pCa relationships of single hybrid channels are shown in D. Data points represent individual measurements. According to their Ca2+ sensitivities to activation, single hybrid channels could be divided into at least five groups (I–V). Group I (solid circles) and group II (solid triangles) displayed Ca2+ sensitivity to activation similar to that of the wt and E3987A mutant channel, respectively. Group III (solid squares), group IV (open circles), and group V (open squares) showed sensitivities to Ca2+ activation in between those of the wt and E3987A mutant channels. These different Ca2+ sensitivities most probably resulted from hybrid channels with different compositions of wt and E3987A mutant subunits. Note that two single channels are present in recordings shown in C and Po indicates the average open probability.
Mentions: 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.

Bottom Line: Single point mutation of this conserved glutamate to alanine (E3987A) reduced markedly the sensitivity of the channel to activation by Ca(2)+, as measured by using single-channel recordings in planar lipid bilayers and by [(3)H]ryanodine binding assay.However, this mutation did not alter the affinity of [(3)H]ryanodine binding and the single-channel conductance.Coexpression of the wild-type and mutant E3987A RyR2 proteins in HEK293 cells produced individual single channels with intermediate sensitivities to activating Ca(2)+.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Research Group, University of Calgary, Calgary, Alberta, Canada T2N 4N1.

ABSTRACT
Activation of the cardiac ryanodine receptor (RyR2) by Ca(2)+ is an essential step in excitation-contraction coupling in heart muscle. However, little is known about the molecular basis of activation of RyR2 by Ca(2)+. In this study, we investigated the role in Ca(2)+ 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 Ca(2)+, as measured by using single-channel recordings in planar lipid bilayers and by [(3)H]ryanodine binding assay. However, this mutation did not alter the affinity of [(3)H]ryanodine binding and the single-channel conductance. In addition, the E3987A mutant channel was activated by caffeine and ATP, was inhibited by Mg(2)+, 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 Ca(2)+. These results are consistent with the view that glutamate 3987 is a major determinant of Ca(2)+ sensitivity to activation of the mouse RyR2 channel, and that Ca(2)+ 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.

Show MeSH