Limits...
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.

Show MeSH
Mutation E3987A does not alter the affinity of [3H]ryanodine binding to RyR2. [3H]ryanodine binding to cell lysate prepared from HEK293 cells transfected with wt or mutant E3987A cDNA was performed in the presence of 25 mM Tris, 50 mM HEPES, pH 7.4, 500 mM KCl, CaCl2 (0.2 mM for wt and 1.0 mM for mutant E3987A), 0.1–30 nM [3H]ryanodine, and various protease inhibitors at 37°C for 2 h. Data shown are from a representative experiment, which has been repeated three to four times. Inset shows a Scatchard plot.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2233748&req=5

Figure 4: Mutation E3987A does not alter the affinity of [3H]ryanodine binding to RyR2. [3H]ryanodine binding to cell lysate prepared from HEK293 cells transfected with wt or mutant E3987A cDNA was performed in the presence of 25 mM Tris, 50 mM HEPES, pH 7.4, 500 mM KCl, CaCl2 (0.2 mM for wt and 1.0 mM for mutant E3987A), 0.1–30 nM [3H]ryanodine, and various protease inhibitors at 37°C for 2 h. Data shown are from a representative experiment, which has been repeated three to four times. Inset shows a Scatchard plot.

Mentions: 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 (Du and MacLennan 1998). The characteristic functional response to ryanodine of single E3987A mutant RyR2 channel shown in Fig. 3 indicates that the functional high affinity ryanodine binding site is retained in the mutant channel. However, it is possible that the E3987A mutation could affect the properties of ryanodine binding in addition to Ca2+ activation. To test this possibility, we determined the equilibrium dissociation constant of [3H]ryanodine binding to mutant E3987A in the presence of 1 mM Ca2+ (Fig. 4). Scatchard analysis showed that the E3987A mutant RyR2 exhibited high affinity [3H]ryanodine binding with a Kd of 2.1 ± 0.39 nM and a Bmax of 0.76 ± 0.15 pmol/mg (n = 5), similar to the Kd of 2.3 ± 0.63 nM and Bmax of 1.24 ± 0.26 pmol/mg (n = 4) of the wt RyR2. High affinity [3H]ryanodine binding to the corresponding mutant E3885A in RyR3 was also detected (data not shown). Therefore, mutation of this conserved glutamate does not alter the affinity of [3H]ryanodine binding to RyR2.


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

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

Mutation E3987A does not alter the affinity of [3H]ryanodine binding to RyR2. [3H]ryanodine binding to cell lysate prepared from HEK293 cells transfected with wt or mutant E3987A cDNA was performed in the presence of 25 mM Tris, 50 mM HEPES, pH 7.4, 500 mM KCl, CaCl2 (0.2 mM for wt and 1.0 mM for mutant E3987A), 0.1–30 nM [3H]ryanodine, and various protease inhibitors at 37°C for 2 h. Data shown are from a representative experiment, which has been repeated three to four times. Inset shows a Scatchard plot.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233748&req=5

Figure 4: Mutation E3987A does not alter the affinity of [3H]ryanodine binding to RyR2. [3H]ryanodine binding to cell lysate prepared from HEK293 cells transfected with wt or mutant E3987A cDNA was performed in the presence of 25 mM Tris, 50 mM HEPES, pH 7.4, 500 mM KCl, CaCl2 (0.2 mM for wt and 1.0 mM for mutant E3987A), 0.1–30 nM [3H]ryanodine, and various protease inhibitors at 37°C for 2 h. Data shown are from a representative experiment, which has been repeated three to four times. Inset shows a Scatchard plot.
Mentions: 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 (Du and MacLennan 1998). The characteristic functional response to ryanodine of single E3987A mutant RyR2 channel shown in Fig. 3 indicates that the functional high affinity ryanodine binding site is retained in the mutant channel. However, it is possible that the E3987A mutation could affect the properties of ryanodine binding in addition to Ca2+ activation. To test this possibility, we determined the equilibrium dissociation constant of [3H]ryanodine binding to mutant E3987A in the presence of 1 mM Ca2+ (Fig. 4). Scatchard analysis showed that the E3987A mutant RyR2 exhibited high affinity [3H]ryanodine binding with a Kd of 2.1 ± 0.39 nM and a Bmax of 0.76 ± 0.15 pmol/mg (n = 5), similar to the Kd of 2.3 ± 0.63 nM and Bmax of 1.24 ± 0.26 pmol/mg (n = 4) of the wt RyR2. High affinity [3H]ryanodine binding to the corresponding mutant E3885A in RyR3 was also detected (data not shown). Therefore, mutation of this conserved glutamate does not alter the affinity of [3H]ryanodine binding to RyR2.

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