Li P, Chen S R
Cardiovascular Research Group, University of Calgary, Calgary, Alberta, Canada T2N 4N1.
J Gen Physiol. 2001 Jul;118(1):33-44. doi: 10.1085/jgp.118.1.33.
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.
钙离子激活心肌兰尼碱受体（RyR2）是心肌兴奋 - 收缩偶联的关键步骤。然而，关于钙离子激活RyR2的分子机制却知之甚少。在本研究中，我们探究了位于小鼠RyR2预测跨膜片段M2中的保守谷氨酸3987在钙离子感知中的作用。将该保守谷氨酸单点突变为丙氨酸（E3987A），通过平面脂质双分子层单通道记录和[³H]兰尼碱结合试验测定，显著降低了通道对钙离子激活的敏感性。然而，该突变并未改变[³H]兰尼碱结合亲和力和单通道电导。此外，E3987A突变通道可被咖啡因和ATP激活，被镁离子抑制，且被兰尼碱修饰的方式与野生型通道相似。野生型和突变型E3987A RyR2蛋白在HEK293细胞中共表达，产生了对激活钙离子具有中等敏感性的单个单通道。这些结果与以下观点一致：谷氨酸3987是小鼠RyR2通道对钙离子激活敏感性的主要决定因素，且RyR2的钙离子感知涉及兰尼碱受体单体之间的协同作用。本研究结果还为小鼠RyR2的结构和功能特性提供了初步见解，这对于研究转基因小鼠模型中RyR2的功能和调控应具有重要意义。
