Cav1.2钙通道的镁离子依赖性易化与失活作用的新机制研究

项目名称： Cav1.2钙通道的镁离子依赖性易化与失活作用的新机制研究

项目编号： No.31500930

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 生物科学

项目作者： 赵美眯

作者单位： 中国医科大学

项目金额： 20万元

中文摘要： 已知细胞膜电压门控钙通道受钙离子的正负反馈调节，即钙依赖性易化与失活（CDF&CDI）。申请人最近的研究发现，另一二价阳离子镁离子也可双向调节钙通道活性，并提出了镁依赖性易化与失活（MDF&MDI）的新概念。研究认为CDF&CDI是由钙调蛋白（CaM）介导的，并且只有处于被钙调蛋白激酶II（CaMKII）磷酸化的钙通道才会表现出该反应。CaM上同时有钙和镁的结合位点，前期研究结果显示MDF&MDI作用与其调节钙通道对钙的敏感性相关，并提出了以CaMKII通道磷酸化为基础，由镁与CaM结合介导，以镁与钙相互作用为主要表现的分子机制模型。本项目拟采用分子生物学及膜片钳技术观察MDF&MDI作用与钙浓度、CaM与钙通道结合状态及钙通道CaMKII磷酸化水平的关系，明确MDF&MDI的分子机制；并制备心肌缺血再灌注损伤模型，阐明MDF&MDI作用的病理生理意义，为相关心血管疾病的防治提供新思路。

中文关键词： 离子通道；镁离子；钙通道；膜片钳；心肌细胞

英文摘要： By controlling cellular Ca2+ entry, the voltage-gated Ca2+ channels (Cavs) of cell membrane play essential roles in many cellular functions. The openings of Cavs are regulated by Ca2+, including both a positive mechanism and a negative feedback mechanism: Ca2+-dependent facilitation (CDF) and inactivation (CDI). Mg2+ is another abundant divalent cation in the internal milieu of cells. During the investigation of the molecular mechanisms of CDF and CDI, we surprisingly found that Mg2+ is also an important regulator for the maintenance of the basal activity of the cardiac L-type Ca2+ (Cav1.2) channel. Thus, we then further investigated the Mg2+-induced modulation of the Cav1.2 channel over a wide range of Mg2+ concentration (0 - 100 mM), and we have found that Mg2+ has dual effects on the Cav1.2 channel: lower concentration of Mg2+ maintains and then enhances the basal activity of Ca2+ channel, whereas higher concentration of Mg2+ inhibits the channel activity. Based on our previous findings, we have proposed to name this phenomenon as Mg2+-dependent facilitation and inactivation (MDF & MDI). It is believed that calmodulin (CaM), which serves as a resident Ca2+ sensor, mediates the dual regulations of Cav1.2 channel induced by Ca2+ via its binding to the channel. A series of our previous studies have also shown that CaM, which mediates CDF & CDI, may recover the channel activity from its run-down. And we also reported the CaM’s effect on run-down Ca2+ channels requires CaM-dependent protein kinase II (CaMKII)-mediated phosphorylation of the channels, and both CaM and CaMKII are needed to recover the channel activity. Based on these findings, we proposed a new model in which Mg2+ regulates the Cav1.2 channel by adjusting its sensitivity to Ca2+ through the modulation of CaM binding and CaMKII-mediated phosphorylation of the channel. By using molecular biological techniques, we will conduct Mg2+/Ca2+ binding site mutants of CaM and CaMKII phosphorylation site mutants of Cav1.2 channel. Then we will investigate the relationships of the Mg2+ concentrations to the binding of Ca2+ to CaM, the binding of CaM to the Cav1.2 channel, and the CaMKII-mediated phosphorylation level of the channel by patch clamp experiments in guinea-pig cardiac myocytes and Cav1.2 channel and/or CaMs-transfected HEK cells and protein binding assays including pull-down assay and co-immunoprecipitation. We will also observe the changes of MDF & MDI in pathophysiological conditions by using heart ischemia-reperfusion injury model. By performing the above experiments, the project is to verify the hypothesis that Mg2+ has dual effects on the Cav1.2 channels, which modulate the Ca2+ sensitivity, the CaM binding, and phosphoralston level of the channel, and is of great significance in physiological and pathophysiological conditions. This project is also of significance for sparking new thinking and new agents for the prevention and/or treatment of some related myocardial diseases.

英文关键词： Ion channel;Magnesium ion;Calcium channel;Patch clamp;Cardiac myocytes