与心源性猝死相关的钙离子释放通道结构及功能紊乱的分子机理研究

Sudden cardiac death is the leading cause of natural mortality in the world, the proportion of these deaths has been estimated to be 30-40% in cardiovascular disease mortality. In a recent study using four regional populations in China, the estimated Chinese annual incidence of sudden cardiac death is over 500,000. Evidence indicates that abnormal calcium release through dysfunctional cardiac muscle ryanodine receptors is directly involved in two inherited forms of sudden cardiac death. Ryanodine receptor functions as an intracellular calcium release channel in the sarcoplasmic reticulum membrane, and it plays a crucial role in muscle excitation-contraction coupling. A detailed knowledge of its three-dimensional structure and conformational dynamics is essential to develop effective treatment of ryanodine receptor-related heart disease. Our central hypothesis is that abnormal interactions between ryanodine receptor's structural domains, and abnormal conformational changes underlie dysfunction of the calcium release channel. To test our working hypothesis, the following specific aims will be pursued: Mapping the disease causing mutations in the three-dimensional structure of ryanodine receptor; Design FRET pairs, base on the structural information to characterize conformational dynamics in health and disease receptors; Identify drugs that are capable of restore the normal conformation in mutant ryanodine receptor. At the completion of these studies, we expect to have a better understanding of the mechanisms underlying ryanodine receptor channel dysfunction. The information will help us to comprehend the role of ryanodine receptor in excitation-contraction coupling mechanisms, and how ryanodine receptor dysfunction leads to cardiac muscle diseases, which in turn should allow more rational design of novel therapeutic strategies.

研究发现部分心源性猝死与人类RyR2基因突变相关。RyR2基因编码的2型ryanodine受体是心肌胞浆肌质网膜上钙离子释放通道，并在心肌的兴奋收缩偶联中发挥重要作用。但RyR2基因突变对其编码受体的结构以及功能的影响尚不清楚。我们在先前对RyR2上重要序列的定位及重要结构域功能研究的基础上，认为某些结构域上氨基酸的突变会影响ryanodine受体的正常结构和构象变化，引起受体功能上的紊乱，进而诱发心源性猝死。本研究课题将致力于测定ryanodine受体高分辨结构，定位某些突变残基的位点；构建分子探针，用来研究该受体的蛋白结构域与钙通道开启和关闭相关的构象变化；研究氨基酸突变所引起的构象变化异常，并检测某些药物对此类异常可能的修复作用。本研究将探讨钙离子释放通道结构及功能紊乱在心源性猝死中的分子机制，并为抗心源性猝死药物的筛选提供理论依据。

心源性猝死是自然死亡的最常见原因。研究发现部分心源性猝死与人类RYR2基因突变相关。RYR2基因突变对其编码钙通道的结构以及功能的影响尚不清楚。本项研究重点在探讨突变是如何造成通道蛋白结构与构象变化的紊乱，导致心律失常与心源性猝死的分子机制。在基金资助下，主要的研究内容、重要结果、关键数据和科学意义总结如下：(1). 筛查出了一例新发的与心源性猝死和儿茶酚胺敏感性多形性室性心动过速相关的RYR2基因突变；构建了基因工程小鼠，在模式动物层面上验证了该突变为致病基因突变；在小鼠心肌细胞中检测了因RYR2基因突变而导致其编码钙离子释放通道蛋白的功能异常；应用冷冻电镜高分辨结构分析，在三维结构中对突变位点进行定位，揭示了突变导致钙通道开闭的构象变化异常，进而引起蛋白功能异常的分子机制。从根本上揭示了突变引发室速和造成心源性猝死的致病机制。(2). 定位若干突变氨基酸的位点，用分子动态模拟技术来研究钙通道的蛋白结构域与钙通道开启和关闭相关的构象变化; 揭示了一组于心源性猝死和儿茶酚胺敏感性多形性室性心动过速相关的RYR2基因突变共同的致病机制。(3). 对临床上心律失常病人进行基因检测，在一例有心源性猝死家族史的家系中鉴定出另一个全新的RYR2基因突变。(4). 探索了与钾通道Kir2.1 and K2P1相关的心肌细胞双重膜电位研究，揭示了钾通道在低血钾症中的分子机制。在项目执行的四年中，申请立项的研究内容均按计划实施，研究目标已全部完成。发表SCI论文3篇，培养博士研究生4人，硕士研究生2人。课题组成员获得国家自然科学基金面上项目1项、青年项目2项。