Regulation of Normal and Diseased Cardiac Ca2+ Channels

描述(申请人提供)：本项目的总体目标是继续研究人员在正常和疾病心肌中兴奋-收缩偶联的分子机制方面的工作，重点是兰尼定受体的作用。Ryanodine受体(RyR)的重要性体现在RyR的突变会导致各种心脏疾病。在先天性心脏病中，突变位于RyR四聚体每个亚单位的四个区域。调查人员发现，其中两个区域形成了域交换机。它们的拉链关闭钙通道，而拉链打开它；在休息状态下，不完全的结构域拉链会导致钙通道功能障碍，导致心力衰竭(心律失常和肥大)。研究人员最近的数据表明，同样类型的渠道监管也在其他两个地区运行，这表明它们参与了渠道门控机制，担任大门经理。似乎域交换机和门管理器是通过另一组域、发送器的中介来耦合的。因此，受体每个区域内的结构域间相互作用的缺陷将导致RyR连锁的心脏病。为了阐明这三个关键区域中每个相互作用结构域的空间关系，研究人员计划使用多肽作为位点方向载体对目标结构域进行定点荧光标记，并利用肽图和质谱学技术确定结构域-结构域相互作用的位置。为了确定这些结构域在通道调节中的作用，(I)将使用蛋白结合的荧光探针检测这些结构域中的结构域肽或位点特异性抗体引起的局部构象变化，以及(Ii)所产生的通道活性的激活将随后进行[~3H]ryanodine结合、单通道电导和细胞内钙瞬变的分析，以确定(I)和(Ii)之间的相关性。此外，与致力于心律失常和肥大模型系统的研究人员合作，调查者将研究体内RyR中有缺陷的结构域-结构域相互作用如何导致疾病状态，以及如何从药物上控制结构域相互作用和通道功能。来自该项目的新信息将使人们能够更好地了解一些心脏疾病中发生的钙通道功能障碍的发病机制，并将为治疗干预提供新的机会。兰尼定受体是一种钙通道蛋白，在心肌细胞钙水平的节拍调节中起着核心作用。通道功能缺陷会导致各种心脏疾病，如室性心动过速(心脏性猝死)和肥大。该计划旨在确定其缺陷导致通道功能障碍的分子机制(特别是区域间相互作用)，并开发治疗这些疾病的新方法。

DESCRIPTION (provided by applicant): The overall goal of this project is to continue the Investigator's work on the molecular mechanism of excitation- contraction coupling in normal and diseased cardiac muscle focusing on the role of the ryanodine receptor. The importance of the ryanodine receptor (RyR) is shown by the fact that mutations in the RyR lead to various cardiac disorders. In congenital cardiac diseases, mutations are localized in the four regions of each subunit of the RyR tetramer. The Investigator has shown that two of these regions form a Domain Switch. Their zipping closes the calcium channel, while unzipping opens it; incomplete domain zipping in the resting state causes calcium channel dysfunctions, resulting in heart failure (arrhythmias and hypertrophy). The investigator's recent data suggest that the same type of channel regulation is operating also within the other two regions, pointing to their involvement in the channel gating mechanism, serving as Gate Manager. It appears that the Domain Switch and the Gate Manager are coupled by mediation of yet another set of domains, Transmitter. Thus, defect of the inter-domain interaction within each of these regions of the receptor will cause RyR-linked cardiac diseases. To elucidate the spatial relationship of interacting domains in each of these three key regions, the Investigator plans to perform site-directed fluorescence labeling of the target domain using peptides as a site- direction carrier and identify the sites of domain-domain interaction using the peptide mapping and mass spectroscopic techniques. To establish the role of these domains in channel regulation, (i) a local conformational change induced by domain peptides or site-specific antibodies in these domains will be detected using the protein-bound fluorescence probe, and (ii) the resultant activation of the channel activity will be followed by assays of [3H]ryanodine binding, single channel conductance and intra-cellular Ca2+ transients, to determine correlation between (i) and (ii). Further, in collaboration with researchers working on model systems of cardiac arrhythmias and hypertrophy, the Investigator will examine how the defective domain- domain interaction in the in vivo RyR causes disease states, and how one can pharmacologically control the domain interaction and channel function. The new information derived from this program will permit a better understanding of the pathogenic mechanisms of calcium channel dysfunction occurring in some cardiac diseases and will offer new opportunities for therapeutic interventions. The ryanodine receptor is a calcium channel protein playing a central role in the beat to beat regulation of the cellular calcium level in cardiac muscle. Defective channel function causes various heart diseases, such as ventricular tachycardia (sudden cardiac death) and hypertrophy. This program is designed to identify molecular mechanisms (inter-domain interactions in particular), whose defect causes channel dysfunction, and to develop new methods of therapeutic treatment of these disorders.