Regulation of Normal and Diseased Cardiac Ca2+ Channels

描述（由申请方提供）：本项目的总体目标是继续研究者对正常和患病心肌中兴奋-收缩偶联分子机制的研究，重点是兰尼碱受体的作用。RyR的突变导致各种心脏疾病，这一事实表明RyR的重要性。在先天性心脏病中，突变位于RyR四聚体的每个亚基的四个区域中。研究人员已经证明，这些区域中的两个形成了域转换。它们的压缩关闭钙通道，而解压缩打开它;在静息状态下不完全的域压缩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，同样类型的通道调节也在其他两个区域内运行，指出它们参与通道门控机制，充当门控管理器。看来域交换机和门管理器是通过另一组域（发送器）的中介耦合的。因此，受体的这些区域中的每一个内的结构域间相互作用的缺陷将导致RyR相关的心脏疾病。为了阐明这三个关键区域中相互作用结构域的空间关系，研究者计划使用肽作为位点定向载体对靶结构域进行位点定向荧光标记，并使用肽图谱和质谱技术鉴定结构域相互作用的位点。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由这些结构域中的结构域肽或位点特异性抗体诱导的局部构象变化，和（ii）将通过[3 H]兰尼定结合、单通道电导和细胞内Ca 2+瞬变的测定来跟踪所得到的通道活性的激活，以确定（i）和（ii）之间的相关性。此外，与研究心律失常和肥大模型系统的研究人员合作，研究人员将研究体内RyR中有缺陷的结构域-结构域相互作用如何导致疾病状态，以及如何能够控制结构域相互作用和通道功能。从该计划中获得的新信息将使人们更好地了解某些心脏疾病中钙通道功能障碍的致病机制，并为治疗干预提供新的机会。兰尼碱受体是一种钙通道蛋白，在心肌细胞钙水平的搏动调节中发挥核心作用。缺陷的通道功能会导致各种心脏疾病，如室性心动过速（心源性猝死）和肥大。该计划旨在确定分子机制（特别是结构域间相互作用），其缺陷导致通道功能障碍，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.