Regulation of Normal and Diseased Cardiac Ca2+ Channels

描述(申请人提供)：总体目标是阐明正常和病变心肌兴奋-收缩偶联的分子机制。根据Ryanodine受体(RyR)突变在一些心脏病中的独特分布和初步研究，研究人员假设这些可变的结构域组成相互作用的结构域对，而拉开这些结构域对关闭钙通道，而解压打开该通道。结构域间相互作用的减弱将导致结构域解压缩的趋势增加，从而导致通道开放，从而导致心脏病的一般综合征(即胞浆钙升高和心脏损伤)。根据研究人员最近的研究，一组与RyR不同区域相对应的多肽(称为域肽)是识别和表征参与钙通道调节的关键结构域的有力工具。这样的结构域肽将被用来模拟在患病通道中发生的相同类型的通道超激活现象。突变的结构域多肽将作为阴性对照，以检验观察到的多肽超激活效应的生理学意义。通过定点荧光标记定位这些结构域肽结合的RyR区域将识别关键结构域对。通过使用已被结合到结构域对的关键位置的荧光探针，研究人员建议监测相互作用结构域的拉链/解压行为。然后，研究人员和他的合作者将在从单个分子(单通道测量)到整个细胞系统(有皮肤的心脏纤维)的水平上检查结构域肽对心脏通道的影响。此外，与一组研究心肌肥厚动物模型中的通道功能障碍的研究人员合作，研究人员将检查在这种疾病模型中结构域压缩/解压作用是如何改变的，以及人们如何从药物上控制改变的结构域和通道功能。这些新的信息将有助于更好地了解心脏钙通道调节的基本机制，以及某些心脏疾病中钙通道功能障碍的发病机制。该程序还将为心脏RyR通道病变的治疗方法提供新的线索。

DESCRIPTION (provided by applicant): The overall goal is to elucidate the molecular mechanism of excitation-contraction coupling in normal and diseased cardiac muscle. Based upon the unique distribution of the ryanodine receptor (RyR) mutations reported in some cardiac diseases and the pilot studies, the investigator postulates that these mutable domains constitute interacting domain pairs, and that zipping of such domain pairs closes the calcium channel while unzipping opens the channel. Weakening of the inter-domain interaction will cause an increased tendency of domain unzipping, and hence of channel-opening; this leads to the general syndrome of cardiac disease (i.e. increased cytoplasmic Ca 2+ and cardiac injury). According to the investigator's recent studies, a group of peptides corresponding to various regions of the RyR (designated as domain peptides) serves as a powerful tool to identify and characterize key domains involved in Ca 2+ channel regulation. Such domain peptides will be used to mimic the same type of channel hyper-activation phenomena that occur in diseased channels. Mutated domain peptides will be used as a negative control to test the physiologic significance of the observed hyper-activation effect of the peptides. Localizing the regions of the RyR to which these domain peptides bind by means of site-directed fluorescence labeling will identify the key domain pair. By using a fluorescence probe that has been incorporated into the critical site of the domain pair, the investigator proposes to monitor the zipping/unzipping action of the interacting domains. The investigator and his collaborators will then examine the effects of domain peptides on the cardiac channel at levels ranging from the single molecule (single channel measurements) to the whole cell system (skinned cardiac fibers). Further, in collaboration with a group of researchers working on channel dysfunction in a cardiac hypertrophy animal model, the investigator will examine how the domain zipping/unzipping action is altered in this disease model and how one can pharmacologically control the altered domain and channel functions. The new information derived from this program will permit a better understanding of the fundamental mechanism of cardiac calcium channel regulation as well as the pathogenic mechanism of Ca 2+ channel dysfunction occurring in some cardiac diseases. This program will also provide one with new clues for the method of treatments of diseased cardiac RyR channels.