STRUCTURE AND FUNCTION OF SARCOPLASMIC RETICULUM

描述：这个项目的总体目标是解决分子 正常人和正常人的兴奋-收缩-松弛耦合机制 有病的骨骼肌。E-C-R偶联的整个反应周期可以 按以下顺序发生的三件大事描述：(一) 二氢吡啶受体之间的电压依赖性相互作用 (DHPR)和连接足蛋白(JFP)； JFP打开SR钙通道；以及(Iii)释放失活/恢复 涉及几个假定的反应中间体和调节剂的过程。 这些重大事件中的每一个都将利用体外E-C 用于研究问题的偶联分析系统(TRIAD)或纯化的蛋白质 在分子水平上。 目标1信号中蛋白质-蛋白质相互作用的详细模式 DHPR向JFP的传播问题将得到解决。一系列 来源于DHPRα1亚基II-III胞质环的多肽 将对触发和SR钙释放(激活剂)的能力进行筛选 阻断激活肽在体外培养的E-C细胞中的作用 联用分析系统和多肽结合分析。适当的多肽将 用于定位JFP中的信号接收点(S)。 第二个目标将探索JFP构象的变化，涉及 肌质网钙释放的激活和失活。这将涉及到 退偏信号与JFP相关性的研究 构象变化以及激活肽与JFP的结合 构象变化与肌质网钙释放及其时间关系 JFP构象变化与通道开放之间的关系。准确的位置 构象探针在JFP中的连接位置将是 进行了调查，以描述所涉及的实际结构变化。本地 将通过荧光监测JFP结构的变化和全球变化 记者团体和圆二色性分别。 第三个目的将试图阐明E-C-R的完整反应循环 耦合。将努力解决个别反应中间体 通过平行分析五个参数。这些措施包括：(1)联合方案 构象变化；(Ii)钙释放速率；(Iii)对第二次反应 激活刺激；(Iv)SR管腔内的Ca~(2+)；和(V)Ca~(2+)重摄取率。

DESCRIPTION: The overall goal of this project is to resolve the molecular mechanism of excitation-contraction-relaxation coupling in normal and diseased skeletal muscles. The whole reaction cycle of E-C-R coupling could be described by three major events occurring in the following sequence: (I) the voltage-dependent interaction between the dihydropyridine receptor (DHPR) and the junctional foot protein (JFP); (II) conformational changes in the JFP to open the SR Ca2+ channel; and (III) release inactivation/recovery processes involving several putative reaction intermediates and modulators. Each of these major events will be investigated utilizing the in vitro E-C coupling assay system (triad) or purified proteins for studying the problems at the molecular level. In Aim 1 the detailed modes of protein-protein interactions in the signal transmission from the DHPR to the JFP will be resolved. A series of peptides derived from the II-III cytoplasmic loop of the DHPR alpha1 subunit will be screened for the ability to trigger and SR Ca2+ release (Activator) and to block (Blocker) the actions of Activator peptides in the in vitro E-C coupling assay system and peptide binding assays. Appropriate peptides will be used to localize the signal-receiving site(s) in the JFP. The second aim will explore changes in JFP conformation involved in activation and inactivation of SR Ca2+ release. This will involve investigations of the correlations between the depolarization signal and JFP conformational change, and between the binding of Activator peptide, JFP conformational change and SR Ca2+ release, and of the temporal relation between JFP conformational change and channel opening. The exact location of the site of attachment of conformational probes in the JFP will be investigated to characterize the actual structural changes involved. Local changes and global changes in JFP structure will be monitored by fluorescent reporter groups and circular dichroism, respectively. The third aim will attempt to elucidate the complete reaction cycle of E-C-R coupling. Efforts will be made to resolve individual reaction intermediates by analyzing five parameters in a parallel. These include: (i) JFP conformational changes; (ii) Ca2+ release rate; (iii) response to a second activation stimulus; (iv) Ca2+ in the SR lumen; and (v) Ca2+ re-uptake rate.