STRUCTURE AND FUNCTION OF SARCOPLASMIC RETICULUM

描述：该项目的总体目标是解决分子 正常和正常状态下的兴奋-收缩-松弛耦合机制 diseased skeletal muscles. The whole reaction cycle of E-C-R coupling could 可以用按以下顺序发生的三个主要事件来描述： (I) 二氢吡啶受体之间的电压依赖性相互作用 (DHPR) and the junctional foot protein (JFP); (II) conformational changes in the JFP to open the SR Ca2 channel; and (III) release inactivation/recovery 涉及几种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 进行研究 用于研究问题的偶联分析系统（三联体）或纯化蛋白质 在分子水平上。 在目标 1 中，信号中蛋白质-蛋白质相互作用的详细模式 从 DHPR 到 JFP 的传输将得到解决。 一系列 源自 DHPR α1 亚基 II-III 细胞质环的肽 将筛选触发和 SR Ca2 释放（激活剂）的能力 并阻断（Blocker）激活肽在体外 E-C 中的作用 偶联测定系统和肽结合测定。 Appropriate peptides will 用于定位 JFP 中的信号接收站点。 第二个目标将探索 JFP 构象的变化，涉及 activation and inactivation of SR Ca2 release. 这将涉及到 去极化信号与 JFP 之间相关性的研究 构象变化以及激活肽 JFP 的结合 构象变化和 SR Ca2 释放以及时间关系 JFP构象变化和通道开放之间的关系。 确切位置 JFP 中构象探针的附着位点将是 进行调查以表征所涉及的实际结构变化。 本地 JFP 结构的变化和全球变化将通过荧光监测 分别是报告基团和圆二色性。 第三个目标将尝试阐明E-C-R的完整反应循环 耦合。 将努力解决个别反应中间体 by analyzing five parameters in a parallel. 其中包括： (i) JFP 构象变化； (ii) Ca2释放率； (iii) response to a second 激活刺激； (iv) Ca2 in the SR lumen; and (v) Ca2 re-uptake rate.

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.