Structure and Function of Sarcoplasmic Reticulum

描述(由申请人提供)：本项目的总体目标是 解析兴奋(E)-收缩(C)松弛的分子机制 (R)正常肌肉和病变肌肉的偶联。骨骼肌型E-C-R 耦合似乎是在几个连续步骤中发生的。拟议中的实验 目的阐明上述各个步骤的作用机制。 (1)在表面膜去极化(肌细胞兴奋)时， 二氢吡啶受体II-III环的激活域与 其阻滞域与Ryanodine受体(RyR)/钙解离 释放通道蛋白；T小管极化逆转这些过程 (假设)。调查员将测试此模型(以及其他模型，如 嗯)通过检查与这些结构域(激活剂)相对应的多肽 和阻滞剂)在E-C偶联过程中与体内的对应物竞争 三合一和去皮或透气纤维。为了进一步定义该机制， 多肽的激活/抑制模式将与 多肽结合的模式。(2)这些II-III环结构域与 它们在RyR上的特定结合部位会引起局部构象变化 在信号接收区。调查员将对绑定进行本地化 这些环结构域的位置，并将监测动态构象 在E-C耦合期间信号接收区中发生的变化 特定部位的荧光探针。(3)信号的构象变化 接收区与RyR的全局构象变化相耦合，并且 钙的释放(收缩)。这个过程似乎涉及到一种 RyR内受监管的子域的数量。使用一种新型的多肽探针 技术，这位调查员已经发现了几个子域涉及到 RyR钙通道的调节。将努力发现一种 足够数量的子域来推断 分子内通信网络。(4)钙诱导后不久 释放(收缩)，激活钙ATPase以促进重新摄取 释放的钙(松弛)。调查员假设 RyR和钙ATPase之间的通讯是由 腔内钙离子发生一过性变化。这将通过以下方式进行测试 钙离子活性变化的时程相关性 ATPase与管腔内钙浓度的关系。这一计划将 可能解决了控制E-C耦合的各个步骤的基本机制， 这将有助于更好地了解 骨骼肌和心肌。

DESCRIPTION (provided by applicant): The overall goal of this project is to resolve the molecular mechanism of Excitation (E)-Contraction (C) Relaxation (R) coupling in normal and diseased muscles. Skeletal muscle-type E-C-R coupling appears to occur in several sequential steps. The proposed experiments aim to elucidate the mechanism for each of these steps. (1) Upon depolarization of the surface membrane (excitation of muscle cell), the activator domain of the dihydropyridine receptor II-III loop binds to, and its blocker domain dissociates from, the ryanodine receptor (RyR)/calcium release channel protein; T-tubule polarization reverses these processes (hypothesis). The investigator will test this model (and alternative models as well) by examining how the peptides corresponding to these domains (activator and blocker) compete with their in vivo counterparts during E-C coupling in triads and skinned or permeabilized fibers. To further define the mechanism, the pattern of peptide activation/inhibition will be correlated with the pattern of peptide binding. (2) The binding of these II-III loop domains to their specific binding sites on the RyR produces local conformational changes in the signal reception region. The investigator will localize the binding sites of these loop domains, and will monitor the dynamic conformational changes occurring in the signal reception region during E-C coupling using the site-specific fluorescence probe. (3) The conformational change in the signal reception region is coupled with a global conformational change in the RyR and calcium release (contraction). This process seems to involve interactions of a number of regulatory sub-domains within the RyR. Using a novel peptide probe technique, this investigator has uncovered several sub-domains involved in the regulation of the RyR calcium channel. Efforts will be made to uncover a sufficient number of sub-domains to deduce the global structure of the intra-molecular communication network. (4) Soon after the induction of calcium release (contraction), the calcium ATPase is activated to facilitate re-uptake of the released calcium (relaxation). The investigator hypothesizes that the communication between the RyR and the calcium ATPase is mediated by the transient changes occurring in the luminal calcium. This will be tested by correlating the time course of the changes in the activity of the calcium ATPase with those in the luminal calcium concentration. This program will likely resolve the basic mechanisms governing individual steps of E-C coupling, and will provide a better understanding of abnormal channel regulation in skeletal and cardiac muscles.