Structure and Function of Sarcoplasmic Reticulum

描述（由申请人提供）：本项目的总体目标是 解析激发（E）-收缩（C）-松弛的分子机制 (R)在正常和患病肌肉中的耦合。骨骼肌型E-C-R 偶联似乎以几个连续步骤发生。拟议的实验 旨在阐明这些步骤中的每一个的机制。 (1)在表面膜去极化（肌细胞的兴奋）时， 二氢吡啶受体II-III环的激活剂结构域结合， 其阻断剂结构域与兰尼碱受体（RyR）/钙离子解离 释放通道蛋白; T-小管极化逆转这些过程 （假设）。研究者将测试该模型（以及替代模型， 通过检查对应于这些结构域的肽（激活剂） 和阻断剂）在E-C偶联过程中与它们的体内对应物竞争， 三元组和剥皮或渗透纤维。为了进一步定义该机制， 肽激活/抑制的模式将与 肽结合的模式。(2)这些II-III环结构域与 它们在RyR上的特异性结合位点产生局部构象变化 在信号接收区域。研究者将定位绑定 这些环域的位点，并将监测动态构象 在使用该方法的E-C耦合期间在信号接收区域中发生的变化 位点特异性荧光探针。(3)信号中的构象变化 受体区域与RyR中的全局构象变化偶联， 钙释放（收缩）。这个过程似乎涉及到一个 RyR内的调节子域的数量。使用新型肽探针 技术，这位调查员已经发现了几个涉及的子域， RyR钙通道的调节。将努力揭露一个 足够数量的子域来推断全局结构， 分子内通讯网络(4)钙诱导后不久 释放（收缩）时，钙ATP酶被激活以促进再摄取 释放的钙（松弛）。研究人员假设， RyR和钙ATP酶之间的通讯是由 管腔钙发生短暂变化。这将由以下人员进行测试： 将钙离子活性变化的时间过程与 ATP酶与管腔内钙离子浓度有关。此程序将 可能解决了控制E-C耦合各个步骤的基本机制， 并将提供对异常通道调节的更好理解， 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.