STRUCTURE AND FUNCTION OF SARCOPLASMIC RETICULUM

这项计划的目的是阐明机制的细节 肌浆网中(钙镁)ATPase对钙的摄取 (Sr)，T小管系统与sR导联的耦合研究 对钙的快速释放，以及细胞的鉴定和特性 CA2通道。具体地说，a)回答一些未解决的问题 关于ATPase反应的机制，将由 急流、快速过滤和荧光探头的联合使用 技巧。努力的目的是解决以下问题 寡聚体ATPase相邻亚基的作用机制 在钙泵和钙释放反应中相互作用。 在钙释放过程中可能发生的酶构象变化也将 被调查。B)通过以下方式解决分子机制问题 在T小管膜中诱发的去极化信号是 传递到SR导致快速的钙释放，体外模型 在本实验室建立的兴奋-收缩耦合将是 使用。具体地说，实验控制的T管的效果 肌质网钙释放的膜电位及其关键成分和关键 将研究T-微管/锶偶联的反应步骤。这个 结构组件的拓扑将由以下因素确定 免疫组织化学在电子显微镜水平上的定位 抑制偶联机制的抗体。C)钙的释放 SR的渠道将通过以下几种方式进行识别和表征 独立方法：共价放射-光亲和标记通道 阻滞剂，信号平行的构象探针的定位 钙释放动力学与钙通道功能的重建 从提纯的成分中提取。

The aims of this program are the elucidation of details of the mechanism of the Ca2+ uptake by the (Ca2++Mg2+) ATPase in the sarcoplasmic reticulum (SR), the study of the coupling between the T-tubule system and SR leading to rapid Ca2+ release, and the identification and chaacteriztion of the Ca2+ channel. Specifically, a) answers to a number of unsettled quetions concerning the mechanism of the ATPase reaction will be sought by the combined use of quench flow, rapid filtration and fluorescence probe techniques. Efforts will be aimed at the solution of questions concerning the mechanism by which neighboring subunits of the oligomeric ATPase interact with each other during the Ca2+ pump and Ca2+ release reactions. The possible changes of enzyme conformation during Ca2+ release will also be investigated. b) To solve the problem of the molecular mechanism by which the depolarization signal elicited in the T-tubule membrane is transmitted to the SR leading to rapid Ca2+ release, the in vitro model of excitation-contraction coupling established in this laboratory will be used. Specifically, the effects of experimentally controlled T-tubule membrane potential on Ca2+ release from SR, and key components and key reaction steps involved in T-tubule/Sr coupling will be investigated. The topology of the structural components will be determined by immuno-histochemical localization at the electron microscopic level of antibodies that inhibit the coupling mechanism. c) The Ca2+ release channel of the SR will be identified and characterized through several independent approaches: covalent radio-photoaffinity labeling with channel blockers, localization of the conformational probe whose signal parallels Ca2+ release kinetics, and reconstitution of the Ca2+ channel functions from purified components.