STRUCTURE AND FUNCTION OF SARCOPLASMIC RETICULUM

该计划的目的是阐明的机制的细节， 肌浆网（Ca ~（2+）+Mg ~（2+））ATP酶对Ca ~（2+）的摄取 (SR)研究了T-小管系统与SR引导之间的耦合， 快速释放Ca ~（2+）的能力，以及对 钙通道。 具体而言，a）回答一些悬而未决的问题 关于ATP酶反应的机制，将由 骤冷流、快速过滤和荧光探针的联合使用 技术. 将努力解决下列问题： 寡聚ATP酶的相邻亚基 在Ca 2+泵和Ca 2+释放反应期间相互作用。 Ca ~（2+）释放过程中酶构象可能发生的变化也将 追究 B）解决分子机制问题， 其中在T-小管膜中引起的去极化信号是 传递到SR导致快速Ca 2+释放，体外模型 在这个实验室建立的兴奋-收缩耦合将是 采用 具体来说，实验控制的T-小管 膜电位对SR中Ca 2+释放的影响，以及SR中的关键成分和关键 将研究T-小管/Sr偶联中涉及的反应步骤。 的 结构部件的拓扑结构将由 免疫组织化学定位在电子显微镜水平， 抑制偶联机制的抗体。 C. Ca 2+释放 SR的通道将通过以下几个方面进行识别和表征： 独立的方法：用通道进行共价放射-光亲和标记 阻断剂，其信号平行于 Ca 2+释放动力学和Ca 2+通道功能的重建 纯化的成分。

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.