CALCIUM RELEASE UNITS IN CARDIAC MUSCLE

描述(改编自申请者的描述)：总体目标 这项建议是为了阐明组织结构的基础 心肌和骨骼肌钙释放单位(CRU) 兴奋-收缩耦合。CRU由连接的肌浆组成 网状结构(JSR)由一个腔钙结合蛋白组成， 钙调蛋白(CSQ)和几种膜蛋白，包括 钙释放通道(Ryanodine受体；RyRs)和支持蛋白 连接素和三聚氰胺。邻近的肌膜区域包括L型钙 通道(二氢吡啶受体；DHPR)。若要确定 组装、组织和维护这些单位的结构完整性， 结构将通过电子显微镜进行评估，使用薄片和 冷冻破碎法，蛋白质组成将用 免疫荧光结合共聚焦显微镜和Western blotting。 将使用三种策略来评估改变蛋白质的效果 在CRU结构上的组成。1)过度表达One的转基因小鼠 或者更多的CRU蛋白将被研究。2)肌肉特异性CRU蛋白 将在非肌肉细胞中表达，以确定蛋白质 是形成CRU所必需的。3)骨骼肌中CRU的组成 将利用分子生物学和超微结构对文昌鱼进行研究 接近了。

DESCRIPTION (adapted from the applicant's description): The overall goal of this proposal is to elucidate the basis for structural organization of the calcium release units (CRUs) of cardiac and skeletal muscle which underlie excitation-contraction coupling. CRUs are comprised of junctional sarcoplasmic reticulum (jSR) which are composed of a luminal calcium-binding protein, calsequestrin (CSQ), and several membrane proteins which include the calcium-release channel (ryanodine receptor; RyRs), and supporting proteins junctin and triadin. The adjacent areas of sarcolemma include L-type calcium channels (dihydropyridine receptors; DHPRs). To determine the basis for the assembly, organization, and maintenance of structural integrity of these units, structure will be assessed by electron microscopy, using thin sections and freeze fracture methods, and protein composition will be assessed using immunofluorescence coupled to confocal microscopy and by Western blotting. Three strategies will be used to assess the effect of altered protein composition on the structure of CRUs. 1) Transgenic mice which overexpress one or more of the CRU proteins will be studied. 2) Muscle specific CRU proteins will be expressed in non-muscle cells in order to determine the proteins necessary for formation of CRUs. 3) The composition of CRUs in skeletal muscle of Amphioxus will be examined using molecular biology and ultrastructural approaches.