Regulation of Membrane Excitability

项目摘要/摘要 骨骼肌兴奋-收缩(EC)偶联依赖于L的三联体连接的相互作用。 质膜上的钙通道(二氢吡啶受体，DHPR)和1型兰尼定 肌浆网(SR)的受体(1型RyRs，RyR1)。DHPR，对血浆的反应 膜去极化，激活RyR1从SR释放钙，这被认为是通过 两种蛋白质之间的构象偶联。这种耦合受到冻结断裂的有力支持 电子显微镜显示，DHPR排列成四组，与四个亚基相对 所有其他的RyR1。然而，尽管有丰富的功能、生化和结构证据，但有 对于连接DHPR和RyR1的蛋白质-蛋白质相互作用的同一性，几乎没有共识。这项建议 提出了四个具体目标，长期目标是确定这些互动的身份。目标1是 使用链霉亲和素和FRET的定点结合来确定DHPR的构象变化 对于EC联结来说是必不可少的。含有生物素受体结构域(BAD)的编码DHPR亚基的构建 和/或感兴趣部位的荧光蛋白将在肌管中表达后进行分析 内源性亚基。目标2是确定三维结构中特定位置的定位 DHPR的。将从转基因小鼠的肌肉中提纯含有生物素的DHPR，并对其进行 电子显微镜分析为单一的冷冻水化颗粒。目标3是定义特定的方向 DHPR的结构域与RyR的关系。将使用冷冻断裂法和薄层电子显微镜 以可视化结合到DHPR目标位置的金链霉亲和素的处置。目标4是识别交界处 通过利用蛋白质组学和代谢学来邻近DHPR中功能重要位置的蛋白质 生物素化作用。实现这些特定的目标应该揭示关于基本肌肉的基本新信息 功能，EC耦合。拟议的实验还将提供必要的知识，以了解 遗传性人类肌肉疾病低血钾性周期性麻痹(由1s-DHPR突变引起)和 恶性高热和中枢性核心病(由RyR1突变引起)。项目说明： 这项提议将描述两种蛋白质(DHPR和RyR1)之间的相互作用，这两种蛋白质是必不可少的 用于肌肉收缩。DHPR和RyR1的突变会导致遗传性的人类肌肉疾病。因此， 拟议的研究将对理解正常的肌肉功能和糖尿病的病理机制具有重要意义。 人类肌肉疾病。

Project Summary / Abstract Skeletal muscle excitation-contraction (EC) coupling depends upon interactions at triad junctions between L- type Ca2+ channels (dihydropyridine receptors, DHPRs) in the plasma membrane and type 1 ryanodine receptors (type 1 RyRs, RyR1) in the sarcoplasmic reticulum (SR). The DHPR, in response to plasma membrane depolarization, activates RyR1 to release Ca2+ from the SR, which is thought to occur via conformational coupling between the two proteins. Such coupling is strongly supported by freeze-fracture electron microscopy, which reveals that DHPRs are arranged into groups of four apposed to the four subunits of every other RyR1. However, despite a wealth of functional, biochemical and structural evidence, there is little consensus on the identity of the protein-protein interactions linking the DHPR and RyR1. This proposal advances four specific aims with the long-range goal of establishing the identity of these interactions. Aim 1 is to use site-directed binding of streptavidin and FRET to identify conformational changes of the DHPR that are essential for EC coupling. Constructs encoding DHPR subunits containing a biotin acceptor domain (BAD) and/or fluorescent proteins at sites of interest will be analyzed after expression in myotubes null for the endogenous subunit. Aim 2 is to identify the localization of specific sites within the three-dimensional structure of the DHPR. Biotin-containing DHPRs will be purified from muscle of transgenic mice and subjected to electron-microscopic analysis as single, frozen-hydrated particles. Aim 3 is to define the orientation of specific domains of the DHPR in relation to the RyR. Freeze-fracture and thin-section electron microscopy wiil be used to visualize the disposition of gold-streptavidin bound to targeted sites of DHPRs. Aim 4 is to identify junctional proteins that neighbor functionally important sites within the DHPR by exploiting both proteomics and metabolic biotinylation. Accomplishing these specific aims should reveal essential new information about a basic muscle function, EC coupling. The proposed experiments will also provide knowledge essential for understanding the inherited human muscle diseases hypokalemic periodic paralysis (caused by mutations of ¿1S-DHPR) and malignant hyperthermia and central core disease (caused by mutations of RyR1). PROJECT NARRATIVE: This proposal will characterize the interaction between two proteins (the "DHPR" and "RyR1") that are essential for muscle contraction. Mutations in the DHPR and RyR1 cause inherited, human muscle diseases. Thus, the proposed research will be important for understanding both normal muscle function and the pathology of human muscle diseases.