Regulation of Membrane Excitability

描述（由申请人提供）：骨骼肌兴奋-收缩（EC）耦合依赖于质膜中L-型Ca2+通道（二氢吡啶受体，dhpr）和肌浆网（SR）中1型ryanodine受体（1型RyRs， RyR1）之间的三联结的相互作用。DHPR响应质膜去极化，激活RyR1从SR释放Ca2+，这被认为是通过两种蛋白之间的构象偶联发生的。这种耦合得到了冷冻断裂电镜的强烈支持，这表明dhpr与其他RyR1的4个亚基相对排列成4组。然而，尽管有大量的功能、生化和结构证据，但对于连接DHPR和RyR1的蛋白-蛋白相互作用的身份却几乎没有共识。该应用程序推进了四个具体目标，其长期目标是建立这些交互的身份。目的1是利用链霉亲和素和FRET的定点结合来确定DHPR的构象变化，这是EC偶联所必需的。编码DHPR亚基的构建体含有生物素受体结构域（BAD）和/或在感兴趣的位点上的荧光蛋白，在肌管中表达后分析内源性亚基。目的2是确定DHPR三维结构中特定位点的定位。含生物素的dhpr将从转基因小鼠的肌肉中纯化，并作为单一的冷冻水合颗粒进行电子显微镜分析。目的3是确定DHPR与RyR相关的特定结构域的取向。冷冻断裂和薄层电子显微镜将用于可视化金链霉亲和素结合到dhpr的目标位点的处置。目的4是通过利用蛋白质组学和代谢生物素化来鉴定DHPR中功能重要位点附近的连接蛋白。完成这些特定的目标应该揭示一个基本的肌肉功能，EC耦合的重要的新信息。本实验还将为了解遗传性人类肌肉疾病低钾血症性周期性麻痹（由11S-DHPR突变引起）和恶性高热症和中央核心疾病（由RyR1突变引起）提供必要的知识。本应用将描述两种对肌肉收缩至关重要的蛋白（“DHPR”和“RyR1”）之间的相互作用。DHPR和RyR1的突变会导致遗传性的人类肌肉疾病。因此，所提出的研究对于理解正常肌肉功能和人类肌肉疾病的病理都是重要的。

DESCRIPTION (provided by applicant): 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 application 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 will 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 11S-DHPR) and malignant hyperthermia and central core disease (caused by mutations of RyR1). PUBLIC HEALTH RELEVANCE This application 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.