Regulation of Membrane Excitability

描述(申请人提供)：骨骼肌兴奋-收缩(EC)耦合依赖于质膜上的L类钙通道(二氢吡啶受体，DHPR)和肌浆网(SR)中的1型Ryanodine受体(类型1 RyRs，RyR1)之间的三联体相互作用。DHPR响应质膜去极化，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耦合发生的。这种偶联得到了冷冻断裂电子显微镜的有力支持，它表明DHPR被排列成四个基团，而不是每隔一个RyR1的四个亚基。然而，尽管有丰富的功能、生化和结构证据，但对于连接DHPR和RyR1的蛋白质-蛋白质相互作用的同一性，人们几乎没有达成共识。这个应用程序提出了四个具体目标，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，这些变化是EC偶联所必需的。编码含有生物素受体结构域(BAD)和/或在感兴趣部位的荧光蛋白的DHPR亚基的构建将在肌管中表达内源性亚基后进行分析。目标2是确定DHPR三维结构中特定位置的定位。含有生物素的DHPR将从转基因小鼠的肌肉中纯化出来，并作为单个冷冻水合颗粒进行电子显微镜分析。目标3是定义DHPR的特定结构域相对于RyR的方向。冷冻断口和薄片电子显微镜将用于显示结合到DHPR目标位置的金-链霉亲和素的处置情况。目标4是通过利用蛋白质组学和代谢生物素化来识别与DHPR中功能重要位置相邻的连接蛋白。实现这些特定的目标应该会揭示有关一种基本肌肉功能--EC偶联的基本新信息。拟议的实验还将为理解遗传性人类肌肉疾病、低血钾性周期性瘫痪(由11S-DHPR突变引起)、恶性高热和中枢核心疾病(由RyR1突变引起)提供必要的知识。公共卫生相关性本应用程序将描述肌肉收缩所必需的两种蛋白质(“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，这项拟议的研究对于理解正常的肌肉功能和人类肌肉疾病的病理都将是重要的。

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.