Regulation of Membrane Excitability

描述（由申请人提供）：骨骼肌兴奋-收缩（EC）偶联依赖于质膜中L型Ca 2+通道（二氢吡啶受体，DHPR）和肌浆网（SR）中1型兰尼碱受体（1型RyR，RyR 1）之间三联体连接处的相互作用。响应于质膜去极化，DHPR激活RyR 1以从SR释放Ca 2+，这被认为是通过两种蛋白质之间的构象偶联发生的。冷冻断裂电子显微镜强烈支持这种耦合，它揭示了DHPR被排列成四个与每隔一个RyR 1的四个亚基并列的组。然而，尽管有丰富的功能，生物化学和结构证据，但在连接DHPR和RyR 1的蛋白质-蛋白质相互作用的身份上几乎没有共识。本申请提出了四个具体目标，其长期目标是建立这些相互作用的身份。目的1是使用链霉亲和素和FRET的定点结合来鉴定EC偶联所必需的DHPR的构象变化。编码在感兴趣的位点含有生物素受体结构域（BAD）和/或荧光蛋白的DHPR亚基的构建体将在内源性亚基无效的肌管中表达后进行分析。目的2是确定DHPR三维结构内特定位点的定位。将从转基因小鼠的肌肉中纯化含生物素的DHPR，并作为单个冷冻水合颗粒进行电子显微镜分析。目的3是确定DHPR的特定结构域相对于RyR的方向。将使用冷冻断裂和薄片电子显微镜观察结合至DHPR靶位点的金-链霉亲和素的分布。目的4是通过利用蛋白质组学和代谢生物素化来识别DHPR内邻近功能重要位点的连接蛋白。实现这些特定的目标应该揭示一个基本的肌肉功能，EC耦合的重要新信息。拟议的实验还将为理解遗传性人类肌肉疾病低钾性周期性麻痹（由11 S-DHPR突变引起）和恶性高热和中央核心疾病（由RyR 1突变引起）提供必要的知识。公共卫生相关性本申请将描述肌肉收缩所必需的两种蛋白质（“DHPR”和“RyR 1”）之间的相互作用。DHPR和RyR 1的突变导致遗传性人类肌肉疾病。因此，拟议的研究对于了解正常肌肉功能和人类肌肉疾病的病理学都很重要。

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.