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Regulation of Membrane Excitability

膜兴奋性的调节

基本信息

批准号：
8292044
负责人：
KURT G BEAM
金额：
$ 61.25万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-09-05 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8292044
关键词：
Affinity Behavior Binding Binding Sites Biochemical Biological Assay Biotin Biotinylation Calcium Cell membrane Central Core Myopathy Complex Consensus Coupling Cryoelectron Microscopy Data Dihydropyridine Receptors Electron Microscopy Electrons Engineering Fluorescence Resonance Energy Transfer Freeze Fracturing Freezing Goals Gold Human Human Pathology Hypokalemic periodic paralysis Imagery Inherited Knowledge Label Ligand Binding Link Malignant hyperpyrexia due to anesthesia Maps Mass Spectrum Analysis Measurement Membrane Metabolic Microscopic Microtomy Muscle Muscle Contraction Muscle Fibers Muscle function Mutation Myopathy Peptides Probability Proteins Proteomics Recombinants Regulation Research RyR1 Ryanodine Receptors Sarcoplasmic Reticulum Signal Transduction Site Skeletal Muscle Streptavidin Techniques Transgenic Mice Triad Acrylic Resin abstracting crosslink disease-causing mutation immunoreactivity insight interest novel particle protein protein interaction research study response spatial relationship three dimensional structure voltage

项目摘要

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.

项目总结/摘要骨骼肌兴奋-收缩（EC）偶联依赖于L- 型Ca 2+通道（二氢吡啶受体，DHPR）和1型兰尼碱受体（1型RyR，RyR 1）在肌浆网（SR）。DHPR对血浆的反应膜去极化，激活RyR 1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。这种耦合得到了冷冻断裂的有力支持电子显微镜，显示DHPR排列成四个与四个亚基并列的组每一个RyR 1然而，尽管有丰富的功能，生物化学和结构证据，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。这项建议提出了四个具体目标，长期目标是建立这些相互作用的身份。目标1是使用链霉亲和素和FRET的定点结合来鉴定DHPR的构象变化，对EC耦合至关重要。编码含有生物素受体结构域（BAD）的DHPR亚基的构建体和/或荧光蛋白在不表达荧光蛋白的肌管中表达后进行分析。内源亚基目的2是确定三维结构中特定位点的定位的DHPR。将从转基因小鼠的肌肉中纯化含有生物素的DHPR，并进行免疫处理。电子显微镜分析为单个冷冻水合颗粒。目标3是确定具体的与RyR相关的DHPR结构域。将使用冷冻断裂和薄片电子显微镜可视化结合至DHPR靶位点的金-链霉亲和素的处置。目的4是识别连接通过利用蛋白质组学和代谢，生物素化完成这些特定的目标应该揭示基本肌肉的基本新信息功能，EC耦合。拟议的实验还将提供了解遗传性人类肌肉疾病低钾性周期性麻痹（由1 S-DHPR突变引起），恶性高热和中央核心疾病（由RyR 1突变引起）。项目叙述：该提案将描述两种蛋白质（“DHPR”和“RyR 1”）之间的相互作用，用于肌肉收缩。DHPR和RyR 1的突变导致遗传性人类肌肉疾病。因此拟议的研究将是重要的了解正常的肌肉功能和病理，人类肌肉疾病