Structure of Channels in Excitation-Contracting Coupling

励磁收缩耦合通道的结构

• 批准号: 7656625
• 负责人: TERENCE C WAGENKNECHT
• 金额: $ 41.47万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-10 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7656625
• 关键词: Amino Acids; Binding; Binding Sites; Calcium Channel; Calcium ion; Calmodulin; Calmodulin 1; Cell membrane; Central Core Myopathy; Communication; Complex; Contracts; Coupling; Cryoelectron Microscopy; Cultured Cells; Dihydropyridine Receptors; Disease; Generations; Grant; Green Fluorescent Proteins; Heart; Heart failure; In Vitro; Ligands; Location; Malignant hyperpyrexia due to anesthesia; Maps; Methodology; Modeling; Mutate; Mutation; Myocardium; Myopathy; Neurons; Pacemakers; Play; Process; Proteins; Reagent; Regulation; Resolution; Role; Ryanodine Receptor Calcium Release Channel; Ryanodine Receptors; Sarcolemma; Sarcoplasmic Reticulum; Site; Skeletal Muscle; Source; Structure; Surface; Techniques; Testing; Time; image processing; image reconstruction; particle; receptor; receptor function; reconstruction; skeletal; skeletal disorder; sorcin; sudden cardiac death; time use

DESCRIPTION (provided by applicant): Ryanodine receptors (RyRs) are intracellular calcium channels that are particularly important in skeletal and cardiac muscle where they play a key role in excitation-contraction (E-C) coupling, the process by which neuron-induced depolarization of the plasma membrane (sarcolemma) causes release of calcium ions from the sarcoplasmic reticulum (via RyRs). Mutated and malfunctioning RyRs have been implicated in certain diseases of skeletal (malignant hyperthermia, central-core disease) and, quite recently, cardiac muscle (two forms of sudden cardiac death, heart failure). In cardiac muscle RyRs also play important roles in pacemaker activity and in the generation of arrhythmogenesis. By characterizing the structure of RyRs and their interactions with associated proteins we expect to advance our understanding of RyRs' roles in E-C coupling mechanisms and in heart and skeletal muscle diseases. Purified RyRs from either natural sources (mammalian skeletal & cardiac muscle) or from transfected cultured cells that express mutated receptors will be characterized by cryo-electron microscopy and single-particle image processing to generate three- dimensional reconstructions (3D-cryoEM) at 10-30 Angstrom resolution. The specific aims of this proposal are to: (1) Determine the location of surface-exposed amino acids and regulatory sites by tagging them with green fluorescent protein or other macromolecular ligands, and (2) Characterize dynamical aspects of RyR function at increasing resolution by 3D cryo-EM and time-resolved cryo-EM. Specifically, the mechanisms by which the numerous domains that comprise the structure interact with each other to regulate channel gating by means of very long-range (>100 Angstroms) communication will be determined. Additionally, a hypothesized disruption of interdomain interactions by mutations that cause skeletal and sudden cardiac death diseases will be characterized, as well as the structural transitions that occur upon RyR regulation by calmodulin.

描述(申请人提供)：兰尼定受体(RyRs)是细胞内的钙通道，在骨骼肌和心肌中特别重要，它们在兴奋-收缩(E-C)耦合中发挥关键作用，即神经元诱导质膜(肌膜)的去极化导致钙离子从肌浆网(通过RyRs)释放。突变和功能失常的RyRs与某些骨骼疾病(恶性体温过高，中枢核心疾病)和最近的心肌疾病(两种形式的心源性猝死，心力衰竭)有关。在心肌中，RyRs也在起搏器活动和心律失常的发生中发挥重要作用。通过研究RyRs的结构及其与相关蛋白的相互作用，我们期望进一步了解RyRs在E-C偶联机制以及在心脏和骨骼肌疾病中的作用。从自然来源(哺乳动物骨骼肌和心肌)或从表达突变受体的转基因培养细胞中纯化的RyR将通过冷冻电子显微镜和单粒子图像处理来表征，以产生分辨率为10-30埃的三维重建(3D-CryoEM)。这项提议的具体目的是：(1)通过用绿色荧光蛋白或其他大分子配体标记表面暴露的氨基酸和调控位点来确定它们的位置，以及(2)通过3D低温EM和时间分辨低温EM来表征RyR功能在提高分辨率时的动力学特性。具体地说，将确定构成该结构的许多结构域相互作用以通过超长距离(&gt；100埃)通信来调节通道选通的机制。此外，还将描述由导致骨骼和心脏猝死疾病的突变导致的域间相互作用的假想中断，以及由钙调蛋白调节RyR时发生的结构转变。