Structure of Channels in Excitation-Contracting Coupling

励磁收缩耦合通道的结构

• 批准号: 8128520
• 负责人: TERENCE C WAGENKNECHT
• 金额: $ 41.04万
• 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 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8128520
• 关键词: Algorithms; Architecture; Binding; Calcium ion; Calmodulin; Cell membrane; Central Core Myopathy; Chemicals; Complex; Contracts; Coupling; Cryoelectron Microscopy; Cytoplasm; Detection; Dihydropyridine Receptors; Electron Microscopy; Elements; Freezing; Frozen Sections; Glean; Goals; Green Fluorescent Proteins; Image; Image Analysis; In Vitro; Inherited; Ions; Knowledge; Label; Ligands; Location; Malignant hyperpyrexia due to anesthesia; Maps; Membrane Proteins; Methodology; Microscope; Microtomy; Modeling; Molecular; Morphologic artifacts; Muscle; Muscle function; Mutation; Myopathy; Nature; Phase; Play; Preparation; Process; Proteins; Resolution; Role; Ryanodine Receptor Calcium Release Channel; Ryanodine Receptors; Sarcoplasmic Reticulum; Site; Skeletal Muscle; Striated Muscles; Structure; System; Techniques; Technology; Testing; Tissues; Tomogram; Ultramicrotomy; Use of New Techniques; Visual; base; crosslink; density; electron tomography; genetic regulatory protein; image processing; improved; interest; new technology; particle; protein distribution; public health relevance; reconstruction; research study; sensor; voltage

DESCRIPTION (provided by applicant): Excitation-contraction (EC) coupling in skeletal muscle refers to the process by which depolarization of the muscle plasma membrane leads to release of calcium ions into the cytoplasm from the sarcoplasmic reticulum (SR). EC coupling is performed by an array of voltage-sensor proteins (dihydropyridine receptors (DHPRs) in the plasma membrane/transverse tubule system that physically interact with and control an array of SR- embedded calcium-release channels (ryanodine receptors (RyRs), in conjunction with numerous other proteins. This assemblage of proteins (termed a couplon) occurs at regular intervals in muscle, and elucidating its structural architecture, ultimately at the atomic level, is essential to understanding the molecular mechanism of EC coupling in healthy and diseased muscle. Current models of the couplon structure are based largely upon visual interpretation of classical electron microscopy (EM) images, and are highly schematized and qualitative. The first aim of this proposal is to use the latest quantitative cryo-EM and tomographic reconstruction techniques to determine actual mass density distributions of the proteins and membranes comprising the couplon. Focused-ion-beam milling, a new technique for cutting sections of frozen-hydrated tissue that is being developed at our Center, will be used to prepare muscle for cryo-EM; the micrographs and tomograms obtained using this new technique will be compared to those obtained by the more standard technique of cryo-ultramicrotomy. Once optimal methodology is established, hundreds of tomograms will be obtained, and RyRs, which, being large 2.3 MDa complexes, are easily detected in tomograms, will be computationally extracted, classified, and averaged to maximize the contrast and resolution (the goal is 3-4.5 nm). The averaged RyRs are expected to resolve RyR-associated proteins, such as the RyR-DHPR complex which has never been observed. For aim 2, in vitro assembly experiments will be done to make complexes of purified RyR with its natural ligands, such as components of the DHPR and the regulatory protein calmodulin. Cryo-EM and 3D single-particle image analysis will be applied to these complexes to determine in more detail the nature of the interactions and dynamics of RyR and its binding partners. PUBLIC HEALTH RELEVANCE: Muscle diseases such as malignant hyperthermia and central-core disease result from inherited mutations of protein components of the excitation-contraction apparatus, including the ryanodine receptor and the dihydropyridine receptor. This proposal will use advanced electron microscopy technology to characterize the structural organization and interactions among these proteins. The findings will be important for understanding normal and diseased muscle function.

描述（由申请人提供）：骨骼肌中的兴奋-收缩（EC）耦合是指肌肉质膜的去极化导致钙离子从肌浆网（SR）释放到细胞质中的过程。 EC 耦合是通过质膜/横管系统中的一系列电压传感器蛋白（二氢吡啶受体 (DHPR)）进行的，这些蛋白与许多其他蛋白质结合，与一系列 SR 嵌入的钙释放通道（兰尼碱受体 (RyR)）物理相互作用并控制它们。这种蛋白质的组合（称为 EC耦合）在肌肉中定期发生，最终在原子水平上阐明其结构体系对于理解健康和患病肌肉中EC耦合的分子机制至关重要。当前的耦合子结构模型主要基于经典电子显微镜 (EM) 图像的视觉解释，并且高度模式化和定性。该提案的首要目标是使用最新的定量 冷冻电镜和断层扫描重建技术确定构成耦合子的蛋白质和膜的实际质量密度分布。聚焦离子束铣削是我们中心正在开发的一种用于切割冷冻水合组织切片的新技术，将用于为冷冻电镜准备肌肉；使用这种新技术获得的显微照片和断层照片将与通过更标准技术获得的显微照片和断层照片进行比较 冷冻超薄切片术。一旦建立了最佳方法，将获得数百张断层图，并且 RyR 作为大型 2.3 MDa 复合物，很容易在断层图中检测到，将通过计算提取、分类和平均，以最大化对比度和分辨率（目标是 3-4.5 nm）。平均 RyR 有望解析 RyR 相关蛋白，例如 作为从未被观察到的 RyR-DHPR 复合物。对于目标 2，将进行体外组装实验，以制备纯化的 RyR 与其天然配体（例如 DHPR 和调节蛋白钙调蛋白的成分）的复合物。 Cryo-EM 和 3D 单粒子图像分析将应用于这些复合物，以更详细地确定 RyR 及其结合伙伴的相互作用和动力学的性质。 公共健康相关性：恶性高热和中枢核心疾病等肌肉疾病是由兴奋收缩装置的蛋白质成分（包括兰尼碱受体和二氢吡啶受体）的遗传突变引起的。该提案将使用先进的电子显微镜技术来表征这些蛋白质之间的结构组织和相互作用。这些发现对于了解正常和患病的肌肉功能非常重要。