Structure of Channels in Excitation-Contracting Coupling

描述（由申请人提供）：骨骼肌中的兴奋-收缩（EC）偶联是指肌肉质膜去极化导致钙离子从肌浆网（SR）释放到细胞质中的过程。EC偶联通过质膜/横小管系统中的电压传感器蛋白（二氢吡啶受体（DHPR））阵列与许多其他蛋白质结合进行，所述电压传感器蛋白与SR嵌入的钙释放通道（兰尼碱受体（RyR））阵列物理相互作用并控制SR嵌入的钙释放通道阵列。这种蛋白质的组合（称为耦合子）在肌肉中以规则的间隔发生，并最终在原子水平上阐明其结构架构，对于理解健康和患病肌肉中EC耦合的分子机制至关重要。目前的模型的耦合子结构主要是基于经典的电子显微镜（EM）图像的视觉解释，并高度系统化和定性。本建议的第一个目的是使用最新的定量冷冻EM和断层重建技术，以确定实际的质量密度分布的蛋白质和膜组成的耦合。聚焦离子束铣削，一种新的技术，用于切割部分的冷冻水合组织，正在开发中，在我们的中心，将被用来准备肌肉冷冻EM的显微照片和断层扫描，使用这种新技术获得的将比较那些由冷冻超微切片的更标准的技术。一旦建立了最佳方法，将获得数百个断层图像，并且RyR（其是大的2.3MDa复合物，在断层图像中容易检测到）将被计算提取、分类和平均以最大化对比度和分辨率（目标是3- 4.5nm）。平均RyR预计将解析RyR相关蛋白，例如从未观察到的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组分）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性质。 公共卫生相关性：肌肉疾病，如恶性高热和中央核心疾病，是由兴奋-收缩装置的蛋白质组分的遗传突变引起的，包括兰尼碱受体和二氢吡啶受体。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。

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.