Defining architecture of EC coupling machinery in situ

项目摘要/摘要 这一建议的重点是骨骼肌的兴奋-收缩偶联(ECC)。ECC由一个 肌肉细胞质膜去极化与钙离子释放相关的一系列生理事件 从肌浆网(SR)进入细胞质，导致肌肉收缩。ECC在空间上受到限制 到肌肉细胞的一个亚室(“三联体连接”)，并通过 质膜电压门控钙通道(二氢吡啶受体，DHPR)与钙释放 通道(1型Ryanodine受体，RyR1)。许多目前正在使用的治疗肌肉疾病的药物 瞄准这两个钙离子通道。尽管最近在这些化合物的结构表征方面取得了显著进展 这两个通道的相互作用的分子机制仍然难以捉摸，因为缺乏详细的 ECC机制的3D架构，包括通道和相关的调节蛋白。确定 这种多蛋白复合体的结构是一个巨大的挑战，因为它们在脂质中的天然位置 膜和缺乏一种通用的手段，以保持复杂的完整性时，用洗涤剂提取 他们的脂质双层环境。在这个项目中，我们将通过利用先进的低温技术来应对这一挑战 骨骼肌冷冻水合三联体的电子断层扫描研究(目标1)AS 以及在EM网格上培养的肌管内(目标2)。为了完成这些研究，我们努力发展 ECC复合体的原位低温ET分析的实验工作流程。此工作流将包括以下内容 主要步骤：制备适合于低温ET分析的膜包埋ECC络合物； 收集、图像分析、断层重建和亚断层图像平均；可视化和 低温断层图像中密度的注释。确定的结构将揭示机械信息 ECC钙离子释放复合体中潜在的蛋白质-蛋白质相互作用的特征将允许重要的 对ECC流程的功能洞察。未来，我们将把这里开发的工作流应用到结构- 不同类型肌肉和病理条件下ECC的功能特征。总体而言， 拟议的研究具有非常重要的意义，因为它们将提供对ECC的机械性结构洞察 阐明去调控的钙信号的病理后果的机制，这最终将有助于 寻找针对神经肌肉疾病的新疗法。作为这项研究的一部分，开发的工作流程 对其他整体膜蛋白复合体的研究具有广泛的适用性。

Project Summary/Abstract The focus of this proposal is on excitation-contraction coupling (ECC) in skeletal muscle. The ECC consists of a series of physiological events linking the depolarization of muscle cell’s plasma membrane to the release of Ca2+ from the sarcoplasmic reticulum (SR) into cytoplasm, resulting in muscle contraction. ECC is restricted spatially to a subcompartment of muscle cells (‘triad junction’) and regulated precisely via a physical interaction between the voltage-gated Ca2+ channel (dihydropyridine receptor, DHPR) on the plasma membrane and the Ca2+-release channel (type 1 ryanodine receptor, RyR1) in the SR. Many drugs currently in use to treat muscle disorders target these two Ca2+ channels. Despite recent remarkable advances in the structural characterization of these two channels, the molecular mechanisms underlying their interactions remain elusive due to the lack of detailed 3D architecture of the ECC machinery comprising both channels and associated regulatory proteins. Determining architecture of such multiprotein complexes is a formidable challenge given their native location in lipid membranes and the lack a general means to preserve the complex integrity upon extraction with detergents from their lipid bilayer environment. In this project, we will address this challenge by utilizing advanced cryogenic electron tomography (cryoET) to study frozen-hydrated triad junctions isolated from skeletal muscle (aim 1) as well as within myotubes cultured on EM grids (aim 2). To accomplish these studies, we endeavor to develop the experimental workflow for in situ cryoET analysis of the ECC complex. This workflow will consist of the following major steps: preparation of the membrane-embedded ECC complexes suitable for cryoET analysis; cryoET data collection, image analysis, tomographic reconstruction and subtomogram averaging; visualization and annotation of densities in cryo-tomograms. The determined structures will reveal mechanistically informative features underlying protein-protein interactions in the ECC Ca2+ release complex that will allow important functional insights into the ECC process. In the future, we will apply the workflow developed here to structure- functional characterization of ECC in different types of muscle and under pathological conditions. Overall, the proposed studies are highly significant, as they will provide mechanistic structural insights into the ECC machinery illuminating the pathological consequences of deregulated Ca2+ signaling, that will ultimately aid in search for novel therapies targeting neuromuscular diseases. The workflow developed, as part of this research will have broad applicability to studies of other integral membrane protein complexes.