Defining architecture of EC coupling machinery in situ

现场定义 EC 耦合机械的架构

• 批准号: 10711223
• 负责人: Irina I Serysheva
• 金额: $ 20.59万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10711223
• 关键词: 3-Dimensional; Address; Affect; Architecture; Binding; Biochemical; Cell membrane; Cellular Membrane; Central Core Myopathy; Complex; Contractile Proteins; Coupled; Coupling; Crowding; Cryoelectron Microscopy; Cytoplasm; Data; Data Collection; Defect; Detergents; Dihydropyridine Receptors; Disease model; Electrons; Elements; Environment; Event; Foundations; Freezing; Functional disorder; Future; Geometry; Goals; Hydration status; Hypokalemic periodic paralysis; Image; Image Analysis; Impairment; In Situ; Integral Membrane Protein; Ions; Knowledge; Link; Lipid Bilayers; Location; Malignant hyperpyrexia due to anesthesia; Measurement; Membrane; Membrane Lipids; Methodology; Molecular; Morphologic artifacts; Multiminicore disease; Multiprotein Complexes; Muscle; Muscle Cells; Muscle Contraction; Muscle Fibers; Mutation; Myopathy; Neuromuscular Diseases; Outcome; Pathologic; Pharmaceutical Preparations; Physiological; Positioning Attribute; Preparation; Process; Proteins; Receptor Gene; Regulation; Research; Resolution; Ryanodine Receptor Calcium Release Channel; Sampling; Sarcoplasmic Reticulum; Series; Signal Transduction; Skeletal Muscle; Structure; Techniques; Thick; Tomogram; Visualization; convolutional neural network; cryogenics; drug discovery; electron tomography; genetic regulatory protein; insight; new therapeutic target; particle; preservation; protein complex; protein protein interaction; reconstruction; structural determinants; three dimensional structure; tomography; voltage

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.

项目摘要/摘要 该提议的重点是骨骼肌中的兴奋反应耦合（ECC）。 ECC由 将肌肉细胞的质膜沉积与Ca2+释放联系起来的一系列物理事件 从肌质网（SR）到细胞质，导致肌肉收缩。 ECC在空间上受到限制 到肌肉细胞（“三合会连接”）的子组门，并通过物理相互作用进行调节 质膜上的电压门控Ca2+通道（Dihydropyine受体，DHPR）和Ca2+ release SR中的通道（1型Ryanodine受体，RYR1）。目前使用的许多药物用于治疗肌肉障碍 靶向这两个CA2+通道。尽管最近在这些结构表征上取得了显着进步 两个通道，由于缺乏细节，其相互作用的分子机制仍然难以捉摸 ECC机械的3D结构完成了两个通道和相关调节蛋白。确定 鉴于其本地位置在脂质中 机制和缺乏一般的手段，可以在提取时保留复杂的完整性，从而确定 他们的脂质双层环境。在这个项目中，我们将通过使用高级低温来应对这一挑战 电子断层扫描（冷冻）研究从骨骼肌中分离出的冷冻水合的三合会（AIM 1） 以及在EM网格上培养的Myotube中（AIM 2）。为了完成这些研究，我们努力发展 对ECC复合物的原位冷冻分析的实验工作流程。此工作流将包括以下 主要步骤：制备适合冷冻分析的膜包裹的ECC复合物；冷冻数据 收集，图像分析，层析成像重建和亚图平均；可视化和 冷冻图中密度的注释。确定的结构将揭示机械信息丰富 ECC CA2+释放复合物中的蛋白质 - 蛋白质相互作用的基础特征，该复合物将允许重要 对ECC过程的功能见解。将来，我们将在此处运用此处开发的工作流程 - 结构 - 在不同类型的肌肉和病理条件下，ECC的功能表征。总体而言， 拟议的研究非常重要，因为它们将为ECC提供机械性结构见解 机械阐明了放松管制的CA2+信号传导的病理后果，这最终将有助于 寻找针对神经肌肉疾病的新型疗法。作为这项研究的一部分，开发了工作流程 将广泛适用于对其他积分膜蛋白复合物的研究。