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.

项目总结/摘要 本提案的重点是骨骼肌的兴奋-收缩偶联（ECC）。环境投诉委员会由一个 肌细胞质膜去极化与钙离子释放之间的一系列生理事件 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 连接到肌肉细胞的亚隔室（“三联体连接”），并通过肌肉细胞之间的物理相互作用进行精确调节。 质膜上的电压门控性Ca 2+通道（二氢吡啶受体，DHPR）和Ca 2+释放 通道（1型兰尼碱受体，RyR 1）。目前用于治疗肌肉疾病的许多药物 针对这两个Ca 2+通道。尽管最近在这些结构表征方面取得了显着进展， 两个通道，其相互作用的分子机制仍然难以捉摸，由于缺乏详细的 ECC机制的3D结构包括通道和相关的调节蛋白。确定 考虑到这些多蛋白复合物在脂质中的天然位置， 膜和缺乏一个通用的手段，以保持复杂的完整性萃取后，洗涤剂从 它们的脂质双层环境。在这个项目中，我们将利用先进的低温技术来应对这一挑战。 电子断层扫描（cryoET）研究从骨骼肌中分离的冷冻水合三联体连接（目的1）， 以及在EM网格上培养的肌管内（目的2）。为了完成这些研究，我们奋进开发 ECC复合体的原位cryoET分析的实验工作流程。此工作流将包括以下内容 主要步骤：制备适用于cryoET分析的膜包埋ECC复合物; cryoET数据 采集、图像分析、断层重建和子断层图像平均;可视化和 在冷冻断层图像中标注密度。确定的结构将揭示机械信息 在ECC Ca 2+释放复合物中具有潜在的蛋白质-蛋白质相互作用， 对ECC流程的功能性见解。在未来，我们将应用这里开发的工作流程来构建- ECC在不同类型的肌肉和病理条件下的功能表征。总体看 建议的研究是非常重要的，因为它们将提供对ECC的机械结构见解 机械阐明失调的Ca 2+信号传导的病理后果，这将最终有助于 寻找针对神经肌肉疾病的新疗法。作为本研究的一部分， 将具有广泛的适用性，以研究其他完整的膜蛋白复合物。