Structure of Channels in Excitation-Contracting Coupling

描述（由申请人提供）：兰尼碱受体（RyR）是在骨骼肌和心肌中特别重要的细胞内钙通道，在兴奋-收缩（E-C）偶联中起关键作用，E-C偶联是神经元诱导的质膜（肌膜）去极化导致肌浆网（通过RyR）释放钙离子的过程。RyR突变和功能障碍与某些骨骼疾病（恶性高热，中央核心疾病）以及最近的心肌疾病（两种形式的心源性猝死，心力衰竭）有关。在心肌中，RyR在起搏器活动和心肌发生中也起重要作用。通过表征RyRs的结构及其与相关蛋白的相互作用，我们期望推进我们对RyRs在E-C偶联机制以及心脏和骨骼肌疾病中的作用的理解。来自天然来源（哺乳动物骨骼肌和心肌）或来自表达突变受体的转染培养细胞的纯化RyR将通过冷冻电子显微镜和单粒子图像处理来表征，以产生10-30埃分辨率的三维重建（3D-cryoEM）。该提案的具体目标是：（1）通过用绿色荧光蛋白或其他大分子配体标记表面暴露的氨基酸和调节位点来确定它们的位置，以及（2）通过3D cryo-EM和时间分辨cryo-EM在增加分辨率下表征RyR功能的动力学方面。具体地，将确定构成结构的众多域彼此相互作用以通过非常长距离（>100埃）通信来调节通道门控的机制。此外，一个假设的破坏结构域间的相互作用的突变，导致骨骼和心脏性猝死疾病的特点，以及结构转换后发生的RyR调节钙调蛋白。

DESCRIPTION (provided by applicant): Ryanodine receptors (RyRs) are intracellular calcium channels that are particularly important in skeletal and cardiac muscle where they play a key role in excitation-contraction (E-C) coupling, the process by which neuron-induced depolarization of the plasma membrane (sarcolemma) causes release of calcium ions from the sarcoplasmic reticulum (via RyRs). Mutated and malfunctioning RyRs have been implicated in certain diseases of skeletal (malignant hyperthermia, central-core disease) and, quite recently, cardiac muscle (two forms of sudden cardiac death, heart failure). In cardiac muscle RyRs also play important roles in pacemaker activity and in the generation of arrhythmogenesis. By characterizing the structure of RyRs and their interactions with associated proteins we expect to advance our understanding of RyRs' roles in E-C coupling mechanisms and in heart and skeletal muscle diseases. Purified RyRs from either natural sources (mammalian skeletal & cardiac muscle) or from transfected cultured cells that express mutated receptors will be characterized by cryo-electron microscopy and single-particle image processing to generate three- dimensional reconstructions (3D-cryoEM) at 10-30 Angstrom resolution. The specific aims of this proposal are to: (1) Determine the location of surface-exposed amino acids and regulatory sites by tagging them with green fluorescent protein or other macromolecular ligands, and (2) Characterize dynamical aspects of RyR function at increasing resolution by 3D cryo-EM and time-resolved cryo-EM. Specifically, the mechanisms by which the numerous domains that comprise the structure interact with each other to regulate channel gating by means of very long-range (>100 Angstroms) communication will be determined. Additionally, a hypothesized disruption of interdomain interactions by mutations that cause skeletal and sudden cardiac death diseases will be characterized, as well as the structural transitions that occur upon RyR regulation by calmodulin.