RyR architecture and gatekeeping of internal calcium stores

项目总结 细胞内、大电导的RyR钙释放通道是主要的 骨骼肌细胞和心肌细胞钙稳态调节剂。自然发生的突变 在骨骼异构体RyR1中，RyR1导致几种肌肉疾病(中央核心病，多微小核心病， 线状肌病、劳力性横纹肌溶解)和恶性高热，而 心脏RyR2亚型导致心律失常和心力衰竭。一些翻译后的修改 在野生型RyR1和RyR2中也会引起类似的影响。这些RyR变化的绝大多数 通过RyR超敏反应引起钙稳态的破坏，从而导致亚 阈值开放和肌浆网钙渗漏。 该项目应用最先进的结构生物学技术来确定RyR的结构和 原子水平和近自然状态的变构作用，以检查分子 RyR介导的钙泄漏机制，并分析相似的修饰如何影响不同的 两种RyR亚型。我们将确定选定的RyR1和RyR2突变体的原子结构，以及 WT RyR1或RyR2，进行翻译后修改，使用CryoEM和3D图像重建。 由于最近的技术进步，用低温电子显微镜对非晶态样品进行原子分辨现在是可能的 电子显微镜用直接电子探测器的研制。为了进一步建立和 描述数据集、分类和多变量统计分析中可能存在的异质性 将进行数千个RyR低温电磁粒子。将从HEK细胞中纯化突变的RyRs 对从兔中纯化的RyR进行了品系和翻译后修饰的研究。 解决RyR的原子结构与疾病相关的变化将有助于理解 有缺陷的通道关闭的分子机制，并揭示了 骨骼和心脏的异构体。这将推动该领域朝着基于结构的、 针对异构体的药物设计。

PROJECT SUMMARY The intracellular, large conductance intracellular RyR calcium release channel is a major regulator of calcium homeostasis in skeletal and cardiac muscle cells. Naturally occurring mutations in the skeletal isoform RyR1 lead to several myopathies (central core disease, multi-minicore disease, nemaline myopathy, exertional rhabdomyolysis) and malignant hyperthermia, whereas mutations in the cardiac isoform RyR2 lead to arrhythmia and heart failure. Some post-translational modifications can also cause similar effects in wild type RyR1 and RyR2. The vast majority of these RyR alterations cause disruption of calcium homeostasis through RyR hypersensitization, which causes sub- threshold opening and sarcoplasmic reticulum calcium leak. The project applies state-of-the-art structural biology technology to determine RyR's structure and allosterism at the atomic level and in a near-native state, in order to examine the molecular mechanism of RyR-mediated calcium leak and analyze how similar modifications affect differently the two RyR isoforms. We will determine the atomic structure of selected RyR1 and RyR2 mutants, and wt RyR1 or RyR2 with post-translational modifications, using cryoEM and 3D image reconstruction. Reaching atomic resolution of non-crystalline samples by cryoEM is now possible owing to the recent development of direct electron detectors for electron microscopy. To further establish and characterize possible heterogeneity in the dataset, classification and multivariate statistical analysis of several thousand RyR cryoEM particles will be performed. Mutant RyRs will be purified from HEK cell lines, and post-translational modifications will be studied on RyR purified from rabbit. Solving the atomic structure of RyR with disease-related alterations will help to understand the molecular mechanism of defective channel closure, and reveal any differential mechanisms between the skeletal and cardiac isoforms. This will advance the field forward towards structure-based, isoform-specific drug design.