Structure-function analysis for elucidating pathogenicity of cardiac ryanodine receptor genetic variants

结构功能分析阐明心脏兰尼碱受体遗传变异的致病性

• 批准号: 10407960
• 负责人: ANDREW Robert MARKS
• 金额: $ 76.28万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10407960
• 关键词: Arrhythmia; Caffeine; Calcium; Cardiac; Cardiac Myocytes; Catecholaminergic Polymorphic Ventricular Tachycardia; Cell physiology; Cells; Clinical; Clinical Data; Computer software; Coupling; Cryoelectron Microscopy; Cyclic AMP-Dependent Protein Kinases; Data; Databases; Defect; Diagnosis; Diastole; Disease; Egtazic Acid; Electronics; Engineering; Enzymes; Event; Exercise; FKBP1B gene; Functional disorder; Heart failure; In Vitro; Inherited; Ion Channel; Ion Channel Gating; Life; Link; Lipid Bilayers; Macromolecular Complexes; Measurement; Measures; Membrane; Methods; Modeling; Mutation; Myocardium; Oryctolagus cuniculus; Pathogenicity; Pathologic; Patients; Phenotype; Phosphorylation; Post-Translational Protein Processing; Probability; Process; Proteins; Protomer; Publishing; Rare Diseases; Recombinants; Reporting; Research; Research Project Summaries; Resolution; RyR1; RyR3; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Semiconductors; Skeletal Muscle; Stress; Structure; Structure-Activity Relationship; Sudden Death; Surface; System; Technology; Testing; Transgenic Mice; Variant; base; biophysical properties; dalton; design; genetic variant; high throughput screening; improved; innovation; insight; integrated circuit; metal oxide; millisecond; molecular modeling; mutant; mutation screening; nanosecond; new technology; novel; public database; reconstruction; searchable database; sudden cardiac death; therapy development; variant of unknown significance

Project summary The research proposed in this application is designed to elucidate the structure-function relationships of a form of exercise-induced sudden death known as catecholaminergic polymorphic ventricular tachycardia (CPVT), caused by mutations in the Type-2 ryanodine receptor (RyR2)/calcium release channel. RyR2 channels are required for the release of calcium (Ca2+) from intracellular stores, a process that triggers cellular functions including excitation-contraction (EC) coupling in the cardiac muscle. RyR2, along with RyR1 and RyR3, are the largest known ion channels, comprised of the four identical ~565 kDa channel-forming protomers, as well as regulatory subunits, enzymes, and their respective targeting/anchoring proteins in a macromolecular complex that exceeds three million daltons. It is known that RyR2 mutations cause arrhythmias including exercise-induced sudden death, or CPVT, and stress- induced post-translational modifications of RyR2 contribute to both CPVT and heart failure progression. The applicants have recently obtained near-atomic-level resolution cryo-electron microscopy (cryo-EM) reconstructions of Type-1 RyR (RyR1) from highly purified rabbit skeletal muscle in both the closed and the open states, defining the transmembrane pore in unprecedented detail and placing all cytosolic domains as tertiary folds, including a Ca2+ domain. Using modeling software, the structure of RyR2 has been modeled based on homology with RyR1. We propose to study the localization, structural effects, and function of at least 11 representative pathogenic CPVT mutations, in order to develop a system for understanding how pathogenic genetic variants in different regions of the channel cause clinical disease. These studies will be conducted by solving cryo-EM structures of mutant RyR2 channels and by functionally testing these mutations using a novel, high bandwidth, high-throughput lipid bilayer technology developed by our team. This technology is capable of identifying channel opening events with nanosecond resolution (compared to current single channel current resolution of millisecond resolution). We will then develop a database of all known genetic variants CPVT-associated and systematically engineer these mutations into recombinant RyR2 in order to study these channels using our novel high-throughput lipid bilayer measurement system. The data from this project will be useful for understanding the underlying mechanisms of CPVT. It will provide an approach that can be used to develop therapies for CPVT. It will advance our understanding of novel technologies for studying other diseases caused by RyR2 dysfunction and for studying other ion channels.

项目概要 本申请提出的研究旨在阐明结构与功能的关系 一种称为儿茶酚胺能多形性心室的运动引起的猝死 心动过速 (CPVT)，由 2 型兰尼碱受体 (RyR2)/钙释放突变引起 渠道。 RyR2 通道是从细胞内储存释放钙 (Ca2+) 所必需的，这一过程 触发细胞功能，包括心肌中的兴奋-收缩（EC）耦合。 RyR2, 与 RyR1 和 RyR3 一起，是已知最大的离子通道，由四个相同的 ~565 kDa 组成 通道形成原聚体，以及调节亚基、酶及其各自的 在超过三百万道尔顿的大分子复合物中靶向/锚定蛋白质。我们都知道 RyR2 突变会导致心律失常，包括运动诱发的猝死 (CPVT) 和压力- RyR2 诱导的翻译后修饰有助于 CPVT 和心力衰竭进展。 申请人最近获得了近原子级分辨率的冷冻电子显微镜（cryo-EM） 从高度纯化的兔骨骼肌中重建 1 型 RyR（RyR1） 开放状态，以前所未有的细节定义跨膜孔，并将所有胞质结构域放置为 三级折叠，包括 Ca2+ 结构域。使用建模软件对RyR2的结构进行了建模 基于与 RyR1 的同源性。我们建议研究 at 的定位、结构效应和功能 至少 11 种代表性致病性 CPVT 突变，以便开发一个系统来了解如何 通道不同区域的致病基因变异导致临床疾病。这些研究将 通过求解突变型 RyR2 通道的冷冻电镜结构并对其进行功能测试来进行 使用我们团队开发的新型、高带宽、高通量脂质双层技术进行突变。 该技术能够以纳秒分辨率识别通道打开事件（与 当前单通道电流分辨率为毫秒分辨率）。然后我们将开发一个包含所有内容的数据库 已知的遗传变异与 CPVT 相关，并将这些突变系统地改造为重组体 RyR2，以便使用我们新颖的高通量脂质双层测量系统研究这些通道。 该项目的数据将有助于理解 CPVT 的基本机制。它将 提供了一种可用于开发 CPVT 疗法的方法。它将加深我们对 研究由 RyR2 功能障碍引起的其他疾病和研究其他离子的新技术 渠道。