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.

项目摘要 本申请中提出的研究旨在阐明结构-功能关系 一种运动引起的猝死称为儿茶酚胺能多形心室肌 心动过速（CPVT），由2型兰尼碱受体（RyR 2）突变/钙释放引起 频道RyR 2通道是从细胞内储存释放钙（Ca 2+）所必需的，这是一个过程， 其触发细胞功能，包括心肌中的兴奋-收缩（EC）偶联。RyR2， 与RyR 1和RyR 3沿着是已知最大的离子通道，由四个相同的~565 kDa的 通道形成原体，以及调节亚基，酶，和它们各自的 靶向/锚定蛋白质的大分子复合物，超过三百万道尔顿。已知 RyR 2突变会导致心律失常，包括运动诱发的猝死或CPVT，以及压力- 诱导的RyR 2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低温电子显微镜（cryo-EM） 从高度纯化的兔骨骼肌中重建1型RyR（RyR 1）， 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作为 三级折叠，包括Ca 2+结构域。使用建模软件，对RyR 2的结构进行了建模 与RyR 1同源。我们建议研究at的定位，结构效应和功能 至少11个代表性的致病性CPVT突变，以开发一个系统，了解如何 不同区域的致病性遗传变异导致临床疾病。这些研究报告将 通过解决突变体RyR 2通道的cryo-EM结构并通过功能测试这些 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开放事件（与常规技术相比）。 当前单通道当前分辨率为毫秒分辨率）。我们将建立一个数据库， 已知的CPVT相关的遗传变异体，并将这些突变系统地工程改造成重组 RyR 2，以便使用我们的新型高通量脂质双层测量系统研究这些通道。 本研究的数据将有助于了解CPVT的潜在机制。它将 提供了一种可用于开发CPVT治疗方法的方法。它将促进我们对 用于研究由RyR 2功能障碍引起的其他疾病和用于研究其他离子的新技术 渠道