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

基本信息

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
项目类别：
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

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

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项目摘要

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.

项目摘要本应用程序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形式，称为儿茶酚胺能多态性心室心动过速（CPVT），由2型ryanodine受体（RYR2）/钙释放引起渠道。从细胞内存储中释放钙（Ca2+）需要RYR2通道，这是一个过程这会触发细胞功能，包括心脏肌肉中的兴奋 - 收缩（EC）耦合。 ryr2，与RYR1和RYR3一起，是最大的已知离子通道，由四个相同的〜565 kDa组成渠道形成的原始物以及调节亚基，酶及其各自的酶在超过三百万达尔顿的大分子复合物中靶向/锚定蛋白。我们都知道 RYR2突变会导致心律不齐，包括运动引起的猝死或CPVT和应力 - 诱导的RYR2翻译后修饰有助于CPVT和心力衰竭进展。申请人最近获得了接近原子的分辨率冷冻电子显微镜（Cryo-EM）封闭式和开放状态，以前所未有的细节定义跨膜孔，并将所有胞质结构域置于第三折，包括Ca2+域。使用建模软件，RYR2的结构已被建模基于与RYR1的同源性。我们建议研究AT的定位，结构效应和功能至少有11个代表性的致病性CPVT突变，以开发一个系统来理解如何通道不同区域中的致病遗传变异引起临床疾病。这些研究将是通过求解突变体RYR2通道的冷冻EM结构并通过功能测试使用我们团队开发的新型，高带宽，高通量脂质双层技术的突变。该技术能够通过纳秒分辨率识别渠道开放事件（与当前单通道电流分辨率的分辨率）。然后，我们将开发一个数据库已知的遗传变异CPVT相关并系统地将这些突变设计为重组 RYR2是为了使用我们新型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。会提供一种可用于开发CPVT疗法的方法。它将提高我们对用于研究由RYR2功能障碍引起的其他疾病和研究其他离子引起的新技术频道。