Structural Basis of Dysfunctional Calcium Release Channels in Heart Disease

心脏病中钙释放通道功能失调的结构基础

• 批准号: 8429376
• 负责人: Zheng Liu
• 金额: $ 37.8万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-15 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8429376
• 关键词: Amino Acids; Arrhythmogenic Right Ventricular Dysplasia; Binding; Calcium; Calmodulin; Cardiac; Cessation of life; Chimeric Proteins; Coupling; Cryoelectron Microscopy; Dissociation; Fluorescence Resonance Energy Transfer; Functional disorder; Goals; Green Fluorescent Proteins; Heart Diseases; Heart failure; Inherited; Integral Membrane Protein; Investigation; Ion Channel; Knowledge; Laboratories; Location; Maps; Mediating; Membrane; Movement; Muscle; Myocardium; Myopathy; Pharmaceutical Preparations; Play; Proteins; Regulation; Research; Resolution; Roentgen Rays; Role; RyR2; Ryanodine Receptor Calcium Release Channel; Ryanodine Receptors; Sarcoplasmic Reticulum; Signal Transduction; Structural Models; Structure; System; Tacrolimus Binding Proteins; Techniques; Testing; United States; Ventricular Tachycardia; Work; base; design; digital imaging; effective therapy; fluorophore; image processing; innovation; molecular mass; mortality; novel; novel therapeutics; particle; public health relevance; screening; sudden cardiac death; three dimensional structure; tool

DESCRIPTION (provided by applicant): There is a fundamental gap in understanding how dysfunction of cardiac ryanodine receptor (RyR2) leads to certain type of sudden cardiac death and heart failure. RyR2 is an intracellular calcium release channel that plays a crucial role in cardiac muscle excitation-contraction (E-C) coupling. Our long term goals include investigation of the interactions between RyR and its modulators involved in muscle E-C coupling, determination of a detailed high-resolution three-dimensional structural model for RyR, and characterization of RyR's conformational dynamics. The objective of this application is to combine two highly complementary biophysical techniques, cryo- electron microscopy (cryo-EM) and fluorescence resonance energy transfer (FRET), to characterize functional structures and conformational dynamics of RyR2. Our central hypothesis is that abnormal interactions between RyR2's structural domains and between RyR2 and its modulators, and abnormal conformational changes underlie dysfunction of RyR2. The hypothesis will be tested by pursuing two Specific Aims: (1). Generate and characterize structure of RyR2 fusion proteins containing cyan and yellow fluorescent proteins (CFP and YFP) that are suitably located for FRET studies of RyR2's conformational dynamics. Results from our ongoing cryo-EM studies will serve as an essential guide to strategically insert CFP and YFP into RyR2's sequence, we can precisely control the distance between the CFP and YFP, so that they are suitably juxtaposed for FRET studies. (2). Investigate the dynamics of the CFP/YFP tagged RyR2s by FRET. Particular emphasis will be given to proposed interactions between RyR2's structural domains, and interactions of RyR2 with modulators that physiologically regulate RyR2 channel functions, such as FK506-binding protein and calmodulin, which have been implicated to be defective in heart failure and sudden cardiac death. We expect to have a better understanding of the mechanisms underlying RyR2 channel dysfunction. The information will help us to comprehend the role of RyRs in E-C coupling mechanisms, and how RyR2 dysfunction leads to cardiac muscle diseases, which in turn should allow more rational design of novel therapeutic strategies.

描述（由申请人提供）：了解心脏Ryanodine受体（RYR2）的功能障碍如何导致某些类型的心脏死亡和心力衰竭。 RYR2是一个细胞内钙释放通道，在心肌激发收缩（E-C）耦合中起着至关重要的作用。我们的长期目标包括研究RYR及其参与肌肉E-C耦合的调节剂之间的相互作用，确定RYR的详细高分辨率三维结构模型以及RYR构象动力学的表征。该应用的目的是结合两种高度互补的生物物理技术，冷冻电子显微镜（Cryo-EM）和荧光共振能传递（FRET），以表征RyR2的功能结构和构象动力学。我们的中心假设是RYR2的结构结构域与RYR2及其调节剂之间的异常相互作用以及RYR2功能障碍的异常构象变化。该假设将通过追求两个具体目标来检验：（1）。生成和表征含有青色和黄色荧光蛋白（CFP和YFP）的RYR2融合蛋白的结构，这些蛋白适用于RYR2构象动力学的FRET研究。我们正在进行的冷冻EM研究的结果将作为策略性的指南，以策略性地将CFP和YFP插入RYR2的序列中，我们可以精确控制CFP和YFP之间的距离，以便将它们适当地置于FRET研究中。 （2）。研究用FRET标记的RYR2标记的CFP/YFP的动力学。 RYR2的结构结构域之间提出的相互作用以及RYR2与生理调节RYR2通道功能的调节剂的相互作用（例如FK506结合蛋白和钙调蛋白）的相互作用，将特别强调，RYR2的相互作用已与心脏失效和突然的心脏死亡有关。我们希望对RYR2通道功能障碍的基础机制有更好的了解。这些信息将帮助我们理解RYRS在E-C耦合机制中的作用，以及RYR2功能障碍如何导致心脏肌肉疾病，这反过来又应允许对新型治疗策略进行更合理的设计。