Molecular interactions regulating RyR Ca2+ channels

描述（由申请人提供）：本研究的主要目的是解决调节心跳力和速率的分子相互作用。心肌和骨骼肌一样，收缩是由肌浆网（SR）通过胞内通道（ryanodine receptor, RyRs）释放的Ca2+激活的。拟议的研究集中在定义RyR通道s和调节蛋白钙调蛋白（CaM）之间的结构和功能相互作用，CaM被认为是一个移动的Ca2+感应通道亚基。目的1是确定CaM作为RyR Ca2+敏感性的异构体特异性调节剂的机制。目的2是表征纯化的RyR上的CaM和FKBP结合位点，并鉴定保留高亲和力RyR结合的荧光衍生物。目的3是使用荧光CaM和FKBP衍生物，结合荧光RyR融合蛋白，定义大分子RyR内的距离关系和Ca2+依赖的结构变化，以测量荧光共振能量转移（FRET）。新发现的CaM点突变体选择性地消除了与ryr的刺激或抑制相互作用，将为表征CaM- ryr结合模式和解决CaM作为分子开关调节通道活性的机制提供有价值的工具。在初步研究中开发的环境敏感的荧光CaM衍生物将允许直接监测通过与完整的、功能性的RyR1、RyR2和RyR3异构体相互作用介导的CaM Ca2+亲和力的变化。基于诱变、定点标记和FRET的结构数据将通过通道活性的功能分析来支持，包括单通道记录、放射性配体结合和Ca2+通量测定。总之，现有的技术和试剂将允许在工作的RyR通道内进行首次时间分辨结构测量。了解RyR调节蛋白的作用是推动当前研究在兴奋-收缩（EC）耦合期间控制SR Ca2+释放机制的主要挑战，特别是CaM，引起了这些通道对Ca2+响应的明显和明确的变化。拟议的研究将提供通道调节的基本结构和机制见解，从而有助于寻找针对心律失常和心力衰竭中有缺陷的SR Ca2+处理的改进策略。

DESCRIPTION (provided by applicant): The broad objective of this research is to resolve molecular interactions that regulate the force and rate of the heartbeat. In cardiac muscle, as in skeletal muscle, contraction is activated by Ca2+ released from the sarcoplasmic reticulum (SR) via intracellular channels known as ryanodine receptors (RyRs). Proposed studies are focused on defining structural and functional interactions between RyR channel s and the regulatory protein calmodulin (CaM), which is postulated to act as a mobile, Ca2+-sensing channel subunit . Aim 1 is to determine the mechanism by which CaM functions as an isoform-specific modulator of RyR Ca2+ sensitivity. Aim 2 is to characterize CaM and FKBP binding sites on purified RyRs, and identify fluorescent derivatives that retain high-affinity RyR binding. Aim 3 is to define distance relationships and Ca2+ dependent structural changes within the macromolecular RyR using fluorescent CaM and FKBP derivatives, in combination with fluorescent RyR fusion proteins, for measurements of fluorescence resonance energy transfer (FRET). Newly-identified point mutants of CaM that selectively abolish either stimulatory or inhibitory interactions with the RyRs will provide valuable tools for characterizing the modes of CaM-RyR binding and for resolving the mechanism through which CaM functions as a molecular switch modulating channel activity. Environment-sensitive, fluorescent CaM derivatives developed in preliminary studies will allow direct monitoring of changes in CaM Ca2+ affinity mediated via interactions with the intact, functional, RyR1, RyR2, and RyR3 isoforms. Structural data based on mutagenesis, site-directed labeling, and FRET will be supported by functional assays of channel activity, including single channel recordings, radioligand binding, and Ca2+ flux determinations. Together, available techniques and reagents will allow for the first time-resolved structural measurements within working RyR channels. Understanding the role of RyR regulatory proteins is a major challenge driving current research into the mechanisms that control SR Ca2+ release during excitation-contraction (EC) coupling, and CaM, in particular, evokes pronounced and defined changes in these channels' response to Ca2+. Proposed studies will provide fundamental structural and mechanistic insights into channel regulation, and thereby aid in the search for improved strategies targeting defective SR Ca2+ handling in arrhythmias and heart failure.