Molecular interactions regulating RyR Ca2+ channels

描述(由申请人提供)：这项研究的广泛目标是解决调节心跳力量和频率的分子相互作用。在心肌中，就像在骨骼肌中一样，收缩是由肌浆网(SR)通过被称为兰尼定受体(RyRs)的细胞内通道释放的钙激活的。目前的研究集中在确定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被认为是一个可移动的钙敏感通道亚单位。目的1是确定CaM作为RyR钙敏感性的异构体特异性调节剂的作用机制。目的2是鉴定纯化的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。目标3是使用荧光CaM和FKBP衍生物，结合荧光RyR融合蛋白，确定大分子RyR内的距离关系和钙依赖的结构变化，以测量荧光共振能量转移(FRET)。新发现的CaM点突变体可以选择性地取消与RyRs的刺激或抑制相互作用，这将为表征CaM-RyR结合模式和解析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衍生物将允许通过与完整的、功能的、RyR1、RyR2和RyR3亚型的相互作用来直接监测CaM钙亲和力的变化。基于突变、定点标记和FRET的结构数据将得到通道活性的功能分析的支持，包括单通道记录、放射性配基结合和钙离子通量测定。可用的技术和试剂一起，将允许在工作的RyR通道内进行第一次时间分辨的结构测量。了解RyR调节蛋白的作用是推动当前研究的主要挑战，这些研究推动了在兴奋-收缩(EC)耦合过程中控制SR钙释放的机制，特别是CaM，引起了这些通道对Ca~(2+)反应的明显和明确的变化。拟议的研究将为通道调节提供基本的结构和机制方面的见解，从而有助于寻找针对心律失常和心力衰竭中有缺陷的SR钙处理的改进策略。

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.