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心率的分子相互作用。在心肌中，如在骨骼肌中一样，收缩被经由称为兰尼碱受体（RyR）的细胞内通道从肌浆网（SR）释放的Ca 2+激活。建议的研究集中在定义RyR通道s和调节蛋白钙调素（CaM），这是假定作为一个移动的，Ca 2+传感通道亚基之间的结构和功能的相互作用。 目的1是确定CaM作为RyR Ca 2+敏感性的亚型特异性调节剂的机制。目的2是表征纯化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，用于测量荧光共振能量转移（FRET），定义大分子RyR内的距离关系和Ca 2+依赖的结构变化。新发现的点突变体的钙调素，选择性地取消刺激或抑制相互作用的RyR将提供有价值的工具，用于表征模式的钙调素-RyR结合和解决的机制，通过该机制，钙调素作为一个分子开关调节通道活性。 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RyR 1，RyR 2和RyR 3同种型相互作用介导的CaM Ca 2+亲和力的变化。基于诱变、定点标记和FRET的结构数据将得到通道活性功能测定的支持，包括单通道记录、放射性配体结合和Ca 2+通量测定。总之，可用的技术和试剂将允许工作RyR通道内的第一时间分辨结构测量。了解RyR调节蛋白的作用是推动当前研究控制SR Ca 2+释放的机制的主要挑战，特别是在兴奋-收缩（EC）偶联过程中，CaM引起这些通道对Ca 2+反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的见解，从而帮助寻找针对心律失常和心力衰竭中SR Ca 2+处理缺陷的改进策略。

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.