Communication between neighboring ryanodine receptor channels in skeletal muscle

描述(申请人提供)：本项目的目标是了解骨骼肌兰尼定受体通道(RyRs)的偶联功能。RyRs是一种细胞内钙释放通道，位于细胞内钙库的膜上，在那里它们形成有序的阵列。在细胞刺激后，RyRs组短暂开放，并迅速将痉挛肌肉收缩所需的大量钙释放到胞浆中。这些钙释放事件的特征强烈表明，参与的RyR同步工作，它们都在几毫秒内一起激活和停用。目前还不清楚RyRV如何在细胞内同步它们的门控通信。因此，很难理解在各种肌肉疾病中发现的异常兴奋-收缩偶联的起源，包括那些编码RyR1基因突变的肌肉疾病(恶性高热、中央核心病)。由于RyRs是细胞内的通道，电生理学研究需要细胞细分、通道分离和重建为人工脂质双层。这些双层研究的几乎所有报告都描述了单个(单个)RyR1或多个RyR的独立行为。只有A.Marks博士的实验室描述了如果FK506结合蛋白(FKBP)分子与通道相关，相邻的RyR(2-4个通道)以同步协调方式选通。这种离子通道领域的新过程被称为“耦合门”。尽管它对了解钙释放很重要，但对RyR1之间的这种通讯知之甚少。在过去的几年里，首席调查员开发了方法并积累了初步数据，清楚地证明了研究与他的双层系统耦合的渠道的可行性。因此，该提案的目标是定义用于耦合选通的RyR通道之间的通信机制。该提案的具体目标是：具体目标#1：定义RyR1通道之间的通信机制。这一目标被细分为三个子目标：A)确定ATP/Mg~(2+)在RyR1协调中的作用；B)确定Ca~(2+)在协调门控中的作用；以及C)确定FKBP12(钙稳素)和其他辅助蛋白的作用。

DESCRIPTION (provided by applicant): The goal of this project is to understand the coupled function of skeletal muscle ryanodine receptor channels (RyRs). The RyRs are intracellular calcium release channels located in the membranes of intracellular calcium stores, where they form orderly arrays. After cell stimuli, groups of RyRs briefly open and rapidly release to the cytosol the bulk of calcium required for the twitch muscle contraction. The characteristics of these calcium release events strongly indicate that the participating RyRs work in synchrony and they all activate and deactivate together within few milliseconds. It is still unknown how RyRVs communicate for synchronizing their gating in cells. Consequently, it is difficult to understand the genesis of anomalous excitation-contraction coupling as found in various muscle diseases, including those (Malignant Hyperthermia, Central Core Disease) where mutations in the genes encoding RyR1 have been identified. As RyRs are intracellular channels, electrophysiological studies require cell subfractionation, channel isolation and reconstitution into artificial lipid bilayers. Nearly all reports from these bilayers studies describe a single (individual) RyR1 or independent behavior of multiple RyRs. Only Dr. A. Marks's laboratory described neighboring RyRs (2-4 channels) that gated with synchronous coordination if FK506 binding protein (FKBP) molecules were associated with the channels. This process, new for the field of ion channels, was named "coupled gating". Despite its importance to understand calcium release, little else is known about this inter-RyR1 communication. During the last years, the principle investigator has developed methodologies and accumulated preliminary data that clearly evidence the feasibility of studying channels coupling with his bilayer system. Consequently, the goal of this proposal is to define the mechanisms of communication between RyR channels for coupled gating. The specific aims of this proposal are: Specific Aim #1: Define the mechanisms of communication between RyR1 channels. This aim is subdivided in three sub-Aims: A) Define the role of ATP/Mg2+ in RyR1 coordination; B) Define the role of Ca2+ in coordinated gating; and C) Define the role of FKBP12 (Calstabilinl) and other ancillary proteins.