Communication between neighboring ryanodine receptor channels in skeletal muscle

项目描述（由申请人提供）：本项目旨在了解骨骼肌ryanodine受体通道（RyRs）的耦合功能。ryr是位于细胞内钙储存膜上的细胞内钙释放通道，在那里它们形成有序的阵列。在细胞受到刺激后，ryr群会短暂打开，并迅速向胞浆释放抽搐肌收缩所需的大量钙。这些钙释放事件的特征强烈表明参与的ryr是同步工作的，它们都在几毫秒内一起激活和失活。目前还不清楚ryrv是如何进行通信以同步细胞中的门控的。因此，很难理解在各种肌肉疾病中发现的异常兴奋-收缩耦合的起源，包括那些已经确定编码RyR1基因突变的疾病（恶性高热，中央核心疾病）。由于ryr是细胞内通道，电生理研究需要细胞亚切片、通道分离和重构成人工脂质双分子层。几乎所有来自这些双层研究的报告都描述了单个（个体）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RyRs（2-4个通道）会以同步协调进行门控。这是离子通道领域的新工艺，被命名为“耦合门控”。尽管它对理解钙释放很重要，但对ryr1之间的这种交流知之甚少。在过去的几年里，首席研究员已经开发了方法并积累了初步数据，这些数据清楚地证明了研究通道耦合与他的双层系统的可行性。因此，本提案的目标是定义用于耦合门控的RyR通道之间的通信机制。具体目标1：定义RyR1通道之间的通信机制。该目标可细分为三个子目标：A)确定ATP/Mg2+在RyR1协调中的作用；B)明确Ca2+在协调门控中的作用；C)确定FKBP12 （Calstabilinl）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.