MODULATORY MECHANISMS OF RYANODINE RECEPTOR ADAPTATION

描述(申请人的逐字描述)：心脏和骨骼 肌肉、外膜二氢吡啶受体(DHPR)和 肌浆网钙释放通道/兰尼定受体(RyR)是关键 兴奋-收缩(EC)耦合的组成部分，一系列事件 将电刺激(去极化)与机械收缩联系起来。 Ca~(2+)诱导的Ca~(2+)释放(CICR)是一个Ca~(2+)放大过程。 越来越明显的参与骨骼肌和久负盛名的 在心肌中的作用。然而，一个根本性的问题阻碍了我们的 对CICR的理解。因为钙离子是触发信号和输出 CICR的信号，预计这一过程将自我延续和 要么全有，要么全不。然而，在完整的细胞中，CICR分级很好，可能会被抑制 通过抑制内向钙电流ICa。因此，自我限制机制的 体内Ca~(2+)释放稳定CICR，防止Ca~(2+)耗竭 来自SR的。这些机制的身份尚不清楚。 在这项提案中，我们将研究适应和钙依赖 失活是RyRs固有的两种机制，它调节RyR对 Ca~(2+)和CICR固有的正反馈。这些不同的和 可能互补的机制可能赋予RYR以功能性 灵活性，可暂时提高对来电的响应级别 Ca~(2+)刺激(适应)并在关键的Ca~(2+)一次关闭其活性 在胞浆表面已经达到水平(依赖于钙的失活)。 使用允许快速和校准更改的单通道记录系统 在RyRs周围的介质中，我们建议研究动态 RyRs与细胞配体的相互作用加速适应和 依赖于钙离子的失活，特别是钙离子、镁离子和cADPr。这个 具体目标是：1)使用整个范围来确定RyR对钙离子的响应 快速和校准的钙离子刺激。2)探寻结构 适应的决定因素和钙离子依赖的失活。3)澄清 CADPr对心脏RyRs的作用机制。 从这些研究中获得的信息对于理解 钙离子和其他胞浆因子控制着任何时候开放的通道的数量 给定时间，以及RYR打开、适应、停用和恢复的速率 从非激活状态。

DESCRIPTION (the applicant's description verbatim): In cardiac and skeletal muscle, the dihydropyridine receptor (DHPR) of the external membrane and the Ca2+ release channel/ryanodine receptor (RyR) of sarcoplasmic reticulum are key components of excitation-contraction (EC) coupling, the series of events that link an electrical stimulus (depolarization) to a mechanical contraction. Ca2+ induced Ca2+ release (CICR) is a Ca2+-amplification process with an increasingly evident participation in skeletal muscle and a well-established role in cardiac muscle. Nevertheless, a fundamental problem hinders our understanding of CICR. Because Ca2+ is the triggering signal and the output signal of CICR, the process is expected to be self-perpetuating and all-or-none. In intact cells however, CICR is finely graded and may be arrested by suppressing the inward Ca2+ current, ICa. Thus, self-limiting mechanisms of Ca2+ release operate in vivo to stabilize CICR and to prevent depletion of Ca2+ from the SR. The identity of these mechanisms is unknown. In this proposal, we will investigate if adaptation and Ca2+-dependent inactivation, two mechanisms intrinsic to RyRs, modulate the RyR response to Ca2+ and counter the inherently positive feedback of CICR. These different and probably complementary mechanisms may endow the RyRs with the functional flexibility to both transiently increase their level of response to incoming Ca2+ stimuli (adaptation) and to turn offtheir activity once a critical Ca2+ level has been reached at their cytosolic face (Ca2+-dependent inactivation). Using single-channel recording systems that allow fast and calibrated changes of [Ca2+] in the medium around RyRs, we propose to study the dynamic interaction of RyRs with cellular ligands that accelerate adaptation and Ca2+-dependent inactivation, specifically, Ca2+ ions, Mg2+ ions, and cADPr. The specific aims are: 1) To determine the RyR response to Ca2+ using a whole range of fast and calibrated Ca2+ stimuli. 2) To probe for the structural determinants of adaptation and Ca2+ dependent inactivation. 3) To elucidate the mechanism of action of cADPr on cardiac RyRs. The information derived from these studies will be important to understand how Ca2+ and other cytosolic factors control the number of open channels at any given time, and the rate at which RyRs open, adapt, inactivate, and recover from the inactivated state.