MODULATORY MECHANISMS OF RYANODINE RECEPTOR ADAPTATION

在心肌和骨骼肌中，钙离子诱导的钙离子释放(CICR) 肌浆网(SR)是兴奋收缩的中枢 耦合，将膜去极化与机械作用联系起来的过程 收缩。然而，由于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，并全部- 或者-什么都不做。然而，在完整的细胞中，一种自限机制稳定下来。 CICR，防止肌浆网钙离子的耗竭。这件事的身份 机制尚不清楚。钙离子释放的一个基本性质 通道/Ryanodine受体(RyR)被称为适应可能是其机制 这与CICR固有的积极反馈形成反差。适应允许 RYR关闭(适应)，即使通道周围[Ca~(2+)]增加 是保持的。然而，在体外测量的适应时间常数 (秒)比负反馈机制慢得多 控制体内的CICR(毫秒)。因此，有必要建立 RyR适应的生理学相关性。 这项建议试图确定心脏和骨骼RyR的机制 以确定其生理意义。单一的RYR将 在脂质双层中重组和微环境中的(Ca2+) 通过笼状钙离子的激光闪光光解来改变通道的大小。 这个系统产生的(钙)变化可以通过改变 笼中钙离子浓度，激光灯输出功率，以及 钙离子扩散速率。此外，NP-EGTA的使用，一种新型的笼状钙离子 对镁离子的低亲和力，将使我们能够建立RyR对 Ca~(2+)在存在生理浓度的镁和三磷酸腺苷时， RyR函数的关键调制器。我们建议：(1)界定 心脏和骨骼RYR适应的基本特性；(2) 在所有相关钙离子调节剂存在的情况下测量RyR适应性 释放；(3)评估RyR的功能后果 磷酸化对适应动力学的影响，以及(4)确定作用 FKBP12在RyR适应机制中的作用 FKBP12和RYRs与FKBP12共表达。 这些研究对于理解钙离子和其他胞浆是如何 因素控制在任何给定时间打开的通道的数量，并且 RYR打开、适应和从适应状态恢复的速率。这个 因此，这项工作将提供关于 心肌和骨骼肌收缩的调节。

In cardiac and in skeletal muscle, Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) is central to excitation-contraction coupling, the process that links membrane depolarization to mechanical contraction. However, 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, a self-limiting mechanism stabilizes CICR and prevents depletion of Ca2+ from the SR. The identity of this mechanism is unknown. A fundamental property of Ca2+ release channels/ryanodine receptors (RYR) termed adaptation may be the mechanism that counters the inherent positive feedback of CICR. Adaptation allows RYRs to close (adapt) even though an increased [Ca2+] around the channel is maintained. However, the time constant of adaptation measured in vitro (seconds) is much slower than that of the negative feedback mechanism that controls CICR in vivo (milliseconds). Thus, it is essential to establish the physiological relevance of RYR adaptation. This proposal seeks to determine mechanisms of cardiac and skeletal RYR adaptation to establish its physiological significance. Single RYRs will be reconstituted in lipid bilayers and the (Ca2+) in the microenvironment of the channel will be changed by laser flash photolysis of caged Ca2+. The (Ca2+) change produced by this system may be controlled by varying the concentration of caged Ca2+, the power output of the laser lamp, and the rate of Ca2+ diffusion. Moreover, the use of NP-EGTA, a novel caged Ca2+ with low affinity for Mg2+, will allow us to establish the RYR response to Ca2+ in the presence of physiological concentrations of Mg and ATP, two critical modulators of RYR function. We propose: (1) to define the elementary properties of adaptation in cardiac and skeletal RYRs; (2) to measure RYR adaptation in the presence of all relevant modulators of Ca2+ release; (3) to evaluate the functional consequences of RYR phosphorylation on the kinetics of adaptation, and (4) to identify a role for FKBP12 in the mechanism of RYR adaptation using RYRs expressed without FKBP12 and RYRs coexpressed with FKBP12. These studies are 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, and recover from the adapted state. The work will consequently provide fundamental new information on the regulation of contraction in cardiac and skeletal muscle.