MODULATORY MECHANISMS OF RYANODINE RECEPTOR ADAPTATION

在心脏和骨骼肌中，Ca 2+诱导的Ca 2+释放（CICR）从 肌浆网（SR）是兴奋-收缩中枢 耦合，将膜去极化与机械 收缩。然而，由于Ca 2+是触发信号， CICR的信号，该过程预计将自我延续，所有- 或者没有然而，在完整的细胞中， CICR并防止SR中Ca 2+的耗尽。 机制不明。Ca 2+释放的一个基本性质 通道/兰尼碱受体（RYR）称为适应可能是机制 这与CICR固有的积极反馈相反。适应允许 RYR关闭（适应），即使通道周围的[Ca 2 +]增加 保持不变。然而，在体外测量的适应时间常数 （秒）比负反馈机制慢得多， 对照体内CICR（毫秒）。因此，必须建立 RYR适应的生理相关性。 这项建议旨在确定心脏和骨骼RYR的机制 适应，以建立其生理意义。单个RYR将 在脂质双层和微环境中的（Ca 2+）中重构 激光闪光光解笼状Ca ~（2+）将改变通道的结构。 由该系统产生的（Ca 2+）变化可以通过改变 笼状Ca 2+的浓度、激光灯的功率输出和 Ca 2+扩散速率。此外，使用NP-EGTA，一种新的笼状Ca 2+， 与Mg 2+的亲和力低，将使我们能够建立RYR反应， Ca 2+在生理浓度的Mg和ATP的存在下，两个 RYR功能的关键调节剂。我们建议：（1）定义 心脏和骨骼RYR适应的基本特性;（2） 在所有相关的Ca 2+调节剂存在下测量RYR适应 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）确定磷酸化在适应动力学中的作用 FKBP 12在RYR适应机制中的作用， FKBP 12和与FKBP 12共表达的RYR。 这些研究对于理解Ca 2+和其他细胞溶质 在任何给定的时间，控制开放通道的数量的因素， RYR打开、适应和从适应状态恢复的速率。的 因此，这项工作将提供关于 调节心脏和骨骼肌的收缩。

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.