Skeletal Muscle. Ca2+ release control inside the SR

描述(由申请人提供)：CA2信号将质膜事件与代谢变化联系起来。兴奋-收缩(EC)耦合--在肌肉中传递动作电位以增加细胞内钙浓度和收缩--是公认的钙信号的第一个例子。它的关键分子(电压传感器或DHPR和释放通道或RYR)越来越多地存在于其他细胞和组织中。它在越来越多的遗传性疾病中的参与被认识到。它的变化，在长时间的运动后，关键是导致肌肉疲劳。传统上，EC偶联是通过测量Ca~(2+)的瞬变来探讨的，这一共识是基于这样的共识，即Ca~(2+)的释放是由RyRs胞液面上的配体(如Ca~(2+)或DHPr中的环)实施的。现在，支持SR内部关键监管的证据越来越多。这是一项对这种控制进行系统研究的建议。将检验四个工作假说，它们阐述了从SR内部调节的想法：1)Ca~(2+)释放激活在很大程度上由SR Ca~(2+)含量决定。2)Ca~(2+)释放终止在很大程度上取决于SR内局部的Ca~(2+)消耗。3)一种新的通道激活协同模式是由SR Ca控制的。4)肌质网内钙结合蛋白Calequestrin在钙释放中具有动态调节作用，可能涉及与其他肌质网蛋白的相互作用。这些假设将通过1)监测单个细胞上的细胞平均和局部水平的钙释放(导致钙火花)，同时操纵和监测SR的钙含量来检验。2)在SR中引入外源性Ca~(2+)缓冲液，其中硫酸盐已被证明具有非凡的效果。3)改变钙调素的浓度和功能，通过其在培养细胞中的过度或低表达，或通过将基因转移到成年肌肉中。4)为了更好地了解分子需求，将研究钙调素对钙调控单个RyRs的影响。5)现有的钙释放模型将有助于检验这些假说的各种量化版本的有效性。技术创新在AIMS 1(同时记录SR内和胞浆[Ca~(2+)])、2(缓冲液和极限缓冲液的区别)、3(向成体肌肉转移calequestrin基因)和4(在双分子层的通道中联合应用calequestrin和光释放的Ca)方面有实质性的技术创新。

DESCRIPTION (provided by applicant): Ca2+ signaling links plasma membrane events to metabolic change. Excitation-contraction (EC) coupling -- which in muscle transduces the action potential to increase in intracellular Ca2+ concentration, and contraction-- was the first recognized example of Ca2+ signaling. Its key molecules (voltage sensors or DHPrs and release channels or RyRs) are increasingly found in other cells and tissues. Its involvement is recognized in an increasing number of inheritable diseases. Its changes, upon prolonged exercise, crucially contribute to muscle fatigue. EC coupling has traditionally been probed by measuring Ca2+ transients, based on the consensus that control of Ca2+ release is exerted by ligands (like Ca2+, or loops in the DHPr) at the cytosolic face of the RyRs. Evidence is now increasing for crucial regulation from within the SR. This is a proposal for the systematic study of such control. Four working hypotheses will be tested, which elaborate the idea of regulation from within the SR: 1) Ca2+ release activation is largely determined by SR Ca2+ content. 2) Ca2+ release termination is largely determined by local Ca2+ depletion within the SR. 3) A novel concerted mode of channel activation is controlled by SR Ca. 4) The intra-SR Ca2+-binding protein calsequestrin has a dynamic role in regulation of Ca2+ release, which may involve interactions with other SR proteins. These hypotheses will be tested by 1) monitoring Ca2+ release on single cells, at the cell-averaged and at the local level (where it results in Ca2+ sparks), while manipulating and monitoring the Ca2+ content of the SR. 2) Introducing extrinsic Ca2+ buffers in the SR, one of which, sulfate, has already proved to have extraordinary effects. 3) Changing the concentration and functionality of calsequestrin, by its over- or under-expression in cells in culture, or by gene transfer to adult muscle. 4) To better understand molecular requirements, effects of calsequestrin will be sought on the control of single RyRs by Ca2+. 5) An existing model of Ca2+ release will help test the validity of various quantitative versions of these hypotheses. Technical innovation is substantial in aims 1 (simultaneous recording of intra-SR and cytosolic [Ca2+]), 2 (distinction of buffers and limit-buffers), 3 (transfer of calsequestrin genes to adult muscle) and 4 (combined application of calsequestrin and photoreleased Ca to channels in bilayers).