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-在肌肉中传导动作电位以增加细胞内Ca2+浓度和收缩-是第一个公认的Ca2+信号传导的例子。它的关键分子（电压传感器dhpr和释放通道ryr）越来越多地出现在其他细胞和组织中。在越来越多的遗传性疾病中认识到它的作用。在长时间的运动中，这种变化对肌肉疲劳起着至关重要的作用。EC偶联传统上是通过测量Ca2+瞬态来探测的，基于对Ca2+释放的控制是由RyRs细胞质表面的配体（如Ca2+，或DHPr中的环）施加的共识。现在越来越多的证据表明，在sr内部进行关键的监管。这是对这种控制进行系统研究的建议。将测试四个工作假设，它们阐述了SR内部调节的想法：1)Ca2+释放激活在很大程度上取决于SR Ca2+含量。2) Ca2+释放终止主要由SR内局部Ca2+耗尽决定。3)一种新的协同通道激活模式由SR Ca控制。4)SR内Ca2+结合蛋白calsequestrin在Ca2+释放调控中具有动态作用，可能涉及与其他SR蛋白的相互作用。这些假设将通过1)监测Ca2+释放在单个细胞上，在细胞平均水平和局部水平（在那里它导致Ca2+火花），同时操纵和监测SR的Ca2+含量进行测试。2)在SR中引入外源性Ca2+缓冲液，其中之一，硫酸盐，已经被证明具有非凡的效果。3)改变钙调雌激素的浓度和功能，通过其在培养细胞中的过表达或过低表达，或通过基因转移到成人肌肉。4)为了更好地了解分子需求，我们将寻求钙sequestrin对Ca2+控制单个RyRs的影响。现有的Ca2+释放模型将有助于测试这些假设的各种定量版本的有效性。技术创新在目标1（同时记录sr内和细胞质内[Ca2+]）， 2（缓冲液和极限缓冲液的区分），3（钙sequestrin基因转移到成人肌肉）和4（钙sequestrin和光释放的钙在双层通道中的联合应用）中是实质性的。

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).