Skeletal Muscle. Ca2+ release control inside the SR

描述（由申请方提供）：Ca 2+信号传导将质膜事件与代谢变化联系起来。兴奋-收缩（EC）偶联-在肌肉中转导动作电位以增加细胞内Ca 2+浓度和收缩-是Ca 2+信号传导的第一个公认的例子。它的关键分子（电压传感器或DHPrs和释放通道或RyR）越来越多地在其他细胞和组织中发现。越来越多的遗传性疾病都与此有关。它的变化，在长时间的运动，至关重要的是有助于肌肉疲劳。EC偶联传统上通过测量Ca 2+瞬变来探测，基于这样的共识，即Ca 2+释放的控制是由RyR的胞质面处的配体（如Ca 2+或DHPr中的环）施加的。现在越来越多的证据表明，关键的监管从内部SR。这是一个系统的研究这种控制的建议。将测试四个工作假设，其阐述了SR内调节的想法：1）Ca 2+释放激活主要由SR Ca 2+含量决定。2)钙释放的终止主要取决于SR内的局部Ca ~（2+）耗尽。3）SR Ca ~（2+）控制一种新的协同通道激活模式。4)SR内钙结合蛋白calsequestrin在调节钙释放中具有动态作用，这可能涉及与其他SR蛋白的相互作用。这些假设将通过以下方式进行测试：1）在细胞平均水平和局部水平（其导致Ca 2+火花）监测单个细胞上的Ca 2+释放，同时操纵和监测SR的Ca 2+含量。2）在SR中引入外源性Ca 2+缓冲液，其中之一硫酸盐已被证明具有非凡的效果。3)改变钙螯合蛋白的浓度和功能，通过其在培养的细胞中的过度表达或表达不足，或通过基因转移到成人肌肉。4)为了更好地理解分子的要求，将寻求钙螯合蛋白对Ca 2+控制单个RyR的影响。 5)现有的Ca 2+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内和胞质[Ca 2 +]）、2（缓冲液和限制缓冲液的区别）、3（将钙螯合蛋白基因转移到成人肌肉）和4（将钙螯合蛋白和光释放的Ca结合应用于双层通道）中具有实质性意义。

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).