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).
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