PHYSIOLOGICAL ROLE OF THE MYOSIN REGULATORY LIGHT CHAINS

拟议研究的长期目标是确定 肌球蛋白调节轻链（RLC）在 骨骼肌收缩的调节和/或调制。 的 要检验的中心假设是，Ca 2+和/或Mg 2+结合到 RLC上的单个Ca 2+结合位点在调节中起作用 和/或收缩的调节。为了验证这一假设， 将追求以下具体目标：一。 什么是Ca 2+和Mg 2 + 结合特性的单一钙离子结合位点上的RLC在肌肉？ 为了充分理解金属（Ca 2+和/或Mg 2+）如何与RLC结合， 影响收缩，RLC在肌肉中的金属结合特性将 被衡量。 二. Ca ~（2+）与RLC的结合是否影响力的速率 发展和/或放松，如果是，如何？若干条证据 表明Ca 2+与RLC Ca 2+结合位点的结合影响了 力的发展，通过某种方式改变跨桥动力学。 的 提出的实验将通过确定Ca 2+是否 结合影响力发展和/或松弛的速率。 如果， 正如预期的那样，与RLC结合的金属在收缩中起作用， 改变RLC的金属结合性质应当改变金属 任何受影响的收缩过程的依赖性。 一系列RLC Ca 2 + 结合位点突变体（例如，失活位点，较高的Ca 2+亲和力/ 特异性位点等）将被整合到皮肤肌肉纤维中 并测试它们对a）稳态力发展，B） 力发展的Ca 2+依赖性，c）激活/ 松弛，和d）力发展速率的Ca 2+依赖性。 MLCK对RLC的磷酸化是否影响钙依赖性 和/或力发展和松弛的动力学，并且RLC 磷酸化与RLC金属结合功能相互作用？ 我们 初步研究表明，RLC主要是磷酸化的 在孤立的纤维中，改变磷酸化水平 对力发展的Ca 2+依赖性的影响比 以前曾受到赞赏。 我们的研究结果还表明，Ca 2 + 为了观察RLC的这些效应，需要与RLC绑定 磷酸化 这些结果将得到证实，并延伸到问 以下问题：1）磷酸化对 力发展的Ca 2+依赖性，伴随着 力激活/松弛的动力学; 2）是金属结合 受磷酸化影响的RLC的性质，反之亦然，以及3） 磷酸化的影响是直接的，由于RLC Ca 2+的变化 结合，或间接，通过跨桥效应对肌钙蛋白钙离子亲和力？ 这些问题的答案将决定机制和 MLCK磷酸化RLC在骨骼肌中重要性 收缩。 总之，我们的实验将详细定义 RLC在调节和/或调制 骨骼肌收缩。

The long-term goal of the proposed studies is to determine the physiological role(s) of the regulatory light chains of myosin (RLC) in the regulation and/or modulation of skeletal muscle contraction. The central hypothesis to be tested is that Ca2+ and/or Mg2+ binding to the single Ca2+ binding site on the RLC plays a role in the regulation and/or modulation of contraction. In order to test this hypothesis, the following Specific Aims will be pursued: I. What are the Ca2+ and Mg2+ binding properties of the single Ca2+ binding site on the RLC in muscle? To fully understand how metal (Ca2+ and/or Mg2+) binding to RLC might affect contraction, the metal binding properties of RLC in muscle will be measured. II. Does Ca2+ binding to the RLC affect the rate of force development and/or relaxation and, if so, how? Several lines of evidence suggest that Ca2+ binding to the RLC Ca2+ binding site affects the rate of force development, by somehow altering cross-bridge kinetics. The proposed experiments will test this idea by determining whether Ca2+ binding influences the rate of force development and/or relaxation. If, as expected, metal binding to the RLC plays a role in contraction, then changing the metal binding properties of the RLC should change the metal dependency of any affected contractile process. A series of RLC Ca2+ binding site mutants (e.g., inactivated site, higher Ca2+ affinity/ specificity site, etc.) will be incorporated into skinned muscle fibers and tested for their effects on a) steady state force development, b) the Ca2+-dependence of force development, c) the rate of activation/ relaxation, and d) the Ca2+ dependence of the rate of force development. III: Does phosphorylation of RLC by MLCK affect the Ca2+ dependence and/or kinetics of force development and relaxation, and does RLC phosphorylation interact functionally with RLC metal binding? Our Preliminary Studies have shown that the RLC are mostly phosphorylated in isolated fibers, and that changing the level of phosphorylation has a much larger effect on the Ca2+ dependence of force development than had been previously appreciated. Our results also suggest that Ca2+ binding to the RLC is required to observe these effects of RLC phosphorylation. These results will be confirmed and extended to ask the following questions: 1) are the effects of phosphorylation on the Ca2+-dependence of force development accompanied by changes in the kinetics of force activation/relaxation; 2) are the metal binding properties of the RLC affected by phosphorylation and vice versa and 3) are the effects of phosphorylation direct, due to changes in RLC Ca2+ binding, or indirect, through cross-bridge effects on Tn Ca2+ affinity? The answers to these questions will determine the mechanism and the importance of MLCK phosphorylation of RLC in skeletal muscle contraction. In summary, our experiments will define in detail the potential role of the RLC in the regulation and/or modulation of skeletal muscle contraction.