PHYSIOLOGICAL ROLE OF THE MYOSIN REGULATORY LIGHT CHAINS

拟议研究的长期目标是确定 肌球蛋白调节轻链的生理作用(S) 骨骼肌收缩的调节和/或调节。这个 有待检验的中心假设是，钙离子和/或镁离子与细胞 RLC上的单个Ca~(2+)结合部位在调节中起作用 和/或收缩的调制。为了检验这一假设， 将追求以下具体目标：一、什么是钙和镁 肌肉RLC上单个钙结合部位的结合特性？ 要充分了解金属(钙和/或镁)与RLC的结合情况 影响收缩，RLC在肌肉中的金属结合特性将 被测量。钙离子与RLC的结合是否影响RLC的作用力 发展和/或放松，如果是，如何发展和/或放松？有几条证据 提示钙离子与RLC钙结合部位的结合影响这一速率 力的发展，通过某种方式改变跨桥动力学。这个 拟议中的实验将通过确定钙离子是否 束缚会影响力量发展和/或松弛的速度。如果， 正如预期的那样，金属与RLC的结合在收缩中起作用，然后 改变RLC的金属结合属性应该会改变金属 任何受影响的收缩过程的依赖性。一系列RLC钙离子 结合位点突变体(例如，失活位点、更高的钙亲和力/ 特定站点等)会被结合到皮肤的肌肉纤维中 并测试了它们对a)稳态力发展的影响，b) 钙离子依赖的力量发展，c)激活率/ 松弛，和d)钙离子依赖性的力量发展速度。 MLCK对RLC的磷酸化是否影响RLC对钙的依赖性 和/或力发展和松弛的动力学，并且RLC 磷酸化与RLC金属结合在功能上相互作用？我们的 初步研究表明，RLC主要是磷酸化的 在分离的纤维中，这改变了磷酸化水平 力发育对钙离子依赖性的影响远大于 之前就受到了好评。我们的结果还表明，钙离子 需要与RLC结合才能观察RLC的这些效果 磷酸化。这些结果将得到证实并推广到询问 以下问题：1)是磷酸化对 伴随着钙离子变化的力量发育对钙的依赖 力激活/松弛的动力学；2)是金属结合 RLC的性质受磷酸化的影响，反之亦然。 由于RLC钙离子的变化，磷酸化的影响是直接的吗 结合，或间接，通过跨桥效应对TN的钙亲和力？ 这些问题的答案将决定机制和 RLC的MLCK磷酸化在骨骼肌中的重要性 收缩。总而言之，我们的实验将详细定义 RLC在调节和/或调节中的潜在作用 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.