Physiological Role of the Myosin Regulatory Light Chain

描述（由申请人提供）：拟议研究的长期目标是确定肌球蛋白（RLC）的调节轻链在骨骼肌收缩的调节和/或调节中的生理作用-中心假设是RLC在横纹肌收缩的调节和/或调节中起重要作用。具体来说，需要验证的假设是1)Ca2+和/或Mg+结合到RLC上的单个Ca2+- mg2 +结合位点，2)Ser的磷酸化（通过Ca2+-钙调蛋白激活的肌球蛋白轻链激酶，MLCK）在调节和/或调节收缩中发挥重要作用。在这笔赠款的头三年里，在了解区域协调委员会在收缩方面的作用方面取得了相当大的进展。我们已经证明a) RLC影响过桥循环；b) RLC的磷酸化增加了力发育和细丝活化肌球蛋白atp酶活性的Ca2+敏感性；c) RLC的磷酸化增加了最大的力产生，d) Ca2+- mg2 +结合位点是磷酸化诱导Ca2+对力发展的敏感性转移所必需的。后者表明Ca2+-Mg2+结合位点与磷酸化位点之间存在偶联。此外，我们已经表明，内源性RLC磷酸化水平，除了肌钙蛋白所起的作用，是骨骼肌Ca2+敏感性力发展的关键决定因素，其幅度以前没有被认识到。尽管这些体外实验结果告诉了我们很多关于RLC的功能，但它们在体内的功能仍不完全清楚。RLC在骨骼肌中的作用一直充满争议，这也是事实，其中很大一部分来自于研究RLC功能的方法还没有可用的事实。不幸的是，在肌球蛋白或更复杂的系统（如肌原纤维和皮肤肌纤维）中，没有无伪迹的选择性提取/替代RLC的方法，这导致了争议。接近RLC角色的一种更明确的方式是开发可以在本地设置中操纵RLC的系统。目前有几种强大的方法可以使这成为可能，其中包括转基因和敲入/敲出小鼠模型。为了验证上述假设，我们将利用转基因和敲入/敲出动物模型，利用各种RLC突变体替代内源性小鼠RLC。将对这些动物的完整肌肉和剥皮纤维进行复杂的生理学研究，以确定RLC在骨骼肌收缩和调节中的作用。提出的研究将使我们能够独特地研究RLC在横纹肌收缩中的作用，并确定它们在体内的作用。

DESCRIPTION (provided by the applicant): 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 is that the RLC play an important role in the regulation and/or modulation of striated muscle contraction. Specifically, the hypotheses to be tested are that 1) Ca2+ and/or Mg+ binding to the single Ca2+-Mg2+ binding site on the RLC and 2) the phosphorylation of Ser (by Ca2+- calmodulin activated myosin light chain kinase, MLCK) play important roles in the regulation and/or modulation of contraction. In the first three years of this grant considerable progress has been made in understanding the role of the RLC in contraction. We have shown that a) the RLC affect crossbridge cycling; b) phosphorylation of the RLC increases the Ca2+- sensitivity of both force development and thin filament activated myosin ATPase activity; c) phosphorylation of the RLC increases maximal force production and d) the Ca2+-Mg2+ binding site is required for the phosphorylation induced shift in the Ca2+- sensitivity of force development. The latter suggests that there is coupling between the Ca2+-Mg2+ binding site and the phosphorylation site. In addition, we have shown that the level of endogenous RLC phosphorylation, in addition to the role played by Troponin, is a crucial determinant of the Ca2+- sensitivity of force development in skeletal muscle, the magnitude of which was not previously appreciated. Although these in vitro results have told us much about the function of the RLC, it is still not totally clear what their in vivo function is. It is also true that the role of the RLC in skeletal muscle has been fraught with controversy and much of this has come from the fact that methods to study the function of the RLC have not been available. Unfortunately there have been no artifact-free methods for the selective extraction/replacement of the RLC in either myosin or in more complex systems, e.g., myofibrils and skinned muscle fibers and this has contributed to the controversy. A more unequivocal way of approaching the role of the RLC would be to develop systems whereby the RLC can be manipulated in a native setting. Several powerful approaches are available today that make this possible and these include transgenic and knock-in/out mouse models. To test the above hypotheses, transgenic and knock-in/out animal models, where various mutants of RLC will replace the endogenous mouse RLC, will be utilized. Sophisticated physiological studies on both intact muscle and skinned fibers from these animals will be performed to determine the role of the RLC in skeletal muscle contraction and regulation. The proposed studies will allow us to uniquely study the role of the RLC in striated muscle contraction and to determine their in vivo role.