Physiological Role of the Myosin Regulatory Light Chain

描述(申请人提供)：拟议研究的长期目标是确定肌球蛋白调节轻链在调节和/或调节骨骼肌收缩中的生理作用(S)--中心假设是肌球蛋白调节轻链在调节和/或调节横纹肌收缩中发挥重要作用。具体来说，要检验的假设是：1)钙和/或镁+与RLC上单一的钙-镁结合部位结合；2)丝氨酸的磷酸化(由钙-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，在理解区域合作委员会在收缩中的作用方面取得了相当大的进展。我们已经证明：a)RLC影响交叉桥循环；b)RLC的磷酸化增加了力发育的钙敏感性和细丝激活的肌球蛋白ATPase活性；c)RLC的磷酸化增加了最大的力产生，以及d)磷酸化诱导的力发育的钙敏感性的改变需要钙-镁结合位点。后者提示钙-镁结合部位和磷酸化部位之间存在偶联。此外，我们还表明，内源性RLC的磷酸化水平，除了肌钙蛋白所起的作用外，也是骨骼肌力发育对钙的敏感性的关键决定因素，其大小以前没有被认识到。虽然这些体外结果已经告诉了我们许多关于RLC功能的信息，但仍然不完全清楚它们在体内的功能是什么。RLC在骨骼肌中的作用也确实充满了争议，其中很大一部分是因为还没有研究RLC功能的方法。不幸的是，无论是在肌球蛋白中还是在更复杂的系统中，例如肌原纤维和去皮的肌肉纤维中，都没有无伪影的方法来选择性地提取/替换RLC，这就引起了争议。一种更明确地处理区域LC的作用的方法是开发可以在本地环境中操纵区域LC的系统。今天有几种强大的方法可以实现这一点，其中包括转基因和敲入/敲除小鼠模型。为了验证上述假设，将利用转基因和敲入/敲除动物模型，其中各种RLC突变体将取代内源性小鼠RLC。将对这些动物的完整肌肉和皮肤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。拟议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.