Physiological Role of the Myosin Regulatory Light Chain

描述（由申请方提供）：拟定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/或调制中的生理作用。中心假设是RLC在横纹肌收缩的调节和/或调制中起重要作用。具体而言，待检验的假设是：1）Ca 2+和/或Mg+结合至RLC上的单个Ca 2 +-Mg 2+结合位点，以及2）Ser的磷酸化（通过Ca 2 +-钙调蛋白激活的肌球蛋白轻链激酶，MLCK）在收缩的调节和/或调节中起重要作用。在第一个三年的补助金相当大的进展已经取得了理解的RLC在收缩中的作用。我们已经表明：a）RLC影响跨桥循环; B）RLC的磷酸化增加了力发展和细丝激活的肌球蛋白ATP酶活性的Ca 2+敏感性; c）RLC的磷酸化增加了最大力的产生; d）Ca 2 +-Mg 2+结合位点是磷酸化诱导的力发展的Ca 2+敏感性的转变所必需的。后者表明Ca 2 +-Mg 2+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磷酸化的水平，除了由肌钙蛋白所发挥的作用，是一个关键的决定因素的Ca 2+敏感性的力量发展骨骼肌，其幅度是以前没有赞赏。虽然这些体外实验结果告诉我们很多关于RLC的功能，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，其中大部分来自于研究RLC功能的方法还不可用的事实。不幸的是，还没有无人为因素的方法来选择性提取/替换肌球蛋白或更复杂系统中的RLC，例如，肌原纤维和皮肤肌纤维，这引起了争议。接近RLC的作用的更明确的方式将是开发系统，由此RLC可以在本机设置中被操纵。如今，有几种强大的方法可以使这成为可能，其中包括转基因和基因敲入/敲除小鼠模型。为了测试上述假设，将使用转基因和敲入/敲除动物模型，其中RLC的各种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，并确定其在体内的作用。

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.