CROSS BRIDGE KINETICS AND REGULATION

血管平滑肌是血流分布的重要调节因子，其异常可导致高血压、动脉粥样硬化、冠状动脉再狭窄和休克。其他平滑肌也起着同样重要的作用，调节气道功能、胃肠道、子宫和膀胱的运动。本课题将确定横桥循环的规律和动力学，确定所有平滑肌收缩的根本机制，确定横桥循环的动力学，其结构基础是可变的、平滑肌特异性表达必需的轻链和重链肌球蛋白亚型。将分别对含有或不含重链插入肌球蛋白的平滑肌进行功能研究，并通过靶向表达或交换轻链异构体进行选择性修饰。交叉桥ADP和无机磷酸盐的脱落率将用The Core开发的新型荧光探针来测定，并将与力的发展速度和缩短速度有关。我们将确定参与微调肌球蛋白轻链激酶和磷酸酶活性之间平衡的激酶、磷酸盐和其他蛋白质（与T. Haystead合作），并通过对缺乏（敲除）或受到该蛋白靶向过表达的小鼠模型的功能研究，确定磷酸酶调节蛋白、端粒蛋白的生理作用（与G. Owens合作）。

Vascular smooth muscle is an essential regulator of the distribution of blood flow and its abnormalities contribute to high blood pressure, atherosclerosis, coronary restenosis and shock. Other smooth muscles play similarly important roles and regulate airway function and gastrointestinal, uterine and bladder movements. This project will determine the regulations and kinetics of crossbridge cycling, the fundamental mechanism of contraction of all smooth muscles, determines the kinetics of crossbridge cycling, and its structural basis is the variable, smooth muscle-specific expression of essential light chain and heavy chain myosin isoforms. Functional studies will be conducted on smooth muscles containing, respectively, myosins with or without the heavy chain insert and selectively modified by targeted expression or exchange of light chain isoforms. The off-rates of ADP and inorganic phosphate from crossbridges will be determined with novel fluorescent probes developed by The Core, and will be related to the rates of force development and shortening velocity. We will identify the kinases, phosphates and other proteins involved in fine-tuning the balance between myosin light chain kinase and phosphatase activities (collaboration with T. Haystead) and determine the physiological role of the phosphatase- regulating protein, telokin, through functional studies of mouse models deficient (knockout) or subjected to targeted over-expression of this protein (collaboration with G. Owens).