CATCH: Mechanochemistry and Regulation in Smooth muscle

描述（由申请人提供）：本研究的目的是了解在平滑肌中以非常低的能量使用和缓慢的横桥循环速率调节张力维持的机制。 这是贻贝前足丝牵开肌（ABRM）的特征，是脊椎动物平滑肌“闩锁”状态的原型。 我们发现，磷酸化状态的微型肌联蛋白，tickin，位于粗丝，是一个主要的网站的调节力的产生和维持。 捕捉力与tickin的去磷酸化有关，而捕捉的释放或松弛与其蛋白激酶A介导的磷酸化有关。 我们还确定了不同的机械状态和它们的调控tickin磷酸化，并确定了贻贝tickin的序列。 ABRM是理想的模型，因为（a）可以在完整和透化的肌肉中容易地产生不同的机械状态（钙驱动的循环、非循环或捕获横桥、松弛），以及（B）控制调节机制，即，tickin磷酸化和钙的状态。 保守部分的蛋白质，如肌联蛋白，projectin，C-蛋白和caldesmon，涉及的调节过程中的收缩是类似的部分tickin，这表明其在调节过程中的作用一般。 具体目标是：（1）确定tickin磷酸化介导的ADP与肌球蛋白结合和从肌球蛋白释放的变化在控制抓肌的机械状态中的作用。 (2)通过测量分子的重组部分对透化肌肉中机械参数的影响，确定tickin调节捕获的机制。(3)tickin和tickin重组部分的粗细丝结合特性的测定。 (4)通过测量附着在调节轻链上的荧光探针的方向变化来确定肌球蛋白横桥参与捕获力维持的程度。

DESCRIPTION (provided by applicant): The goal of this research is to understand the mechanisms that regulate tonic force maintenance with a very low energy usage and slow crossbridge cycling rate in smooth muscle. These are characteristics of a "catch" muscle, the anterior byssus retractor (ABRM) muscle of the mussel Mytilus edulis, the prototype of the "latch" state of vertebrate smooth muscle. We showed that the phosphorylation state of the mini-titin, twitchin, located on the thick filament, is a major site of regulation of force production and maintenance. Catch force is associated with the dephosphorylation of twitchin, and release of catch, or relaxation, is associated with its protein kinase A mediated phosphorylation. We have also characterized distinct mechanical states and their regulation by twitchin phosphorylation, and determined the sequence of Mytilus twitchin. The ABRM is an ideal model because of the ease (a) by which distinct mechanical states (calcium-driven cycling, non-cycling or catch crossbridges, relaxation) can be produced in intact and permeabilized muscles, and (b) of controlling the regulatory mechanisms, i.e., the state of twitchin phosphorylation and calcium. Conserved portions of proteins such as titin, projectin, C-protein and caldesmon, implicated in the regulatory processes of contraction are similar to portions of twitchin, suggesting its role in regulatory processes in general. The SPECIFIC AIMS are: (1) Determination of the role of twitchin phosphorylation-mediated changes in ADP binding to and release from myosin in controlling the mechanical states of catch muscle. (2) Determination of the mechanism by which twitchin regulates catch through measurement of the effects of recombinant portions of the molecule on mechanical parameters in permeabilized muscle.(3) Determination of the thick and thin filament binding characteristics of twitchin and recombinant portions of twitchin. (4) Determination of the extent of involvement of myosin crossbridges in catch force maintenance by measurements of changes in orientation of fluorescent probes attached to the regulatory light chain.