CATCH: Mechanochemistry and Regulation in Smooth muscle

描述（由申请人提供）：本研究的目的是了解调节强直力维持的机制，以非常低的能量消耗和缓慢的平滑肌过桥循环速率。这些都是贻贝（Mytilus edulis）的前足牵开肌（ABRM）的特征，它是脊椎动物平滑肌“闩锁”状态的原型。我们发现，位于粗丝上的微丝蛋白（micro -titin, twitchin）的磷酸化状态是调节力产生和维持的主要位点。捕获力与抽搐的去磷酸化有关，而捕获的释放或松弛与其蛋白激酶A介导的磷酸化有关。我们还通过抽动蛋白磷酸化表征了不同的机械状态及其调控，并确定了贻贝抽动蛋白的序列。ABRM是一个理想的模型，因为(a)在完整的和渗透的肌肉中可以容易地产生不同的机械状态（钙驱动的循环，非循环或捕获交叉桥，松弛），以及(b)控制调节机制，即抽搐磷酸化和钙的状态。与收缩调节过程相关的蛋白质如titin、投影蛋白、c蛋白和caldesmon的保守部分与抽动蛋白的部分相似，表明其在一般调节过程中的作用。具体目的是：(1)确定抽搐磷酸化介导的ADP与肌球蛋白结合和释放的变化在控制肌肉机械状态中的作用。(2)通过测量分子重组部分对渗透肌肉力学参数的影响，确定抽搐调节捕获的机制。(3)测定抽动菌粗、细丝结合特性及抽动菌重组部分。(4)通过测量附着在调节光链上的荧光探针的取向变化来确定肌球蛋白交叉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.