CATCH: Mechanochemistry and Regulation in Smooth muscle

描述(申请人提供)：这项研究的目的是了解调节紧张力维持的机制，在非常低的能量使用量和缓慢的跨桥循环速度的情况下，在平滑肌中。这是一种“抓钩”肌肉的特征，即贻贝的前骨骨牵引器(ABRM)肌肉，脊椎动物肌肉的“闩锁”状态的原型。我们发现，位于粗丝上的迷你肌动蛋白twittin的磷酸化状态是力量产生和维持的主要调节点。捕获力与肌动蛋白的去磷酸化有关，而捕获力的释放或松弛与其蛋白激酶A介导的磷酸化有关。我们还表征了不同的机械状态及其通过twitin磷酸化的调节，并确定了Mytilus twitin的序列。ABRM是一个理想的模型，因为它容易(A)在完整和通透性的肌肉中产生不同的机械状态(钙驱动的循环、非循环或捕捉交叉桥、放松)，以及(B)控制调节机制，即抽动蛋白磷酸化和钙的状态。在收缩的调节过程中涉及的蛋白质的保守部分，如肌动蛋白、投射蛋白、C蛋白和钙调蛋白，与twitin的部分相似，表明它在一般的调节过程中发挥作用。其具体目的是：(1)确定肌球蛋白与肌球蛋白结合和释放过程中，肌动蛋白磷酸化介导的ADP变化在控制肌肉机械状态中的作用。(2)通过测定肌球蛋白分子重组部分对通透性肌肉力学参数的影响，确定其调控捕获量的机制。(3)测定肌球蛋白及其重组部分的粗丝和细丝结合特性。(4)通过测量连接在调节轻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.