CATCH--MECHANOCHEMISTRY AND REGULATION IN SMOOTH MUSCLE

所有平滑肌（哺乳动物和无脊椎动物）的一个独特而重要的特征是能够在等长收缩的时间过程中改变力产生和维持的能量成本。 控制平滑肌中的力维持或张力的基本机制尚不清楚。 我们的发现，tickin（一种微型肌联蛋白）的磷酸化状态调节捕获和力的生产在前足丝牵开肌（ABRM）的紫贻贝，开辟了新的研究在这里描述的分子基础上的功能。 我们的总体目标是确定tickin控制肌动蛋白-肌球蛋白相互作用和产生的机械输出的机制。 无脊椎动物的横纹肌，平滑捕捉和相位肌中普遍存在的tickin表明，tickin可以作为所有这些肌肉类型的调节器。 这种调节的基本机制的理解可能会很好地提供控制其他肌肉类型，特别是哺乳动物的平滑肌，它显示出非常相似的机械特性，从无脊椎动物的平滑肌的新见解。本文研究了M. edulis和扇贝（P. magellanicus）的纹状闭壳肌作为实验模型。 根据需要，将使用完整和透化的无脊椎动物肌肉。 具体目的是：（1）确定tickin磷酸化引起肌球蛋白跨桥分离速率常数增加的机制;（2）确定完整和透化ABRM中tickin磷酸化程度与其机械效应之间的关系;（3）检验肌球蛋白运动肌动蛋白的固有速率因tickin的存在而减弱，以及tickin的磷酸化恢复固有快速率的假设，利用体外运动研究肌动蛋白运动的天然粗丝从ABRM和体壁的C。优雅 将测量粗丝上的核苷酸周转，以了解当钙浓度变化时，抽搐蛋白磷酸化如何改变ATP酶活性。(4)确定tickin磷酸化对快纹扇贝收肌的影响，以了解这是否是无脊椎动物肌肉中跨桥动力学调节的普遍机制。(5)对tickin的生化研究将描述tickin分子的特征，并确定其通过蛋白激酶A磷酸化控制收缩蛋白相互作用的机制。

A unique and important characteristic of all smooth muscles (mammalian and invertebrate) is the ability to vary the energy cost of force production and maintenance during the time course of isometric contractions. The basic mechanisms controlling force maintenance, or tone in smooth muscle are not well understood. Our discovery that the phosphorylation state of twitchin (a mini-titin) regulates catch and force production in the anterior byssus retractor muscle (ABRM) of Mytilus edulis, has opened the way to new studies described here on the molecular basis of its function. Our overall goal is to determine the mechanisms whereby twitchin controls actin-myosin interaction and resulting mechanical output. The ubiquitous presence of twitchin in invertebrate striated, smooth catch and phasic muscles suggests that twitchin may serve as a regulator in all of these muscle types. The understanding of the basic mechanisms underlying this regulation may very well provide new insights on the control of other muscle types, especially mammalian smooth muscle, which shows very similar mechanical characteristics as the smooth muscles from invertebrates. In the proposed studies the smooth ABRM of M. edulis, and striated adductor of the sea scallop (P. magellanicus) will serve as experimental models. Intact and permeabilized invertebrate muscles will be used, as needed. The Specific Aims are to (1) Determine the mechanism by which twitchin phosphorylation gives rise to an increase in the detachment rate constant of the myosin crossbridge; (2) Determine the relationship between the degree of phosphorylation of twitchin and its mechanical effect in intact and permeabilized ABRM; (3) Test the hypothesis that the intrinsic rate of actin movement by myosin is attenuated by the presence of twitchin, and that the phosphorylation of twitchin restores the intrinsic fast rate, using in vitro motility studies of actin movement on native thick filaments from ABRM and the body wall of C. elegans. Nucleotide turnover on the thick filaments will be measured in order to learn how twitchin phosphorylation alters the ATPase activity when calcium concentration is varied. (4) Determine of the effect of twitchin phosphorylation on fast striated scallop adductor muscle in order to learn whether this is a generalized mechanism for the modulation of crossbridge kinetics in invertebrate muscle. (5) Biochemical studies on twitchin will characterize the twitchin molecule and determine the mechanism by which its phosphorylation by protein kinase A controls the interaction of contractile proteins.