CATCH--MECHANOCHEMISTRY AND REGULATION IN SMOOTH MUSCLE

所有平滑肌肉(哺乳动物和无脊椎动物)的一个独特而重要的特征是，在等长收缩的时间过程中，能够改变力量产生和维持的能量成本。控制肌力维持或肌张力的基本机制尚不清楚。我们的发现是，肌动蛋白(一种微型肌动蛋白)的磷酸化状态调节了紫贻贝前部牵引肌(ABRM)的抓力和作用力的产生，这为从分子基础上对其功能进行研究开辟了道路。我们的总体目标是确定twitin控制肌动蛋白-肌球蛋白相互作用和由此产生的机械输出的机制。在无脊椎动物的横纹肌、滑钩肌和相肌中普遍存在抽动蛋白，这表明抽动蛋白可能在所有这些肌肉类型中都起到了调节作用。对这种调控的基本机制的理解可以很好地为其他肌肉类型的控制提供新的见解，特别是哺乳动物的平滑肌，它表现出与无脊椎动物的平滑肌肉非常相似的力学特性。在拟议的研究中，蓝鳍金枪鱼和扇贝的横纹内收肌将作为实验模型。根据需要，将使用完整和通透性良好的无脊椎动物肌肉。这些研究的具体目的是：(1)确定肌球蛋白磷酸化导致肌球蛋白交叉桥的脱落速率常数增加的机制；(2)确定肌球蛋白磷酸化程度与其在完整和通透性ABRM中的机械效应之间的关系；(3)利用体外对线虫ABRM天然粗丝和体壁上肌动蛋白运动的运动学研究，验证肌球蛋白的固有移动速度可被肌球蛋白减弱的假设，以及肌动蛋白的磷酸化可恢复固有的快速速率的假设。将测量粗丝上的核苷酸周转率，以了解当钙浓度变化时，肌动蛋白磷酸化如何改变ATPase活性。(4)测定快速横纹扇贝内收肌中twitin磷酸化的作用，以了解这是否是无脊椎动物肌肉交叉桥动力学调节的一种普遍机制。(5)对肌萎缩素的生化研究将确定肌萎缩素分子的特征，并确定其被蛋白激酶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.