CATCH--MECHANOCHEMISTRY AND REGULATION IN SMOOTH MUSCLE

Catch--平滑肌的机械化学和调节

• 批准号: 6374992
• 负责人: MARION Joyce SIEGMAN
• 金额: $ 35.36万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-12-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6374992
• 关键词: Bivalvia; actins; alternatives to animals in research; biomechanics; cyclic AMP; cytoskeletal proteins; enzyme activity; gel electrophoresis; high performance liquid chromatography; mechanical stress; muscle contraction; muscle function; muscle proteins; muscle tone; myosins; phosphorylation; protein kinase; protein structure function; radiotracer; smooth muscle; striated muscles; tissue /cell culture

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.

所有平滑肌（哺乳动物和无脊椎动物）的一个独特且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消耗。 控制平滑肌力量维持或张力的基本机制尚不清楚。 我们发现，twitchin（一种微型肌联蛋白）的磷酸化状态可调节贻贝前足丝牵拉肌 (ABRM) 的捕获和力的产生，这为本文所述的基于其功能的分子基础的新研究开辟了道路。 我们的总体目标是确定抽搐控制肌动蛋白-肌球蛋白相互作用以及由此产生的机械输出的机制。 无脊椎动物的横纹肌、平滑肌和相位肌中普遍存在抽动蛋白，这表明抽动蛋白可以作为所有这些肌肉类型的调节器。 了解这种调节的基本机制很可能为其他肌肉类型的控制提供新的见解，特别是哺乳动物平滑肌，它表现出与无脊椎动物平滑肌非常相似的机械特性。在拟议的研究中，M. edulis 的光滑 ABRM 和海扇贝 (P. magellanicus) 的条纹内收肌将作为实验模型。 根据需要，将使用完整且透化的无脊椎动物肌肉。 具体目标是 (1) 确定抽动蛋白磷酸化导致肌球蛋白横桥脱离速率常数增加的机制； (2)确定完整和透化ABRM中twitchin的磷酸化程度与其机械效应之间的关系； (3) 使用来自 ABRM 的天然粗丝和线虫体壁上的肌动蛋白运动的体外运动研究，检验肌球蛋白引起的肌动蛋白运动的内在速率因 twitchin 的存在而减弱，并且 twitchin 的磷酸化恢复内在的快速速率的假设。 将测量粗丝上的核苷酸周转率，以了解当钙浓度变化时，抽搐磷酸化如何改变 ATP 酶活性。 (4)确定twitchin磷酸化对快速横纹扇贝内收肌的影响，以了解这是否是无脊椎动物肌肉中横桥动力学调节的通用机制。 (5) 对 twitchin 的生化研究将表征 twitchin 分子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。