CATCH: Mechanochemistry and Regulation in Smooth Muscle

CATCH：平滑肌的机械化学和调节

• 批准号: 6371068
• 负责人: MARION Joyce SIEGMAN
• 金额: $ 23.29万
• 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/6371068
• 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。控制平滑肌力维持或张力的基本机制尚不清楚。我们发现twitchin（一种微型titin）的磷酸化状态调节了Mytilus edulis前足牵开肌（ABRM）的捕获和力的产生，为本文描述的关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-肌球蛋白相互作用和由此产生的机械输出的机制。抽搐在无脊椎动物横纹肌、平滑肌和相肌中普遍存在，这表明抽搐可能在所有这些肌肉类型中起调节作用。对这种调节的基本机制的理解很可能为其他肌肉类型的控制提供新的见解，特别是哺乳动物的平滑肌，它表现出与无脊椎动物的平滑肌非常相似的机械特性。本研究将以毛蚶的光滑ABRM和麦哲伦扇贝的条纹内收肌为实验模型。如有需要，将使用完整和渗透的无脊椎动物肌肉。具体目的是：(1)确定抽搐磷酸化导致肌球蛋白过桥分离速率常数增加的机制；(2)确定完整和通透性ABRM中抽动蛋白磷酸化程度与其机械效应的关系；(3)通过体外对秀丽隐杆线虫ABRM粗丝和体壁上肌动蛋白运动的动态研究，验证肌球蛋白的内在运动速率因抽动蛋白的存在而减弱，而抽动蛋白的磷酸化恢复了内在的快速运动速率的假设。将测量粗纤维上的核苷酸周转，以便了解当钙浓度变化时抽搐磷酸化如何改变atp酶活性。(4)确定抽搐磷酸化对快速条纹扇贝内收肌的影响，以了解这是否是无脊椎动物肌肉过桥动力学调节的一般机制。(5)对抽动蛋白的生化研究将对抽动蛋白分子进行表征，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。