CALCIUM AND CALMODULIN IN SMOOTH MUSCLE CONTRACTION

钙和钙调蛋白在平滑肌收缩中的作用

• 批准号: 2139143
• 负责人: J DAVID JOHNSON
• 金额: $ 16.1万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2139143
• 关键词: allosteric site; calcium channel blockers; calmodulin; cell cell interaction; chemical binding; fluorescent dye /probe; muscle contraction; myosin light chain kinase; phorbols; phosphorylation; protein kinase C; smooth muscle; stop flow technique; swine

Calmodulin (CaM) is a ubiquitous calcium (Ca) binding protein which bind myosin light chain kinase (MLCK), caldesmon (CaD), calponin (Calp) and a sarcolemmal Ca-ATPase to regulate the contraction of vascular smooth muscle. Ca binding to CaM stimulates it binding to each of these proteins but little is known concerning the rates at which Ca-CaM binds and dissociates from these important target proteins during a Ca transient. We will use fluorescence stopped-flow with our fluorescent CaM's to determine the Kd's and the rates of association and dissociation of each of these CaM-target protein complexes and determine the rate at which each complex is disrupted by Ca removal. We will determine if these target proteins have differential effects on Ca exchange rates with CaM's N- and C-terminal Ca binding sites and relate Ca dissociation from these sites to the rate of EGTA induced complex disruption. We will study naturally occurring mutant CaM's to determine if altered Ca binding to their N- and C-terminal Ca binding sites is responsible for their inability to activate Na and K ion channels, respectively. This should yield important new information on the mechanism by which Ca-CaM binds and activates more than 20 different cellular target proteins. Hormonal activation of the Ca phospholipid dependent protein kinase C (PKC) results in its activation at resting Ca levels and its phosphorylation o MLCK, CaD, Calp, and Ca-ATPase to modulate and maintain smooth muscle contraction. We will determine if PKC phosphorylation of these target proteins alters CaM banding and activation. We have, for the first time directly measured Ca off-rates from purified PKC and the Ca-ATPase. This will allow us to determine the mechanism and extent that physiological activators of these two enzymes modulate their Ca affinity and their activity. These studies should increase our understanding of how hormonal activation modulates these Ca binding proteins and CaM's interaction with its target proteins in smooth muscle.

钙调素（CaM）是一种普遍存在的钙结合蛋白， 肌球蛋白轻链激酶（MLCK）、钙调蛋白（CaD）、钙调蛋白（卡尔普）和一种 肌膜Ca-ATP酶调节血管平滑肌收缩 肌肉. 钙结合钙调素刺激它结合这些蛋白质 但关于Ca-CaM结合的速率知之甚少， 在钙瞬变期间与这些重要的靶蛋白解离。 我们将使用荧光停流与我们的荧光钙调素的， 确定Kd以及每个分子的结合率和解离率 这些钙调素-靶蛋白复合物，并确定每一个 复合物被Ca去除破坏。 我们将确定这些目标 蛋白质对Ca与CaM的N-和 C-末端Ca结合位点，并将Ca从这些位点解离， EGTA诱导的复合物破坏率。 我们自然会学习 发生突变的钙调素，以确定是否改变钙结合到他们的N-和 C-末端Ca结合位点负责它们不能激活 Na和K离子通道。 这将产生重要的新成果， 关于Ca-CaM结合和激活超过 20种不同的细胞靶蛋白。 钙的激素激活 磷脂依赖性蛋白激酶C（PKC）在 静息Ca水平及其对MLCK、CaD、卡尔普和Ca-ATP酶的磷酸化 调节和维持平滑肌收缩。 我们将确定是否 这些靶蛋白的PKC磷酸化改变了CaM带， activation. 我们首次直接测量了钙离子的解离速率 从纯化的PKC和Ca-ATP酶。 这将使我们能够确定 这两种酶的生理激活剂 调节它们的Ca亲和力和它们的活性。 这些研究应 增加我们对激素激活如何调节这些Ca 结合蛋白及其与靶蛋白的相互作用 肌肉.