CALCIUM AND CALMODULIN IN SMOOTH MUSCLE CONTRACTION

钙调蛋白(CaM)是一种普遍存在的钙结合蛋白，它结合 肌球蛋白轻链激酶(MLCK)、钙调蛋白(CAD)、钙蛋白(CALP)和α 肌膜Ca-ATPase对血管平滑肌收缩的调节 肌肉。CA与CaM的结合刺激其与这些蛋白质中的每一种结合 但是关于钙-钙调素结合的速率和 在钙瞬变过程中从这些重要的靶蛋白解离。 我们将使用荧光停流和我们的荧光摄像头来 测定Kd‘s和每个分子的缔合和解离速率 并确定每个CaM-靶蛋白复合体 复合体被钙的去除所破坏。我们将确定这些目标是否 蛋白质与CaM‘s N-和CaM’s N对钙交换率的影响不同 C-末端钙结合部位及与这些部位的钙解离有关 EGTA诱导的复杂破坏的比率。我们会自然地学习 发生突变的CaM‘s以确定是否改变了钙与其N-和 C-末端钙结合位点是导致其不能激活的原因 钠离子通道和钾离子通道。这应该会产生重要的新 关于钙-钙调素结合和激活超过 20种不同的细胞靶蛋白。内分泌激活型心绞痛 磷脂依赖蛋白激酶C(PKC)导致其在 静息钙水平及其对MLCK、CAD、CALP和Ca-ATPase的磷酸化 来调节和维持平滑的肌肉收缩。我们将确定是否 这些靶蛋白的PKC磷酸化改变了CaM条带和 激活。我们第一次直接测量了钙离子的偏离率 从纯化的PKC和Ca-ATPase中提取。这将使我们能够确定 这两种酶的生理激活剂的作用机制和程度 调节它们的钙亲和力和活性。这些研究应该 增加对荷尔蒙激活如何调节这些病例的理解 Smooth中结合蛋白及CaM与靶蛋白的相互作用 肌肉。

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.