CALCIUM REGULATION OF VASCULAR SMOOTH MUSCLE CONTRACTION

钙通过其细胞内受体钙调素（CaM）起作用， 在调节血管张力方面起着关键作用。血管平滑肌 肌肉收缩依赖于钙调素依赖性 平滑肌肌球蛋白轻链激酶（MLCK）。我们想 了解钙调蛋白如何激活MLCK和其他酶， 调节血管平滑肌张力。 我们的重点是结构 钙调素与这些调节酶相互作用的基础。我们 建议产生CaM和心肌肌钙蛋白C（cTnC）的嵌合体， 用它们来研究钙调素与靶酶的相互作用。心肌肌钙蛋白C是 结构上类似于钙调蛋白，但不能激活钙调蛋白 目标酶。通过构建这两个同源基因的嵌合体， 钙结合蛋白，我们将确定cTnC的结构域， 不能替代相应的钙调素区域， 失去激活的能力。这些数据将我们导向特定的氨基 钙调素的酸性残基，可能参与结合和激活 目标酶。然后，我们将集中讨论这些残留物， 诱变研究这项诱变研究将提供精确的 关于CaM-靶酶相互作用的结构信息。 我们的初步数据显示，一些CaM-cTnC嵌合体与MLCK结合，但 无法激活酶。 这些和其他CaM突变体是有效的 竞争性MLCK抑制剂。我们建议将这些研究扩展至 开发最有效的MLCK抑制剂。这种抑制剂 将提供一个很好的结构模型， MLCK激活的分子机制。 这项研究将确定钙调素的功能作用， 结构域中的靶酶激活，并产生更完整的 钙调素-靶酶相互作用的图片。反过来，这将 帮助我们了解钙是如何调节血管张力的。

Calcium, acting through its intracellular receptor calmodulin (CaM), plays a critical role in regulating vascular tone. Vascular smooth muscle contraction depends on the action of a calmodulin-dependent enzyme, smooth muscle myosin light chain kinase (MLCK). We want to understand how calmodulin activates MLCK and other enzymes that regulate vascular smooth muscle tone. Our focus is on the structural basis of calmodulin's interaction with these regulatory enzymes. We propose to generate chimeras of CaM and cardiac troponin C (cTnC) and use them to study CaM-target enzyme interactions. Cardiac troponin C is structurally similar to calmodulin, but cannot activate calmodulin target enzymes. By constructing chimeras of these two homologous calcium binding proteins, we will determine the domains of cTnC that cannot substitute for the corresponding region of CaM without causing loss of ability to activate. This data directs us to specific amino acid residues of CaM that may participate in binding and activation of target enzymes. We will then focus on these residues in an intensive mutagenesis study. This mutagenesis study will provide precise structural information about CaM-target enzyme interactions. Our preliminary data show that some CaM-cTnC chimeras bind to MLCK, but fail to activate the enzyme. These and other CaM mutants are potent competitive MLCK inhibitors. We propose to extend these studies to develop the most potent MLCK inhibitor possible. Such an inhibitor would provide an excellent structural model for understanding the molecular mechanism of MLCK activation. The proposed study will define the role of calmodulin's functional domains in target enzyme activation, and produce a more complete picture of calmodulin-target enzyme interactions. This, in turn, will help us understand how calcium regulates vascular tone.