CALCIUM REGULATION OF VASCULAR SMOOTH MUSCLE CONTRACTION

钙通过其细胞内受体钙调蛋白 (CaM) 发挥作用， 在调节血管张力方面发挥着重要作用。血管光滑 肌肉收缩取决于钙调蛋白依赖性物质的作用 酶，平滑肌肌球蛋白轻链激酶（MLCK）。我们想要 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 钙调蛋白与这些调节酶相互作用的基础。我们 提议生成 CaM 和心肌肌钙蛋白 C (cTnC) 的嵌合体，并且 用它们来研究 CaM 与靶酶的相互作用。心肌肌钙蛋白 C 是 结构与钙调蛋白相似，但不能激活钙调蛋白 目标酶。通过构建这两个同源物的嵌合体 钙结合蛋白，我们将确定 cTnC 的结构域 不能替代 CaM 的相应区域而不引起 失去激活能力。该数据引导我们找到特定的氨基 CaM 的酸性残基可能参与结合和激活 目标酶。然后我们将重点关注这些残留物 诱变研究。这项诱变研究将提供精确的 有关 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，但是 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到 开发出最有效的 MLCK 抑制剂。这样的抑制剂 将为理解 MLCK激活的分子机制。 拟议的研究将定义钙调蛋白功能的作用 靶酶激活域，并产生更完整的 钙调蛋白-靶酶相互作用的图片。反过来，这将 帮助我们了解钙如何调节血管张力。

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.