CALCIUM REGULATION OF VASCULAR SMOOTH MUSCLE CONTRACTION

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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.