Acting Based Regulation of Smooth Muscle Contraction

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DESCRIPTION (provided by applicant): The regulation of contraction of smooth muscle differs in several respects from other muscle types. A detailed understanding of these differences could lead to new therapies for disorders of smooth muscle including hypertension, asthma, and disorders of the digestive system and urogenital system. In contrast to striated muscle, smooth muscle contains caldesmon, calponin and other actin-binding proteins, that inhibit ATPase activity and force production in model systems. We have identified a novel actin binding protein, fesselin that stimulates actin polymerization and inhibits actin activation of myosin ATPase activity. The various smooth muscle actin-binding proteins may function by altering (a) actin-myosin binding, (b) a transition between two actin-myosin complexes, (c) cell signaling, or (d) the plasticity of the cytoskeleton. Our first goal is to finalize the mechanism by which caldesmon inhibits smooth muscle contraction. The key questions with caldesmon are: (1) How much of the inhibition of contraction results from competitive inhibition of myosin binding? This will be answered by using rapid measurements of the fluorescence changes that occur when caldesmon and myosin bind to actin. (2) Does caldesmon also affect cross-bridge kinetics? This will be examined by studying the inhibition of release of fluorescent nucleotide derivatives from actin-S1 by caldesmon. (3) What is the function of caldesmon-myosin binding? We will collaborate with Dr. Gabrielle Pfitzer on the characterization of transgenic mice lacking part of the myosin-binding region of caldesmon. We will also study the structure of the caldesmon-myosin complex in collaboration with Dr. Peter Knight. Our second goal is to investigate the properties and function of fesselin. The relationship of fesselin to the synaptopodin family of actin-binding proteins will be investigated. We will study the mechanism of inhibition of actin-myosin ATPase activity. The most noteworthy activity of fesselin is its large acceleration of actin polymerization. Heavy emphasis will be placed on this activity and its reversal by Cacalmodulin. These studies will be conducted both in solution and in smooth muscle fiber systems.