The human muscle system contains three muscle types: cardiac muscle, skeletal muscle for physical stability and movement, and smooth muscle in the vasculature, digestive, and reproductive systems. To understand disorders affecting particular muscle types it is necessary to understand mechanisms both of contraction of these muscles and of regulation of contraction. Pharmacologic intervention depends on the ability to selectively modify the contractility of each muscle type. To this end we have been studying the regulation of contraction of both smooth and striated (cardiac and skeletal) muscles. We have shown earlier that the Ca2+ regulation of striated muscle, through the proteins troponin and tropomyosin, does not occur by a weakening of the binding of myosin-ATP to actin-tropomyosin-troponin. Rather, in the absence of Ca2+, the rate of a process, presumably product release, is inhibited. In contrast, it has been shown that the binding of myosin to actin-tropomyosin-troponin, in the absence of ATP, is Ca2+ dependent. These data are consistent with a model of contraction where both myosin makes the transition from a weakly bound state (containing bound ATP or ADP + Pi) to a strongly bound state (containing bound ADP). This transition cannot occur at a significant rate unless actin also makes a transition from the inactive state to the active state. We now propose to use a multidisciplinary approach to test this hypothesis and to further characterize the duality of states of actin and myosin. We have shown that the smooth muscle protein, caldesmon, does inhibit the binding of myosin-ATP to actin. This protein is an excellent probe of myosin crossbridge binding and can be used to study the function of the weakly bound state of myosin. We will use binding studies and stopped-flow kinetic studies to investigate the properties of weakly bound myosin crossbridges. These biochemical studies will be accompanied by mechanical and X-ray diffraction measurements of single muscle fibers to determine the importance of weakly bound crossbridges in contraction and in regulation. We will also use the muscle fiber model to study possible regulatory roles of caldesmon and a newly discovered protein, calponin. Finally, we will make a detailed study possible regulatory roles of caldesmon and a newly discovered protein, calponin. Finally, we will make a detailed study of the binding of myosin to actin in the presence of ATPgammaS to test a recent challenge to our hypothesis that regulation of striated muscle occurs at the transition from the weak binding states to the strong crossbridge states.
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