The proposed studies constitute part of a larger effort to understand how the contraction of smooth muscle is regulated at the level of contractile proteins, actin and myosin. The focus of this proposal is to elucidate the mechanism by which the putative regulatory protein calponin interacts with the actin filament to modulate contraction. In particular, thee studies will attempt to determine if calponin regulates a well described state of smooth muscle contraction called a """"""""latch- state"""""""". The latch-state allows smooth muscles to remain contracted for long periods with relatively low expenditure of chemical energy. The high-economy of smooth muscle contraction is essential for normal physiologic function. In spite of the central importance of this contractile state for normal function of smooth muscle, the molecular basis for the regulation of the latch-state is unknown. Our central hypothesis is that calponin slows the rate of cross-bridge dissociation from actin, and this leads to activation of unphosphorylated cross bridges via a thin filament-linked mechanism. To test this hypothesis we will measure 1) actin filament sliding velocity, 2) changes in the level of force exerted on regulated actin filaments by a field of immobilized myosin molecules, 3) the force, displacement (step size), and attachment time for single myosin molecules interact with single actin filaments, and 4) the rate of myosin dissociation from actin using stopped-flow techniques. These measurements will provide insights into the physiologic parameters of isometric force and unloaded shortening velocity that characterize the contractile state of intact smooth muscles. These assays, in conjunction with recent x-ray diffraction data and high resolution electron microscopic images of actin myosin, tropomyosin, and calponin allow us to formulate and test specific molecular models for how calponin might interact with actin, tropomyosin, and/or myosin. The proposed studies will begin to address the issue of how calponin might interact with actin, tropomyosin, and/or myosin. The proposed studies will begin to address the issue of how calponin-mediated regulation interacts with the now well established myosin phosphorylation regulatory system. A major goal of the proposed studies will be to elucidate the role of calponin in thin-filament linked regulation of unphosphorylated myosin (i.e. the latch-state).
| Haeberle, J R (1999) Thin-filament linked regulation of smooth muscle myosin. J Muscle Res Cell Motil 20:363-70 |
