Activation of the calcium-regulated thin filament by myosin strong binding

The current study was undertaken to investigate the relative contribution of calcium and myosin binding to thin. lament activation. Using the in vitro motility assay, myosin strong binding to the thin. lament was controlled by three mechanisms: 1), varying the myosin concentration of the motility surface, and adding either 2), inorganic phosphate (Pi) or 3), adenosine diphosphate (ADP) to the motility solutions. At saturating myosin conditions, Pi had no effect on thin. lament motility. However, at subsaturating myosin concentrations, velocity was reduced at maximal and submaximal calcium in the presence of Pi. Adding ADP to the motility buffers reduced thin. lament sliding velocity but increased the pCa(50) of the thin. lament. Thus by limiting or increasing myosin strong binding (with the addition of Pi and ADP, respectively), the calcium concentration at which half maximal activation of the thin. lament is achieved can be modulated. In experiments without ADP or Pi, the myosin concentration on the motility surface required to reach maximal velocity inversely correlated with the level of calcium activation. Through this approach, we demonstrate that myosin strong binding is essential for thin. lament activation at both maximal and submaximal calcium levels, with the relative contribution of myosin strong binding being greatest at submaximal calcium. Furthermore, under conditions in which myosin strong binding is not rate limiting (i.e., saturating myosin conditions), our data suggest that a modulation of myosin cross-bridge kinetics is likely responsible for the graded response to calcium observed in the in vitro motility assay.