Actin Sliding Velocities are Influenced by the Driving Forces of Actin-Myosin Binding

Unloaded shortening speeds, V, of muscle are thought to be limited by actin-bound myosin heads that resist shortening, or V = a·d·τ^-1 where τ^-1 is the rate at which myosin detaches from actin and d is myosin's step size. The a-term describes the efficiency of force transmission between myosin heads, and has been shown to become less than one at low myosin densities in a motility assay. Molecules such as inorganic phosphate, P_i, and blebbistatin inhibit both V and actin-myosin strong binding kinetics suggesting a link between V and attachment kinetics. To determine whether these small molecules slow V by increasing resistance to actin sliding or by decreasing the efficiency of force transmission, a, we determine how inhibition of V by Pi and blebbistatin changes the force exerted on actin filaments during an in vitro sliding assay, measured from changes in the rate, τ^-1, at which actin filaments break. Upon addition of 30 mM P_i to a low (30 μM) [ATP] motility buffer V decreased from 1.8 to 1.3 μm·sec^-1 and τ^-1 from 0.029 to 0.018 sec^-1. Upon addition of 50 μM blebbistatin to a low [ATP] motility buffer, V decreased from 1.0 to 0.7 μm·sec^-1 and τ^-1 from 0.059 to 0.022 sec^-1. These results imply that blebbistatin and P_i slow V by decreasing force transmission, a, not by increasing resistive forces, implying that actin-myosin attachment kinetics influence V.