ALTERED KINETICS OF CONTRACTION IN SKELETAL-MUSCLE FIBERS CONTAINING A MUTANT MYOSIN REGULATORY LIGHT-CHAIN WITH REDUCED BIVALENT CATION-BINDING

We examined the kinetic properties of rabbit skinned skeletal muscle f ibers in which the endogenous myosin regulatory light chain (RLC) was partially replaced with a mutant RLC (D47A) containing a point mutatio n within the Ca2+/Mg2+ binding site that severely reduced its affinity for divalent cations. We found that when approximately 50% of the end ogenous RLC was replaced by the mutant, maximum tension declined to si milar to 60% of control and the rate constant of active tension redeve lopment (k(tr)) after mechanical disruption of cross-bridges was reduc ed to similar to 70% of control. This reduction in k(tr) was not an in direct effect on kinetics due to a reduced number of strongly bound my osin heads, because when the strongly binding cross-bridge analog N-et hylmaleimide-modified myosin subfragment1 (NEM-S1) was added to the fi bers, there was no effect upon maximum k(tr). Fiber stiffness declined after D47A exchange in a manner indicative of a decrease in the numbe r of strongly bound cross-bridges, suggesting that the force per cross -bridge was not significantly affected by the presence of D47A RLC. In contrast to the effects on k(tr), the rate of tension relaxation in s teadily activated fibers after flash photolysis of the Ca2+ chelator d iazo-2 increased by nearly twofold after D47A exchange. We conclude th at the incorporation of the nondivalent cation-binding mutant of myosi n RLC decreases the proportion of cycling cross-bridges in a force-gen erating state by decreasing the rate of formation of force-generating bridges and increasing the rate of detachment. These results suggest t hat divalent cation binding to myosin RLC plays an important role in m odulating the kinetics of cross-bridge attachment and detachment.