Altered kinetics of contraction in skeletal muscle fibers containing a mutant myosin regulatory light chain with reduced divalent cation binding.
We examined the kinetic properties of rabbit skinned skeletal muscle fibers in which the endogenous myosin regulatory light chain (RLC) was partially replaced with a mutant RLC (D47A) containing a point mutation within the Ca2+/Mg2+ binding site that severely reduced its affinity for divalent cations. We found that when approximately 50% of the endogenous RLC was replaced by the mutant, maximum tension declined to approximately 60% of control and the rate constant of active tension redevelopment (ktr) after mechanical disruption of cross-bridges was reduced to approximately 70% of control. This reduction in ktr was not an indirect effect on kinetics due to a reduced number of strongly bound myosin heads, because when the strongly binding cross-bridge analog N-ethylmaleimide-modified myosin subfragment1 (NEM-S1) was added to the fibers, there was no effect upon maximum ktr. Fiber stiffness declined after D47A exchange in a manner indicative of a decrease in the number 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 ktr, the rate of tension relaxation in steadily activated fibers after flash photolysis of the Ca2+ chelator diazo-2 increased by nearly twofold after D47A exchange. We conclude that the incorporation of the nondivalent cation-binding mutant of myosin RLC decreases the proportion of cycling cross-bridges in a force-generating state by decreasing the rate of formation of force-generating bridges and increasing the rate of detachment. These results suggest that divalent cation binding to myosin RLC plays an important role in modulating the kinetics of cross-bridge attachment and detachment.
Diffee
G M
GM
Department of Physiology, University of Wisconsin, Madison 53706, USA. gmdiffee@facstaff.wisc.edu
Patel
J R
JR
Reinach
F C
FC
Greaser
M L
ML
Moss
R L
RL
D000818:Animals; D001696:Biomechanical Phenomena; D055592:Biophysical Phenomena; D001703:Biophysics; D002413:Cations, Divalent / Q000378:metabolism; D005033:Ethylmaleimide; D066298:In Vitro Techniques; D007700:Kinetics; D009119:Muscle Contraction / Q000235:genetics / Q000502:physiology*; D018482:Muscle, Skeletal / Q000378:metabolism / Q000502:physiology*; D018994:Myosin Light Chains / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D015879:Myosin Subfragments / Q000737:chemistry; D017354:Point Mutation; D011817:Rabbits; D013439:Sulfhydryl Reagents
D002413:Cations, Divalent; D018994:Myosin Light Chains; D015879:Myosin Subfragments; D013439:Sulfhydryl Reagents; D005033:Ethylmaleimide