Effects of a non-divalent cation binding mutant of myosin regulatory light chain on tension generation in skinned skeletal muscle fibers.
Each myosin molecule contains two heavy chains and a total of four low-molecular weight light chain subunits, two "essential" and two "regulatory" light chains (RLCs). Although the roles of myosin light chains in vertebrate striated muscle are poorly understood at present, recent studies on the RLC have suggested that it has a modulatory role with respect to Ca2+ sensitivity of tension and the rate of tension development, effects that may be mediated by Ca2+ binding to the RLC. To examine possible roles of the RLC Ca2+/Mg2+ binding site in tension development by skeletal muscle, we replaced endogenous RLC in rabbit skinned psoas fibers with an avian mutant RLC (D47A) having much reduced affinity for divalent cations. After replacement of up to 80% of the endogenous RLC with D47A RLC, maximum tension (at pCa 4.5) was significantly reduced compared with preexchange tension, and the amount of decrease was directly related to the extent of D47A exchange. Fiber stiffness changed in proportion to tension, indicating that the decrease in tension was due to a decrease in the number of tension-generating cross-bridges. Decreases in both tension and stiffness were substantially, although incompletely, reversed after reexchange of native RLC for D47A. RLC exchange was also performed using a wild-type RLC. Although a small decrease in tension was observed after wild-type RLC exchange, the decrease was not proportional to the extent of RLC exchange and was not reversed by reexchange of the native RLC. D47A exchange also decreased the Ca2+ sensitivity of tension and reduced the apparent cooperativity of tension development. The results suggest that divalent cation binding to myosin RLC plays an important role in tension generation in skeletal muscle fibers.
Diffee
G M
GM
Department of Physiology, University of Wisconsin, Madison 53706, USA.
Greaser
M L
ML
Reinach
F C
FC
Moss
R L
RL
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism; D066298:In Vitro Techniques; D007537:Isometric Contraction / Q000235:genetics / Q000502:physiology; D009119:Muscle Contraction / Q000235:genetics* / Q000502:physiology*; D018482:Muscle, Skeletal / Q000502:physiology*; D009218:Myosins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits
D002413:Cations, Divalent; D009218:Myosins; D002118:Calcium