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MUSCLE CELL BIOLOGY AND CELL MOTILITY

Effect of low pH on single skeletal muscle myosin mechanics and kinetics

Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont

Submitted 25 March 2008 ; accepted in final form 9 May 2008

Acidosis (low pH) is the oldest putative agent of muscular fatigue, but the molecular mechanism underlying its depressive effect on muscular performance remains unresolved. Therefore, the effect of low pH on the molecular mechanics and kinetics of chicken skeletal muscle myosin was studied using in vitro motility (IVM) and single molecule laser trap assays. Decreasing pH from 7.4 to 6.4 at saturating ATP slowed actin filament velocity (V_actin) in the IVM by 36%. Single molecule experiments, at 1 µM ATP, decreased the average unitary step size of myosin (d) from 10 ± 2 nm (pH 7.4) to 2 ± 1 nm (pH 6.4). Individual binding events at low pH were consistent with the presence of a population of both productive (average d = 10 nm) and nonproductive (average d = 0 nm) actomyosin interactions. Raising the ATP concentration from 1 µM to 1 mM at pH 6.4 restored d (9 ± 3 nm), suggesting that the lifetime of the nonproductive interactions is solely dependent on the [ATP]. V_actin, however, was not restored by raising the [ATP] (1–10 mM) in the IVM assay, suggesting that low pH also prolongs actin strong binding (t_on). Measurement of t_on as a function of the [ATP] in the single molecule assay suggested that acidosis prolongs t_on by slowing the rate of ADP release. Thus, in a detachment limited model of motility (i.e., V_actin d/t_on), a slowed rate of ADP release and the presence of nonproductive actomyosin interactions could account for the acidosis-induced decrease in V_actin, suggesting a molecular explanation for this component of muscular fatigue.

acidosis; fatigue; velocity; laser trap