title: Phosphate enhances myosin-powered actin filament velocity under acidic conditions in a motility assay.
abstract: Elevated levels of inorganic phosphate (P(i)) are believed to inhibit muscular force by reversing myosin's force-generating step. These same levels of P(i) can also affect muscle velocity, but the molecular basis underlying these effects remains unclear. We directly examined the effect of P(i) (30 mM) on skeletal muscle myosin's ability to translocate actin (V(actin)) in an in vitro motility assay. Manipulation of the pH enabled us to probe rebinding of P(i) to myosin's ADP-bound state, while changing the ATP concentration probed rebinding to the rigor state. Surprisingly, the addition of P(i) significantly increased V(actin) at both pH 6.8 and 6.5, causing a doubling of V(actin) at pH 6.5. To probe the mechanisms underlying this increase in speed, we repeated these experiments while varying the ATP concentration. At pH 7.4, the effects of P(i) were highly ATP dependent, with P(i) slowing V(actin) at low ATP (<500 μM), but with a minor increase at 2 mM ATP. The P(i)-induced slowing of V(actin), evident at low ATP (pH 7.4), was minimized at pH 6.8 and completely reversed at pH 6.5. These data were accurately fit with a simple detachment-limited kinetic model of motility that incorporated a P(i)-induced prolongation of the rigor state, which accounted for the slowing of V(actin) at low ATP, and a P(i)-induced detachment from a strongly bound post-power-stroke state, which accounted for the increase in V(actin) at high ATP. These findings suggest that P(i) differentially affects myosin function: enhancing velocity, if it rebinds to the ADP-bound state, while slowing velocity, if it binds to the rigor state.
authors.affiliation: Department of Kinesiology, University of Massachusetts, Amherst, Massachusetts 01003, USA. edebold@kin.umass.edu
authors.forename: Edward P
authors.initials: EP
authors.lastname: Debold
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Turner
authors.forename: Jordan C
authors.initials: JC
authors.lastname: Stout
authors.forename: Sam
authors.initials: S
authors.lastname: Walcott
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects* / Q000502:physiology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002645:Chickens; D004305:Dose-Response Relationship, Drug; D006863:Hydrogen-Ion Concentration; D023421:Models, Animal; D008954:Models, Biological; D009119:Muscle Contraction / Q000187:drug effects* / Q000502:physiology; D018482:Muscle, Skeletal / Q000187:drug effects / Q000502:physiology; D010710:Phosphates / Q000493:pharmacokinetics / Q000494:pharmacology*; D024744:Skeletal Muscle Myosins / Q000187:drug effects* / Q000502:physiology
chemical_list: D010710:Phosphates; D000255:Adenosine Triphosphate; D024744:Skeletal Muscle Myosins