Phosphate enhances myosin-powered actin filament velocity under acidic conditions in a motility assay

Abstract

Elevated levels of inorganic phosphate (Pi) are believed to inhibit muscular force by reversing myosin's force-generating step. These same levels of Pican also affect muscle velocity, but the molecular basis underlying these effects remains unclear. We directly examined the effect of Pi(30 mM) on skeletal muscle myosin's ability to translocate actin ( Vactin) in an in vitro motility assay. Manipulation of the pH enabled us to probe rebinding of Pito myosin's ADP-bound state, while changing the ATP concentration probed rebinding to the rigor state. Surprisingly, the addition of Pisignificantly increased Vactinat both pH 6.8 and 6.5, causing a doubling of Vactinat 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 Piwere highly ATP dependent, with Pislowing Vactinat low ATP (<500 μM), but with a minor increase at 2 mM ATP. The Pi-induced slowing of Vactin, 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 Pi-induced prolongation of the rigor state, which accounted for the slowing of Vactinat low ATP, and a Pi-induced detachment from a strongly bound post-power-stroke state, which accounted for the increase in Vactinat high ATP. These findings suggest that Pidifferentially affects myosin function: enhancing velocity, if it rebinds to the ADP-bound state, while slowing velocity, if it binds to the rigor state.