Elevated levels of phosphate (Pi) reduce isometric force, providing support for the notion that the release of Pi from myosin is closely associated with the generation of muscular force. Pi is thought to rebind to actomyosin in an ADP-bound state and reverse the force-generating steps, including the rotation of the lever arm (i.e., the powerstroke). Despite extensive study, this mechanism remains controversial, in part because it fails to explain the effects of Pi on isometric ATPase and unloaded shortening velocity. To gain new insight into this process, we determined the effect of Pi on the force-generating capacity of a small ensemble of myosin (∼12 myosin heads) using a three-bead laser trap assay. In the absence of Pi, myosin pulled the actin filament out of the laser trap an average distance of 54 ± 4 nm, translating into an average peak force of 1.2 pN. By contrast, in the presence of 30 mM Pi, myosin generated only enough force to displace the actin filament by 13 ± 1 nm, generating just 0.2 pN of force. The elevated Pi also caused a >65% reduction in binding-event lifetime, suggesting that Pi induces premature detachment from a strongly bound state. Definitive evidence of a Pi-induced powerstroke reversal was not observed, therefore we determined if a branched kinetic model in which Pi induces detachment from a strongly bound, postpowerstroke state could explain these observations. The model was able to accurately reproduce not only the data presented here, but also the effects of Pi on both isometric ATPase in muscle fibers and actin filament velocity in a motility assay. The ability of the model to capture the findings presented here as well as previous findings suggests that Pi-induced inhibition of force may proceed along a kinetic pathway different from that of force generation.

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Myosins are a family of motor proteins responsible for various forms of cellular motility, including muscle contraction and vesicular transport. The most fundamental aspect of myosin is its ability to transduce the chemical energy from the hydrolysis of ATP into mechanical work, in the form of force and/or motion. A key unanswered question of the transduction process is the timing of the force-generating powerstroke relative to the release of phosphate (P i ) from the active site. We examined the ability of single-headed myosin Va to generate a powerstroke in a single molecule laser trap assay while maintaining P i in its active site, by either elevating P i in solution or by introducing a mutation in myosin's active site (S217A) to slow P irelease from the active site. Upon binding to the actin filament, WT myosin generated a powerstoke rapidly (≥500 s À1 ) and without a detectable delay, both in the absence and presence of 30 mM P i . The elevated levels of P i did, however, affect event lifetime, eliminating the longest 25% of binding events, confirming that P i rebound to myosin's active site and accelerated detachment. The S217A construct also generated a powerstroke similar in size and rate upon binding to actin despite the slower P i release rate. These findings provide direct evidence that myosin Va generates a powerstroke with P i still in its active site.

Elevated levels of the metabolic by-products, including acidosis (i.e., high [H+]) and phosphate (Pi) are putative agents of muscle fatigue; however, the mechanism through which they affect myosin’s function remain unclear. To elucidate these mechanisms, we directly examined the effect of acidosis (pH 6.5 vs. 7.4), alone and in combination with elevated levels of Pi on the force-generating capacity of a mini-ensemble of myosin using a laser trap assay. Acidosis decreased myosin’s average force-generating capacity by 20% (p < 0.05). The reduction was due to both a decrease in the force generated during each actomyosin interaction, as well as an increase in the number of binding events generating negative forces. Adding Pi to the acidic condition resulted in a quantitatively similar decrease in force but was solely due to an elimination of all high force-generating events (>2 pN), resulting from an acceleration of the myosin’s rate of detachment from actin. Acidosis and Pi also had distinct effects on myosin’s steady state ATPase rate with acidosis slowing it by ∼90% (p > 0.05), while the addition of Pi under acidic conditions caused a significant recovery in the ATPase rate. These data suggest that these two fatigue agents have distinct effects on myosin’s cross-bridge cycle that may underlie the synergistic effect that they have muscle force. Thus these data provide novel molecular insight into the mechanisms underlying the depressive effects of Pi and H+ on muscle contraction during fatigue.