Debold EP, Turner MA, Stout JC, Walcott S. Phosphate enhances myosin-powered actin filament velocity under acidic conditions in a motility assay. Am J Physiol Regul Integr Comp Physiol 300: R1401-R1408, 2011. First published February 23, 2011 doi:10.1152/ajpregu.00772.2010.-Elevated levels of inorganic phosphate (Pi) are believed to inhibit muscular force by reversing myosin's force-generating step. These same levels of Pi can 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 Pi to myosin's ADP-bound state, while changing the ATP concentration probed rebinding to the rigor state. Surprisingly, the addition of Pi significantly increased Vactin at both pH 6.8 and 6.5, causing a doubling of Vactin 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 Pi were highly ATP dependent, with Pi slowing Vactin at low ATP (Ͻ500 M), 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 Vactin at low ATP, and a Pi-induced detachment from a strongly bound post-power-stroke state, which accounted for the increase in Vactin at high ATP. These findings suggest that Pi differentially affects myosin function: enhancing velocity, if it rebinds to the ADP-bound state, while slowing velocity, if it binds to the rigor state.fatigue; cross-bridge cycle; muscle contraction MUSCULAR FORCE AND MOTION are generated through the cyclical interaction of actin and myosin, in a process ultimately powered by the hydrolysis of ATP (Fig. 1). In the 1980s, several investigations established that elevated levels of organic phosphate (P i ) reduced muscular force, leading to the notion that P i release by myosin was closely associated with force generation (12) and the rotation of the lever arm (2). This hypothesis postulates that P i rebinds to myosin (M) in a state in which myosin is strongly bound to both actin (A) and ADP (AM.ADP) and, in one or more steps, reverses the forcegenerating step, leading to an increase in the population of the weakly bound myosin, in the M.ADP.P i state (32). While competing theories exist (3), models based on the P i -induced detachment can account for much of the effect on muscular force (26). However, the effects of P i on muscle's shortening velocity have been more difficult to explain using the same model.While the earliest experiments in muscle fibers suggested that P i had no effect on unloaded shortening velocity (V us ) (7), subsequent efforts revealed that P i could induc...

<0.15 mM). To examine the kinetics of NMIIB at single-molecule level we used a dual-beam optical tweezers. A surface-immobilized bead was coated with single-(NMIIB-SH-HMM) and double-headed (NMIIB-HMM) heavy meromyosin-like molecules. We measured the lifetimes of unitary actomyosin interactions and determined the actin-detachment kinetics at two ATP concentrations. Results showed that at physiological ATP concentration (1 mM), the rate of detachment of acto-NMIIB-SH-HMM interactions was~0.51 s-1, similar to the ADP release rate and steady-state ATPase rate reported from solution kinetic studies. Decreasing the ATP concentration to 1 mM did not alter this rate of detachment (~0.43 s-1). In case of NMIIB-HMM the detachment rates werẽ 0.43 s-1 (1 mM ATP) and~0.28 s-1 (1 mM ATP). Also, we found that the power-stroke of NMIIB-SH-HMM and NMIIB-HMM were about 8 nm. No signs of processive stepping were observed in case of NMIIB-HMM. The detachment rates calculated from landing assays using NMIIB-SH-HMM-GFP and NMIIB-HMM-GFP were 0.58 and 0.57 s-1 at 1 mM ATP. The doubleheaded molecules were not motile, but we observed robust motility of minifilaments of full-length NMIIB-GFP. We will discuss our single-molecule results from the perspective of the essential cellular functions of NMIIB in cell locomotion, tension generation and maintenance.

Elevated levels of phosphate (Pi) and decreased pH (i.e. acidosis) are believed to contribute to muscular fatigue by directly inhibiting muscle's molecular motor, myosin. However, the mechanisms of inhibition are not fully understood at a molecular level. We determined the effects of elevated Pi (30mM) and low pH (6.5) at saturating ATP (2mM), separately and in combination, on myosin's ability to move actin in an in vitro motility assay (Vactin). Decreasing the pH without added Pi significantly (p<0.05) slowed Vactin (6.1 ± 0.2 at pH 7.4 vs. 1.6 ± 0.2 um/s at pH 6.5). The addition of Pi slightly increased Vactin at pH 7.4 (6.1 ± 0.2 at 0mM Pi vs. 6.8 ± 0.2 um/s at 30mM Pi), while at pH 6.5, Pi increased Vactin ~2‐fold (1.6 ± 0.2 at 0mM Pi vs. 3.3 ± 0.2 um/s at 30mM Pi). Repeating these experiments at sub‐saturating ATP showed that elevated Pi decreased Vactin below 1mM ATP at pH 7.4. However, at pH 6.5, elevated Pi continued to increase Vactin at all measured ATP concentrations (from 100uM to 2mM). These data indicate that the effects of Pi on myosin depend on both the pH and the ATP concentration. Since ATP affects the rigor lifetime and pH is believed to affect the ADP‐lifetime, these data imply that Pi may be able to rebind to myosin at more than one step in the cross‐bridge cycle. This work was funded by American Heart Association SDG grant (09SDG2260776) to Edward P. Debold