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

Abstract

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...

Effect of Inorganic Phosphate and Low pH on the Force-Velocity Relation of Single Skinned Skeletal Muscle Fibers Studied by Applying Parabolic Fiber Length Changes

“…Based on in vitro motility experiments, Debald et al [15,16] suggest that low pH and high Pi modify rate of breaking of A-M linkages, and can cause inhibition of either P o and V max depending on experimental conditions. As pointed out by Sugi et al [17], however, the state of contractile proteins in vitro experiments differs too far from those in muscle and their explanation is far from being conclusive.…”

“…Experiments with skinned muscle fibers have shown that low pH (~6.5) has only a small effect on Po, but significantly reduces V max [5,9]. Because of the importance of studying the effect of increased Pi and low pH on in vitro ATP-dependent actinmyosin sliding have been performed intensively [12][13][14][15][16] at micromolar range of ATP concentration. Though their work is interesting, the experimental conditions of their experiments differ to far from the physiological condition in muscle.…”

Cardiac Muscle Activation Blunted by a Mutation to the Regulatory Component, Troponin T

“…For the in vitro motility assays described below, both myosin and actin were isolated from chicken pectoralis tissue as described previously (25). Actin was purified from chicken pectoralis muscle using the methods described by Pardee and Spudich (26) with minor modifications as described previously (25).…”

“…Actin was purified from chicken pectoralis muscle using the methods described by Pardee and Spudich (26) with minor modifications as described previously (25). The isolated filamentous actin was stabilized and labeled fluorescently by incubating it overnight at 4°C with TRITC/ phalloidin (Sigma-Aldrich) as described previously (25).…”

Phosphate and ADP Differently Inhibit Coordinated Smooth Muscle Myosin Groups

“…For the in vitro motility of troponin-tropomyosin-regulated thin (i.e., actin-containing) filaments propelled by skeletal muscle myosin, Debold et al (28) found that n max and f mot of fully activated (high Ca 2þ ) thin filaments did not change upon addition of 30 mM P i ([ATP] ¼ 2 mM). While thin filament regulation could affect the regulation of myosin kinetics by P i , full activation by Ca 2þ should return the kinetics to those of unregulated actin filaments.…”