The Effect of Phosphate on the Force Generating Capacity of a Mini-Ensemble of Myosin Measured in a Laser Trap Assay

Abstract

The accumulation of inorganic phosphate (Pi) is believed to cause much of the loss in the force-generating capacity of muscle during fatigue, but the molecular basis of this effect remains poorly understood. To gain insight into this process we determined the effect of a fatiguing level of Pi (30mM) on the force-generating capacity of a mini-ensemble of skeletal myosin (∼8molecules) interacting with a single actin filament using a three bead laser trap assay, at 100uM ATP. In the absence of Pi the average force generated displayed a bimodal Gaussian distribution with a low force peak centered at 0.30 ± 0.02 pN and second higher peak centered at 1.22 ± 0.08 pN. In the presence of Pi a low force peak centered at 0.28 ± 0.02 pN remained but the high force peak was completely absent. The duration of the low force binding events were statistically similar in both the presence (30 ± 4ms) and absence of Pi (28 ± 3ms) as were the average displacements of these events at 14 ± 1nm and 16 ± 1nm respectively, consistent with a mix of single and double molecule binding events. The high force events generated on average 64 ± 4nm displacements lasting 75 ± 8ms, suggesting these resulted from multiple binding events before detachment from the actin filament. The overall frequency of binding events was similar in the presence (3.0/s) and absence of Pi (2.7s). Taken together these data suggest that Pi reduces the force-generating capacity of a myosin by eliminating the ability to make the successive and simultaneous actin attachments required to generate higher forces.