Pi release (single vs. multiple working strokes).
The original evidence for a working stroke came from length-step experiments employing single fibers. These experiments have been repeated with skinned fibers and myofibrils at different phosphate concentrations. The response to quick stretch is accelerated by added phosphate whereas the response to a release step is less [Pi]-sensitive. This asymmetry is predicted by our quantitative modeling of the rates of tension transients if phosphate is released slowly after the working stroke, assuming that actin-attachment transitions are not involved. We used the same method of analysis to eliminate the possibility of alternative mechanisms in which slow Pi-release occurs before the stroke or a slow isomerization precedes rapid Pi-release as part of the working stroke. The rates of tension transients produced by release of caged-Pi were also better fit by the first mechanism. However, high levels of exogenous Pi could also lower the actin affinity of M.ADP.Pi, which would provide more potent mechanisms to reduce tension and ATPase.
The differing ways of modeling Pi release described above reflect the continuing debate over how Pi release is coupled to energy transduction in muscle. There are three competing possibilities for Pi release. The first, suggested by Pate and Cooke is that Pi is released rapidly as part of the working stroke, which causes excess phosphate to lower the equilibrium constant of the stroke transition and thereby reduce tension in a logarithmic manner. However, this mechanism does not predict the slow Pi-dependent kinetics observed by Ranatunga at high Pi, which indicates that Pi is released slowly after the working stroke, possibly in a strain-independent manner. Our modelling suggests that the reduction in net affinity of myosin to actin caused by Pi binding is insufficient to reduce tension as observed, and therefore we postulate a third mechanism, i.e., that high Pi also reduces the strength of the initial myosin-actin binding transition. This mechanism is capable of reducing tension as well as muscle stiffness. The observed percent reduction in stiffness is only slightly less than the reduction in tension; in this regard, the same fractional reduction is observed. A third mechanism for Pi release, which embodies two working strokes, as suggested in our recent publication, cannot be ruled out. It may be that all three mechanisms are operative and which mechanism dominates may depend upon the conditions of the experiment.