Myosin light chain phosphorylation inhibits muscle fiber shortening velocity in the presence of vanadate

Abstract

We have shown that myosin light chain phosphorylation inhibits fiber shortening velocity at high temperatures, 30 degrees C, in the presence of the phosphate analog vanadate. Vanadate inhibits tension by reversing the transition to force-generating states, thus mimicking a prepower stroke state. We have previously shown that at low temperatures vanadate also inhibits velocity, but at high temperatures it does not, with an abrupt transition in inhibition occurring near 25 degrees C (E. Pate, G. Wilson, M. Bhimani, and R. Cooke. Biophys J 66: 1554-1562, 1994). Here we show that for fibers activated in the presence of 0.5 mM vanadate, at 30 degrees C, shortening velocity is not inhibited in dephosphorylated fibers but is inhibited by 37 +/- 10% in fibers with phosphorylated myosin light chains. There is no effect of phosphorylation on fiber velocity in the presence of vanadate at 10 degrees C. The K(m) for ATP, defined by the maximum velocity of fibers partially inhibited by vanadate at 30 degrees C, is 20 +/- 4 microM for phosphorylated fibers and 192 +/- 40 microM for dephosphorylated fibers, showing that phosphorylation also affects the binding of ATP. Fiber stiffness is not affected by phosphorylation. Inhibition of velocity by phosphorylation at 30 degrees C depends on the phosphate analog, with approximately 12% inhibition in fibers activated in the presence of 5 mM BeF(3) and no inhibition in the presence of 0.25 mM AlF(4). Our results show that myosin phosphorylation can inhibit shortening velocity in fibers with large populations of myosin heads trapped in prepower stroke states, such as occurs during muscle fatigue.

Myosin regulatory light chain phosphorylation inhibits shortening velocities of skeletal muscle fibers in the presence of the myosin inhibitor blebbistatin

“…5a. There was no systematic variation in the curvature correlated with myosin phosphorylation, in agreement with previous observations (Sweeney and Kushmerick 1985;Franks-Skiba et al 2007;Karatzaferi et al 2007). Similar differences in shortening velocities were also found using incubations that contained 10 lM blebbistatin.…”

“…Addition of 20 lM blebbistatin inhibited tension equally in phosphorylated and dephosphorylated fibers and to the same extent as it did at 30°C. In the absence of blebbistatin, phosphorylation had no effect on either V max or a/Po, as observed previously under other conditions (Sweeney and Kushmerick 1985;Franks-Skiba et al 2007;Karatzaferi et al 2007). The addition of 20 lM blebbistatin did not affect the force velocity curve in dephosphorylated fibers but inhibited maximal shortening velocity for fibers with phosphorylated RLC by 57%, Fig.…”

“…In the great majority of conditions where tension is inhibited, velocity is also inhibited (Dantzig and Goldman 1985;Chase et al 1993;Seow et al 1997;Regnier et al 1999;Franks-Skiba et al 2007). The inhibition of velocity has been hypothesized to be the result of an internal load imposed by nonworking myosin heads.…”

The molecular effects of skeletal muscle myosin regulatory light chain phosphorylation

“…Since it has been reported that the phosphorylation-induced shift toward submaximal calcium activation becomes more pronounced with increased temperature (18,74), we measured the in vitro regulation of RLC-phosphorylated and dephosphorylated myosin at 35°C as a function of calcium concentrations (Fig. 4).…”

“…Fiber studies have shown conflicting data as to whether phosphorylation changes the unloaded sliding velocity of myosin, with some studies showing no differences in sliding velocity upon phosphorylation (7,45,52,72) and others showing a depression in velocity (11,12). However, the earlier experiments showing a depression in actin filament sliding velocity were performed under fatigue-like conditions (7,74) and, as has been shown, fatigue-like conditions would cause a depression of phosphorylated myosin unloaded shortening velocity (7,18,31).…”