Myosin light chain phosphorylation affects the structure of rabbit skeletal muscle thick filaments

Abstract

To identify the structural basis for the observed physiological effects of myosin regulatory light chain phosphorylation in skinned rabbit skeletal muscle fibers (potentiation of force development at low calcium), thick filaments separated from the muscle in the relaxed state, with unphoshorylated light chains, were incubated with specific, intact, myosin light chain kinase at moderate (pCa 5.0) and low (pCa 5.8) calcium and with calcium-independent enzyme in the absence of calcium, then examined as negatively stained preparations, by electron microscopy and optical diffraction. All such experimental filaments became disordered (lost the near-helical array of surface myosin heads typical of the relaxed state). Filaments incubated in control media, including intact enzyme in the absence of calcium, moderate calcium (pCa 5.0) without enzyme, and bovine serum albumin substituting for calcium-independent myosin light chain kinase, all retained their relaxed structure. Finally, filaments disordered by phosphorylation regained their relaxed structure after incubation with a protein phosphatase catalytic subunit. We suggest that the observed disorder is due to phosphorylation-induced increased mobility and/or changed conformation of myosin heads, which places an increased population of them close to thin filaments, thereby potentiating actin-myosin interaction at low calcium levels.

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

“…One mechanism proposed to explain the results presented here is that phosphorylation of myosin prevents the binding of myosin heads to the core of the thick filament, forming an ordered array (Levine et al 1996). The inability of myosin heads to lie in this configuration means that heads are more able to interact with actin thus generating a population of heads in non-force generating states.…”

“…In contrast phosphorylation of myosin causes disruption of the ordered array, increasing the fraction of myosin heads in a disordered configuration in the space around the thick filament, see Fig. 9 ( Levine et al 1996), which means that these heads can more readily bind with actin in non-force generating states, State 3 in Fig. 9.…”

“…State 7 shows a myosin head bound to the thick filament. The presence of blebbistatin stabilizes this ordered state (Xu et al 2008); whereas myosin phosphorylation disorders it (Levine et al 1996) The observations that dephosphorylated fibers inhibited at 10°C by blebbistatin, are fast, but that phosphorylated fibers are slow, strongly support the stability of the ordered thick filament array as playing a role in the inhibition of velocity. Thus the inhibition of velocity by phosphorylation can be connected to disruption of this array, which is its only established structural effect.…”

“…Binding to the actin binding cleft would prevent this cleft from closing. We have used blebbistatin because it induces a known structure in myosin, with kinetics that have been thoroughly analyzed and it stabilizes the structure of the thick filament (Xu et al 2008); a structure known to be disordered by myosin phosphorylation (Levine et al 1996). In addition Blebbistatin does not bind to the nucleotide site, as do the phosphate analogs used in previous studies.…”

The effect of work cycle frequency on the potentiation of dynamic force in mouse fast twitch skeletal muscle

“…For example, studies by Levine and colleagues using permeabilized rabbit psoas muscle fibres showed that phosphorylation of the RLC reversibly disrupts the normally well-ordered, helical arrangement of myosin heads close to the surface of the thick filament (Levine et al, 1996;Levine et al, 1998). Sweeney and colleagues showed that this disruption was due to charge repulsion between the myosin head and the thick filament, as the addition of a negative charge to the RLC mimicked this effect of phosphorylation (Sweeney et al, 1994).…”