Changes in the Orientation of the Myosin Light Chain Domain (LCD) Associated with Thick Filament-Based Regulation of Skeletal Muscle

Abstract

The dependence of myosin LCD orientation on temperature, myofilament lattice spacing and sarcomere length was determined using fluorescence polarization from bifunctional sulphorhodamine (BSR) probes on the myosin regulatory light chain (RLC) in relaxed skinned fibers from rabbit psoas muscle. Mutants of chicken skeletal RLC, with native cysteines replaced by alanine and new cysteine pairs introduced at positions 95/103, 122/134, 131/138 or 151/158, were labeled by crosslinking cysteine pairs with BSR. BSR-RLCs were exchanged into skinned muscle fibers replacing ∼20% of native RLC. The second- and fourth-rank order parameters of the orientation distribution of each BSR-RLC in the fiber, and respectively, were calculated from the measured polarized fluorescence intensities. At sarcomere length 2.4 μm the order parameters of each probe had a sigmoidal dependence on temperature in the range 3-33°C with half-maximal change at 18°C. Lattice compression by 5% dextran decreased the transition temperature to 13°C and increased the temperature-dependent change in the order parameters. These results show that the LCD becomes more parallel to the filament axis in relaxing solution at higher temperature and that osmotic compression of the myofilament lattice induces further tilting of the heads towards the filament axis, inducing the fully OFF state of the thick filament. At sarcomere lengths above 2.6 μm, slow ramp stretches (4% of fiber length in 0.25 s) applied in relaxing solution at 25°C in the presence of dextran produced large changes in RLC orientation towards the ON conformation seen during calcium activation, with partial reversal during force relaxation after the stretch. The correlation between RLC orientation and passive fiber tension provides evidence that myosin head orientation is sensitive to thick filament strain. Supported by Wellcome Trust, UK.