LAUSR

Recent fluorescence polarization studies in skeletal muscle fibers using bifunctional rhodamine (BSR) probes on the C-lobe of the regulatory light chain (RLC) of myosin showed that the steady-state calcium-sensitivity and co-operativity of the orientation change of the RLC C-lobe are higher than those of force (Fusi et al., Nat Commun 2016,doi:10.1038/ncomms13281). We have now investigated the in situ kinetics of the conformational change of the RLC during isometric contraction of a muscle fiber triggered by a rapid (>104 s−1) increase in [Ca2+] following UV-photolysis of caged-calcium (NP-EGTA). We monitored by polarized fluorescence changes in the orientation of the E-helix of a BSR-labeled RLC exchanged into demembranated fibers from rabbit psoas muscle during activation by photolysis of NP-EGTA, under conditions which preserve the physiological resting structure of the thick filament (T=25°C, 5% Dextran, 2.45 µm sarcomere length). After photolysis force increased with a sigmoidal time course similar to that of an isometric tetanus in an intact muscle fiber. The change in the order parameter P2 of the RLC probe, associated with it becoming more perpendicular to the fiber axis, also had a sigmoidal time course. The fractional change in P2 was faster than that of force. The P2 change was 25% complete 20ms after photolysis when activation of the thin filament is already maximal (T=12°C, Fusi et al., PNAS 2014, 111:4626-31) but force had developed to only 10% of its plateau value (T0). The P2 change was essentially complete by ca 70ms after photolysis when force was only 60% T0. These results show that the change in RLC orientation associated with the activation of the myosin motors is slower than thin-filament activation but faster than force generation. Supported by MRC, UK.