Comparison of Orientation and Rotational Motion of Skeletal Muscle Cross-bridges Containing Phosphorylated and Dephosphorylated Myosin Regulatory Light Chain

• Published in: The Journal of biological chemistry 2013-03, Vol.288 (10), p.7012-7023
• Main Authors: Midde, Krishna, Rich, Ryan, Marandos, Peter, Fudala, Rafal, Li, Amy, Gryczynski, Ignacy, Borejdo, Julian
• Format: Article
• Language: English
• Subjects: Actins - chemistry; Actins - metabolism; Adenosine Triphosphate - metabolism; Adenosine Triphosphate - pharmacology; Animals; Anisotropy; Calcium - chemistry; Calcium - metabolism; Confocal Microscopy; Contractile Protein; Electron Spin Resonance Spectroscopy - methods; Fluorescence Correlation Spectroscopy; Fluorescence Polarization; Fluorescence Polarization - methods; Hydrolysis; Isometric Contraction - drug effects; Mechanotransduction; Models, Biological; Models, Molecular; Molecular Biophysics; Motion; Muscle; Muscle, Skeletal - chemistry; Muscle, Skeletal - metabolism; Muscle, Skeletal - physiology; Myofibrils - chemistry; Myofibrils - metabolism; Myofibrils - physiology; Myosin; Myosin Light Chains - chemistry; Myosin Light Chains - metabolism; Phosphorylation; Phosphorylation of the RLC; Rabbits; Rotation; Sarcomeres - chemistry; Sarcomeres - metabolism; Sarcomeres - physiology; Skeletal Myosin Cross-bridge; Spin Labels
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.434209

Calcium binding to thin filaments is a major element controlling active force generation in striated muscles. Recent evidence suggests that processes other than Ca2+ binding, such as phosphorylation of myosin regulatory light chain (RLC) also controls contraction of vertebrate striated muscle (Cooke, R. (2011) Biophys. Rev. 3, 33–45). Electron paramagnetic resonance (EPR) studies using nucleotide analog spin label probes showed that dephosphorylated myosin heads are highly ordered in the relaxed fibers and have very low ATPase activity. This ordered structure of myosin cross-bridges disappears with the phosphorylation of RLC (Stewart, M. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 430–435). The slower ATPase activity in the dephosporylated moiety has been defined as a new super-relaxed state (SRX). It can be observed in both skeletal and cardiac muscle fibers (Hooijman, P., Stewart, M. A., and Cooke, R. (2011) Biophys. J. 100, 1969–1976). Given the importance of the finding that suggests a novel pathway of regulation of skeletal muscle, we aim to examine the effects of phosphorylation on cross-bridge orientation and rotational motion. We find that: (i) relaxed cross-bridges, but not active ones, are statistically better ordered in muscle where the RLC is dephosporylated compared with phosphorylated RLC; (ii) relaxed phosphorylated and dephosphorylated cross-bridges rotate equally slowly; and (iii) active phosphorylated cross-bridges rotate considerably faster than dephosphorylated ones during isometric contraction but the duty cycle remained the same, suggesting that both phosphorylated and dephosphorylated muscles develop the same isometric tension at full Ca2+ saturation. A simple theory was developed to account for this fact. Background: Myosin cross-bridges containing phosphorylated and dephosphorylated regulatory light chain may be different. Results: Relaxed cross-bridges, but not active ones, are better ordered in muscle containing dephosphorylated RLC than phosphorylated RLC; they both rotate equally slowly. During contraction phosphorylated cross-bridges rotate faster. Conclusion: Both types of cross-bridges are functionally different. Significance: Phosphorylation of skeletal myosin RLC plays a role in regulation of skeletal muscle contraction.