Phosphorylation of myosin regulatory light chain controls myosin head conformation in cardiac muscle

Thomas Kampourakis, Malcolm Irving

Research output: Contribution to journalArticlepeer-review

51 Scopus citations

Abstract

The effect of phosphorylation on the conformation of the regulatory light chain (cRLC) region of myosin in ventricular trabeculae from rat heart was determined by polarized fluorescence from thiophosphorylated cRLCs labelled with bifunctional sulforhodamine (BSR). Less than 5% of cRLCs were endogenously phosphorylated in this preparation, and similarly low values of basal cRLC phosphorylation were measured in fresh intact ventricle from both rat and mouse hearts. BSR-labelled cRLCs were thiophosphorylated by a recombinant fragment of human cardiac myosin light chain kinase, which was shown to phosphorylate cRLCs specifically at serine 15 in a calcium- and calmodulin-dependent manner, both in vitro and in situ. The BSR-cRLCs were exchanged into demembranated trabeculae, and polarized fluorescence intensities measured for each BSR-cRLC in relaxation, active isometric contraction and rigor were combined with RLC crystal structures to calculate the orientation distribution of the C-lobe of the cRLC in each state. Only two of the four C-lobe orientation populations seen during relaxation and active isometric contraction in the unphosphorylated state were present after cRLC phosphorylation. Thus cRLC phosphorylation alters the equilibrium between defined conformations of the cRLC regions of the myosin heads, rather than simply disordering the heads as assumed previously. cRLC phosphorylation also changes the orientation of the cRLC C-lobe in rigor conditions, showing that the orientation of this part of the myosin head is determined by its interaction with the thick filament even when the head is strongly bound to actin. These results suggest that cRLC phosphorylation controls the contractility of the heart by modulating the interaction of the cRLC region of the myosin heads with the thick filament backbone.

Original languageEnglish
Pages (from-to)199-206
Number of pages8
JournalJournal of Molecular and Cellular Cardiology
Volume85
DOIs
StatePublished - Aug 1 2015

Bibliographical note

Publisher Copyright:
© 2015.

Keywords

  • Cardiac muscle regulation
  • Myosin
  • Myosin regulatory light chain
  • Polarized fluorescence
  • Regulatory light chain phosphorylation

ASJC Scopus subject areas

  • Molecular Biology
  • Cardiology and Cardiovascular Medicine

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