Cardiac Calcium ATPase Dimerization Measured by Cross-Linking and Fluorescence Energy Transfer

Biophys J. 2016 Sep 20;111(6):1192-1202. doi: 10.1016/j.bpj.2016.08.005.

Abstract

The cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA) establishes the intracellular calcium gradient across the sarcoplasmic reticulum membrane. It has been proposed that SERCA forms homooligomers that increase the catalytic rate of calcium transport. We investigated SERCA dimerization in rabbit left ventricular myocytes using a photoactivatable cross-linker. Western blotting of cross-linked SERCA revealed higher-molecular-weight species consistent with SERCA oligomerization. Fluorescence resonance energy transfer measurements in cells transiently transfected with fluorescently labeled SERCA2a revealed that SERCA readily forms homodimers. These dimers formed in the absence or presence of the SERCA regulatory partner, phospholamban (PLB) and were unaltered by PLB phosphorylation or changes in calcium or ATP. Fluorescence lifetime data are compatible with a model in which PLB interacts with a SERCA homodimer in a stoichiometry of 1:2. Together, these results suggest that SERCA forms constitutive homodimers in live cells and that dimer formation is not modulated by SERCA conformational poise, PLB binding, or PLB phosphorylation.

MeSH terms

  • Adenosine Triphosphate / chemistry
  • Adenosine Triphosphate / metabolism
  • Animals
  • Blotting, Western
  • Calcium / chemistry
  • Calcium / metabolism
  • Calcium-Binding Proteins / chemistry
  • Calcium-Binding Proteins / metabolism
  • Dogs
  • Fluorescence Resonance Energy Transfer
  • HEK293 Cells
  • Heart Ventricles / chemistry
  • Heart Ventricles / enzymology
  • Humans
  • Immunoprecipitation
  • Luminescent Proteins / chemistry
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Models, Molecular
  • Muscle Cells / chemistry
  • Muscle Cells / enzymology
  • Mutation
  • Phosphorylation
  • Photobleaching
  • Protein Multimerization
  • Rabbits
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases / chemistry*
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases / genetics
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases / metabolism

Substances

  • Calcium-Binding Proteins
  • Luminescent Proteins
  • phospholamban
  • Adenosine Triphosphate
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases
  • Calcium