A phospholamban-tethered cardiac Ca 2+ pump reveals stoichiometry and dynamic interactions between the two proteins

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Abstract

To study PLB (phospholamban) inhibition of the cardiac Ca 2+ pump [SERCA2a (sarcoplasmic/endoplasmic reticulum Ca 2+ - ATPase 2a)], a fusion protein (SER-20G-PLB) was engineered by tethering SERCA2a with PLB through a 20-glycine residue chain, allowing the PLB tether to either bind to or dissociate from the inhibition site on SERCA2a.When expressed in insect cells, SER-20G-PLB produced active Ca 2+ uptake, which was stimulated by the anti-PLB antibody, both similar to that which occurred with the control sample co-expressing WT (wild-type)-SERCA2a and WT-PLB. The K Ca values of Ca 2+ -dependentATPase were similar for SER-20G-PLB (0.29±0.02 μM) and for the control sample (0.30±0.02 μM), both greater than 0.17± 0.01 μM for WTSERCA2a expressed alone. Thus SER-20G-PLB retains a fully active Ca 2+ pump, but its apparent Ca 2+ affinity was decreased intrinsically by tethered PLB at a 1:1 molar stoichiometry. Like WT-PLB, SER-20G-PLB ran as both monomers and homopentamers on SDS/PAGE. As Ca 2+ concentrations increase from 0 to the micromolar range, the proportion of non-inhibiting pentamers increased from 32% to 52%, suggesting that Ca 2+activation of the pump completely dissociates the PLB tether from the inhibition site on SERCA2a, with concurrent association of PLB pentamers. Collectively, the regulation of SERCA2a is achieved through the Ca 2+ -dependent equilibria involving PLB association and dissociation from SERCA2a, and assembling and disassembling of SER-20G-PLB pentamers.

Original languageEnglish (US)
Pages (from-to)313-319
Number of pages7
JournalBiochemical Journal
Volume439
Issue number2
DOIs
StatePublished - Oct 15 2011

Keywords

  • Cardiac Ca -ATPase
  • Enzyme regulation
  • Equilibrium
  • Phospholamban
  • Protein-protein interaction

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

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