Competitive displacement of wild-type phospholamban from the Ca2+-free cardiac calcium pump by phospholamban mutants with different binding affinities

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Phospholamban (PLB) regulates the cardiac Ca2+ pump (SERCA2a). To test how different species of PLB mutants compete to interact with the Ca2+-free, E2 conformation of SERCA2a, using the insect cell expression system, we examined how various exogenous PLB mutants regulated SER-20G-PLB, a chimeric WT-SERCA2a-Gly-WT-PLB construct, which retains a fully catalytic active Ca2+-pump and its intrinsically regulatory PLB-tether. Exogenous gain-of-function PLB mutants dominantly super-inhibited the WT-PLB-tethered SERCA2a. Further, in the Ca2+-free condition, co-expressed normal- or super-inhibitory PLB mutant with either engineered N30C or V49C residue cross-linked to Lys328 or V89C of SER-20G-PLB at the cytoplasmic or transmembrane domain, respectively, suggesting that these freely diffusing PLB mutants completely replaced the WT-PLB-tether and fit into the binding pocket previously occupied by WT-PLB. Micromolar Ca2+ completely inhibited cross-linking, yielding a similar Ca2+-dependency regardless of the presence of the WT-PLB-tether. In contrast, the PLB mutant with the loss-of-function L31A mutation has decreased binding affinity for SERCA2a, thus cross-linking weakly to the WT-PLB-tethered SERCA2a, and only marginally affected the activity of SER-20G-PLB. Thus, there is a reversible equilibrium between different PLB mutants for binding to E2, in which PLB mutants possessing higher binding affinity for SERCA2a produce a more stable E2PLB and lower Ca2+ affinity.

Original languageEnglish (US)
Pages (from-to)130-137
Number of pages8
JournalCurrent Therapeutic Research - Clinical and Experimental
StatePublished - Nov 1 2014


  • Binding affinity
  • Cardiac Ca-ATPase
  • Cross-linking
  • Enzyme regulation
  • Equilibrium
  • Phospholamban

ASJC Scopus subject areas

  • Pharmacology
  • Pharmacology (medical)

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