Characterizing phospholamban to Sarco(endo)plasmic Reticulum Ca 2+-ATPase 2a (SERCA2a) protein binding interactions in human cardiac sarcoplasmic reticulum vesicles using chemical cross-linking

Brandy L. Akin, Larry R. Jones

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

Chemical cross-linking was used to study protein binding interactions between native phospholamban (PLB) and SERCA2a in sarcoplasmic reticulum (SR) vesicles prepared from normal and failed human hearts. Lys 27 of PLB was cross-linked to the Ca 2+ pump at the cytoplasmic extension of M4 (at or near Lys 328) with the homobifunctional cross-linker, disuccinimidyl glutarate (7.7 Å). Cross-linking was augmented by ATP but abolished by Ca 2+ or thapsigargin, confirming in native SR vesicles that PLB binds preferentially to E2 (low Ca 2+ affinity conformation of the Ca 2+-ATPase) stabilized by ATP. To assess the functional effects of PLB binding on SERCA2a activity, the anti-PLB antibody, 2D12, was used to disrupt the physical interactions between PLB and SERCA2a in SR vesicles. We observed a tight correlation between 2D12-induced inhibition of PLB cross-linking to SERCA2a and 2D12 stimulation of Ca 2+-ATPase activity and Ca 2+ transport. The results suggest that the inhibitory effect of PLB on Ca 2+-ATPase activity in SR vesicles results from mutually exclusive binding of PLB and Ca 2+ to the Ca 2+ pump, requiring PLB dissociation for catalytic activation. Importantly, the same result was obtained with SR vesicles prepared from normal and failed human hearts; therefore, we conclude that PLB binding interactions with the Ca 2+ pump are largely unchanged in failing myocardium.

Original languageEnglish (US)
Pages (from-to)7582-7593
Number of pages12
JournalJournal of Biological Chemistry
Volume287
Issue number10
DOIs
StatePublished - Mar 2 2012

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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