Ca2+ binding to site I of the cardiac Ca2+ pump is sufficient to dissociate phospholamban

Zhenhui Chen, Brandy L. Akin, Larry R. Jones

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Abstract

Phospholamban (PLB) inhibits the activity of SERCA2a, the Ca 2+-ATPase in cardiac sarcoplasmic reticulum, by decreasing the apparent affinity of the enzyme for Ca2+. Recent cross-linking studies have suggested thatPLBbinding and Ca2+ binding to SERCA2a are mutually exclusive. PLB binds to the E2 conformation of the Ca 2+-ATPase, preventing formation of E1, the conformation that binds two Ca2+ (at sites I and II) with high affinity and is required for ATP hydrolysis. Here we determined whether Ca2+ binding to site I, site II, or both sites is sufficient to dissociate PLB from the Ca2+ pump. Seven SERCA2a mutants with amino acid substitutions at Ca 2+-binding site I (E770Q, T798A, and E907Q), site II (E309Q and N795A), or both sites (D799N and E309Q/E770Q) were made, and the effects of Ca2+ on N30C-PLB cross-linking to Lys328 of SERCA2a were measured. In agreement with earlier reports with the skeletal muscle Ca 2+-ATPase, none of the SERCA2a mutants (except E907Q) hydrolyzed ATP in the presence of Ca2+; however, all were phosphorylatable by Pi to form E2P. Ca2+ inhibition of E2P formation was observed only in SERCA2a mutants retaining site I. In cross-linking assays, strong cross-linking between N30C-PLB and each Ca2+-ATPase mutant was observed in the absence of Ca2+. Importantly, however, micromolar Ca2+ inhibited PLB cross-linking only to mutants retaining a functional Ca 2+-binding site I. The dynamic equilibrium between Ca2+ pumps and N30C-PLB was retained by all mutants, demonstrating normal regulation of cross-linking by ATP, thapsigargin, and anti-PLB antibody. From these results we conclude that site I is the key Ca2+-binding site regulating the physical association between PLB and SERCA2a.

Original languageEnglish (US)
Pages (from-to)3253-3260
Number of pages8
JournalJournal of Biological Chemistry
Volume285
Issue number5
DOIs
StatePublished - Jan 29 2010

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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