Superinhibitory phospholamban mutants compete with Ca2+ for binding to SERCA2a by stabilizing a unique nucleotide-dependent conformational state.

The Journal of Biological Chemistry
Brandy L AkinLarry R Jones

Abstract

Three cross-linkable phospholamban (PLB) mutants of increasing inhibitory strength (N30C-PLB < N27A,N30C,L37A-PLB (PLB3) < N27A,N30C,L37A,V49G-PLB (PLB4)) were used to determine whether PLB decreases the Ca(2+) affinity of SERCA2a by competing for Ca(2+) binding. The functional effects of N30C-PLB, PLB3, and PLB4 on Ca(2+)-ATPase activity and E1 approximately P formation were correlated with their binding interactions with SERCA2a measured by chemical cross-linking. Successively higher Ca(2+) concentrations were required to both activate the enzyme co-expressed with N30C-PLB, PLB3, and PLB4 and to dissociate N30C-PLB, PLB3, and PLB4 from SERCA2a, suggesting competition between PLB and Ca(2+) for binding to SERCA2a. This was confirmed with the Ca(2+) pump mutant, D351A, which is catalytically inactive but retains strong Ca(2+) binding. Increasingly higher Ca(2+) concentrations were also required to dissociate N30C-PLB, PLB3, and PLB4 from D351A, demonstrating directly that PLB antagonizes Ca(2+) binding. Finally, the specific conformation of E2 (Ca(2+)-free state of SERCA2a) that binds PLB was investigated using the Ca(2+)-pump inhibitors thapsigargin and vanadate. Cross-linking assays conducted in the absence of Ca(2+) showed t...Continue Reading

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Citations

Aug 13, 2011·The Journal of Biological Chemistry·Philip BidwellSeth L Robia
Jan 7, 2015·Journal of Molecular and Cellular Cardiology·Tanja WittmannJoachim P Schmitt
Oct 22, 2013·Biophysical Journal·Sandeep PallikkuthSeth L Robia
Apr 12, 2015·Biophysical Journal·L Michel Espinoza-FonsecaDavid D Thomas
Jul 26, 2018·International Journal of Molecular Sciences·Tatiana M VinogradovaEdward G Lakatta
Sep 3, 2013·The Journal of Biological Chemistry·Brandy L AkinLarry R Jones
Aug 26, 2014·Biological Chemistry·Grant Kemp, Florian Cymer
Sep 13, 2013·The Journal of Biological Chemistry·Irene C MangialavoriJuan Pablo F C Rossi

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