Direct detection of phospholamban and sarcoplasmic reticulum Ca-ATPase interaction in membranes using fluorescence resonance energy transfer

Biochemistry
Benjamin MuellerDavid D Thomas

Abstract

We used fluorescence resonance energy transfer (FRET) to detect and quantitate the interaction of the sarcoplasmic reticulum Ca-ATPase (SERCA) with phospholamban (PLB) in membranes. PLB inhibits SERCA only at submicromolar Ca. It has been proposed that relief of inhibition at micromolar Ca is due to dissociation of the inhibitory complex. To test this hypothesis, we co-reconstituted donor-labeled SERCA and acceptor-labeled I40A-PLB (superinhibitory, monomeric PLB mutant) in membranes of defined lipid and protein composition, with full retention of Ca-dependent ATPase activity and inhibitory regulation by PLB. FRET from SERCA to PLB was measured as a function of membrane concentrations of PLB and SERCA, and functional activity was measured on the same samples. The data revealed clearly that the stoichiometry of binding is one PLB per SERCA, and that binding is a strict function of the ratio of total PLB to SERCA in the membrane. We conclude that the dissociation constant of PLB binding to SERCA is far less than physiological PLB membrane concentrations. Binding at low Ca (pCa 6.5), where I40A-PLB inhibits SERCA, was virtually identical to that at high Ca (pCa 5.0), where no inhibition was observed. However, the limiting energy t...Continue Reading

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Citations

Jul 9, 2011·The Journal of Biological Chemistry·Joseph M AutryDavid D Thomas
Aug 13, 2011·The Journal of Biological Chemistry·Philip BidwellSeth L Robia
Aug 28, 2012·Journal of Biomolecular Screening·Razvan L CorneaDavid D Thomas
Jul 27, 2005·Proceedings of the National Academy of Sciences of the United States of America·Kirill Oxenoid, James J Chou
Mar 23, 2005·Proceedings of the National Academy of Sciences of the United States of America·J ZamoonG Veglia
Feb 22, 2005·Current Opinion in Biotechnology·Horst Wallrabe, Ammasi Periasamy
Apr 1, 2006·Journal of Molecular Biology·Christine B KarimDavid D Thomas
Oct 22, 2013·Biophysical Journal·Sandeep PallikkuthSeth L Robia
Apr 12, 2015·Biophysical Journal·L Michel Espinoza-FonsecaDavid D Thomas
Nov 7, 2006·Journal of the American College of Cardiology·Masahiko HoshijimaKenneth R Chien
Mar 13, 2012·Biochemical and Biophysical Research Communications·Simon J GruberDavid D Thomas
May 13, 2014·Biochemical and Biophysical Research Communications·Xiaoqiong Dong, David D Thomas
Sep 23, 2016·Biophysical Journal·Daniel J BlackwellSeth L Robia
Jan 18, 2014·Journal of Biomolecular Screening·Simon J GruberDavid D Thomas
Apr 3, 2007·Nature Protocols·Christine B KarimDavid D Thomas
Jun 24, 2018·The Journal of Biological Chemistry·Eli Fernández-de Gortari, L Michel Espinoza-Fonseca
Sep 1, 2006·The Journal of Biological Chemistry·Julie BossuytDonald M Bers
Aug 30, 2012·Journal of Muscle Research and Cell Motility·Brett A ColsonDavid D Thomas
Sep 3, 2013·The Journal of Biological Chemistry·Brandy L AkinLarry R Jones
Aug 26, 2014·Biological Chemistry·Grant Kemp, Florian Cymer
Dec 25, 2019·Annual Review of Biochemistry·Mateusz DylaPoul Nissen
Oct 26, 2018·Biosensors·Tory M SchaafGregory D Gillispie
Oct 3, 2007·The Journal of Biological Chemistry·Kim N HaGianluigi Veglia
Oct 7, 2020·International Journal of Molecular Sciences·Rodrigo Aguayo-Ortiz, L Michel Espinoza-Fonseca
Jan 26, 2021·Oxidative Medicine and Cellular Longevity·Jia WangYue Gao

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