title: Cardiac Calcium ATPase Dimerization Measured by Cross-Linking and Fluorescence Energy Transfer.
abstract: The cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA) establishes the intracellular calcium gradient across the sarcoplasmic reticulum membrane. It has been proposed that SERCA forms homooligomers that increase the catalytic rate of calcium transport. We investigated SERCA dimerization in rabbit left ventricular myocytes using a photoactivatable cross-linker. Western blotting of cross-linked SERCA revealed higher-molecular-weight species consistent with SERCA oligomerization. Fluorescence resonance energy transfer measurements in cells transiently transfected with fluorescently labeled SERCA2a revealed that SERCA readily forms homodimers. These dimers formed in the absence or presence of the SERCA regulatory partner, phospholamban (PLB) and were unaltered by PLB phosphorylation or changes in calcium or ATP. Fluorescence lifetime data are compatible with a model in which PLB interacts with a SERCA homodimer in a stoichiometry of 1:2. Together, these results suggest that SERCA forms constitutive homodimers in live cells and that dimer formation is not modulated by SERCA conformational poise, PLB binding, or PLB phosphorylation.
authors.affiliation: Cell and Molecular Physiology, Loyola University Chicago, Chicago, Illinois.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Blackwell
authors.affiliation: Cell and Molecular Physiology, Loyola University Chicago, Chicago, Illinois.
authors.forename: Taylor J
authors.initials: TJ
authors.lastname: Zak
authors.affiliation: Cell and Molecular Physiology, Loyola University Chicago, Chicago, Illinois. Electronic address: srobia@luc.edu.
authors.forename: Seth L
authors.initials: SL
authors.lastname: Robia
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D004285:Dogs; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006352:Heart Ventricles / Q000737:chemistry / Q000201:enzymology; D006801:Humans; D047468:Immunoprecipitation; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D032342:Muscle Cells / Q000737:chemistry / Q000201:enzymology; D009154:Mutation; D010766:Phosphorylation; D038761:Photobleaching; D055503:Protein Multimerization; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000099211:Phospholamban
chemical_list: D000255:Adenosine Triphosphate; D002118:Calcium; D002135:Calcium-Binding Proteins; D008164:Luminescent Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban