title: Phospholamban binds with differential affinity to calcium pump conformers.
abstract: To investigate the mechanism of regulation of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) by phospholamban (PLB), we expressed Cerulean-SERCA and yellow fluorescent protein (YFP)-PLB in adult rabbit ventricular myocytes using adenovirus vectors. SERCA and PLB were localized in the sarcoplasmic reticulum and were mobile over multiple sarcomeres on a timescale of tens of seconds. We also observed robust fluorescence resonance energy transfer (FRET) from Cerulean-SERCA to YFP-PLB. Electrical pacing of cardiac myocytes elicited cytoplasmic Ca(2+) elevations, but these increases in Ca(2+) produced only modest changes in SERCA-PLB FRET. The data suggest that the regulatory complex is not disrupted by elevations of cytosolic calcium during cardiac contraction (systole). This conclusion was also supported by parallel experiments in heterologous cells, which showed that FRET was reduced but not abolished by calcium. Thapsigargin also elicited a small decrease in PLB-SERCA binding affinity. We propose that PLB is not displaced from SERCA by high calcium during systole, and relief of functional inhibition does not require dissociation of the regulatory complex. The observed modest reduction in the affinity of the PLB-SERCA complex with Ca(2+) or thapsigargin suggests that the binding interface is altered by SERCA conformational changes. The results are consistent with multiple modes of PLB binding or alternative binding sites.
authors.affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, Illinois 60153, USA.
authors.forename: Philip
authors.initials: P
authors.lastname: Bidwell
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Blackwell
authors.forename: Zhanjia
authors.initials: Z
authors.lastname: Hou
authors.forename: Aleksey V
authors.initials: AV
authors.lastname: Zima
authors.forename: Seth L
authors.initials: SL
authors.lastname: Robia
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004285:Dogs; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006352:Heart Ventricles / Q000166:cytology; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods; D008954:Models, Biological; D032342:Muscle Cells / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011817:Rabbits; D000099211:Phospholamban
chemical_list: D001426:Bacterial Proteins; D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D008164:Luminescent Proteins; D008565:Membrane Proteins; D000099211:Phospholamban; C054562:yellow fluorescent protein, Bacteria