title: Phospholamban mutants compete with wild type for SERCA binding in living cells.
abstract: We have used fluorescent fusion proteins stably expressed in HEK cells to detect directly the interaction between the sarcoplasmic reticulum Ca-ATPase (SERCA) and phospholamban (PLB) in living cells, in order to design PLB mutants for gene therapy. Ca(2+) cycling in muscle cells depends strongly on SERCA. Heart failure (HF), which contributes to 12% of US deaths, typically exhibits decreased SERCA activity, and several potential therapies for HF aim to increase SERCA activity. We are investigating the use of LOF-PLB mutants (PLB(M)) as gene therapy vectors to increase SERCA activity. Active SERCA1a and WT-PLB, tagged at their N termini with fluorescent proteins (CFP and YFP), were coexpressed in stable HEK cell lines, and fluorescence resonance energy transfer (FRET) was used to detect their interaction directly. Phosphorylation of PLB, induced by forskolin, caused an increase in FRET from CFP-SERCA to YFP-PLB, indicating that SERCA inhibition can be relieved without dissociation of the complex. This suggests that a LOF mutant might bind to SERCA with sufficient affinity to complete effectively with WT-PLB, thus relieving SERCA inhibition. Therefore, we transiently expressed a series of PLB(M) in the CFP-SERCA/YFP-PLB cell line, and found decreased FRET, implying competition between PLB(M) and WT-PLB for binding to SERCA. These results establish this FRET assay as a rapid and quantitative means of screening PLB(M) for optimization of gene therapy to activate SERCA, as needed for gene therapy in HF.
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota Medical School, Minneapolis, MN 55455, USA.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Gruber
authors.forename: Suzanne
authors.initials: S
authors.lastname: Haydon
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
mesh_terms: D001667:Binding, Competitive; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D015316:Genetic Therapy / Q000379:methods; D057809:HEK293 Cells; D006333:Heart Failure / Q000628:therapy; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism*; D000099211:Phospholamban
chemical_list: D002135:Calcium-Binding Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban; C506943:ATP2A1 protein, human