title: Isoform specificity of the Na/K-ATPase association and regulation by phospholemman.
abstract: Phospholemman (PLM) phosphorylation mediates enhanced Na/K-ATPase (NKA) function during adrenergic stimulation of the heart. Multiple NKA isoforms exist, and their function/regulation may differ. We combined fluorescence resonance energy transfer (FRET) and functional measurements to investigate isoform specificity of the NKA-PLM interaction. FRET was measured as the increase in the donor fluorescence (CFP-NKA-alpha1 or CFP-NKA-alpha2) during progressive acceptor (PLM-YFP) photobleach in HEK-293 cells. Both pairs exhibited robust FRET (maximum of 23.6 +/- 3.4% for NKA-alpha1 and 27.5 +/- 2.5% for NKA-alpha2). Donor fluorescence depended linearly on acceptor fluorescence, indicating a 1:1 PLM:NKA stoichiometry for both isoforms. PLM phosphorylation induced by cAMP-dependent protein kinase and protein kinase C activation drastically reduced the FRET with both NKA isoforms. However, submaximal cAMP-dependent protein kinase activation had less effect on PLM-NKA-alpha2 versus PLM-NKA-alpha1. Surprisingly, ouabain virtually abolished NKA-PLM FRET but only partially reduced co-immunoprecipitation. PLM-CFP also showed FRET to PLM-YFP, but the relationship during progressive photobleach was highly nonlinear, indicating oligomers involving >or=3 monomers. Using cardiac myocytes from wild-type mice and mice where NKA-alpha1 is ouabain-sensitive and NKA-alpha2 is ouabain-resistant, we assessed the effects of PLM phosphorylation on NKA-alpha1 and NKA-alpha2 function. Isoproterenol enhanced internal Na(+) affinity of both isoforms (K((1/2)) decreased from 18.1 +/- 2.0 to 11.5 +/- 1.9 mm for NKA-alpha1 and from 16.4 +/- 2.5 to 10.4 +/- 1.5 mm for NKA-alpha2) without altering maximum transport rate (V(max)). Protein kinase C activation also decreased K((1/2)) for both NKA-alpha1 and NKA-alpha2 (to 9.4 +/- 1.0 and 9.1 +/- 1.1 mm, respectively) but increased V(max) only for NKA-alpha2 (1.9 +/- 0.4 versus 1.2 +/- 0.5 mm/min). In conclusion, PLM associates with and modulates both NKA-alpha1 and NKA-alpha2 in a comparable but not identical manner.
authors.affiliation: Department of Pharmacology, University of California, Davis, California 95616, USA.
authors.forename: Julie
authors.initials: J
authors.lastname: Bossuyt
authors.forename: Sanda
authors.initials: S
authors.lastname: Despa
authors.forename: Fei
authors.initials: F
authors.lastname: Han
authors.forename: Zhanjia
authors.initials: Z
authors.lastname: Hou
authors.forename: Seth L
authors.initials: SL
authors.lastname: Robia
authors.forename: Jerry B
authors.initials: JB
authors.lastname: Lingrel
authors.forename: Donald M
authors.initials: DM
authors.lastname: Bers
mesh_terms: D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D047468:Immunoprecipitation; D017136:Ion Transport / Q000187:drug effects; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010042:Ouabain / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D007527:Isoenzymes; D008164:Luminescent Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; C069141:phospholemman; D015240:Phorbol 12,13-Dibutyrate; D010042:Ouabain; D012964:Sodium; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D000254:Sodium-Potassium-Exchanging ATPase; D007545:Isoproterenol