title: Na⁺/K⁺-ATPase E960 and phospholemman F28 are critical for their functional interaction.
abstract: Na(+)-K(+)-ATPase (NKA) establishes the transmembrane [Na(+)] gradient in cells. In heart, phospholemman (PLM) inhibits NKA activity by reducing its apparent Na(+) affinity, an effect that is relieved by PLM phosphorylation. The NKA crystal structure suggests regions of PLM-NKA interaction, but the sites important for functional effects in live cells are not known. We tested wild type (WT) and CFP-NKA-α1 point mutants (alanine substitution at F956, E960, L964, and F967) for fluorescence resonance energy transfer (FRET) with WT-PLM-YFP in HEK293 cells. NKA-PLM FRET was unaltered with F956A or F967A, reduced with L964A, and nearly abolished with E960A. Mutating the PLM site (F28A) identified by structural analysis to interact with E960-NKA also nearly abolished NKA-PLM FRET. In contrast, NKA-PLM coimmunoprecipitation was only slightly reduced by E960A-NKA or F28A-PLM mutants, consistent with an additional interaction site. FRET titrations indicate that the additional site has higher affinity than that between E960-NKA and F28-PLM. To test whether the FRET-preventing mutations also prevent PLM functional effects, we measured NKA-mediated Na(+)-transport in intact cells. For WT-NKA, PLM reduced apparent Na(+)-affinity of NKA and PLM phosphorylation reversed the effect. In contrast, for E960A-NKA the apparent Na(+)-affinity was unaltered by either PLM or forskolin-induced PLM phosphorylation. We conclude that E960 on NKA and F28 on PLM are critical for PLM effects on both NKA function and NKA-PLM FRET, but also there is at least one additional site that is critical for tethering PLM to NKA.
authors.affiliation: Department of Pharmacology, School of Medicine, University of California, Davis, CA 95616, USA.
authors.forename: Mounir
authors.initials: M
authors.lastname: Khafaga
authors.forename: Julie
authors.initials: J
authors.lastname: Bossuyt
authors.forename: Luiza
authors.initials: L
authors.lastname: Mamikonian
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Li
authors.forename: Linda L
authors.initials: LL
authors.lastname: Lee
authors.forename: Vladimir
authors.initials: V
authors.lastname: Yarov-Yarovoy
authors.forename: Sanda
authors.initials: S
authors.lastname: Despa
authors.forename: Donald M
authors.initials: DM
authors.lastname: Bers
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D018360:Crystallography, X-Ray; D004285:Dogs; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D017136:Ion Transport; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012754:Sharks; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C069141:phospholemman; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase