Na+/K+-ATPase E960 and phospholemman F28 are critical for their functional interaction.

Mounir Khafaga, Julie B C Bossuyt, Luiza Mamikonian, Joseph C. Li, Linda L. Lee, Vladimir Yarov-Yarovoy, Sanda Despa, Donald M Bers

Research output: Contribution to journalArticle

10 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)20756-20761
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume109
Issue number50
StatePublished - Dec 11 2012

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