Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
SHR Neuro Krebs Kardio Lipid Stoffw Microb
Khafaga, M; Bossuyt, J; Mamikonian, L; Li, JC; Lee, LL; Yarov-Yarovoy, V; Despa, S; Bers, DM.
Na⁺/K⁺-ATPase E960 and phospholemman F28 are critical for their functional interaction.
Proc Natl Acad Sci U S A. 2012; 109(50):20756-20761
Doi: 10.1073/pnas.1207866109
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- Führende Autor*innen der Med Uni Graz
- Khafaga Mounir
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- 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.
- Find related publications in this database (using NLM MeSH Indexing)
- Amino Acid Sequence -
- Amino Acid Substitution -
- Animals -
- Crystallography, X-Ray -
- Dogs -
- Fluorescence Resonance Energy Transfer -
- HEK293 Cells -
- Humans -
- Ion Transport -
- Kinetics -
- Membrane Proteins - chemistry
- Mice -
- Models, Biological -
- Models, Molecular -
- Molecular Sequence Data -
- Mutagenesis, Site-Directed -
- Phosphoproteins - chemistry
- Protein Conformation -
- Protein Interaction Domains and Motifs -
- Rats -
- Recombinant Fusion Proteins - chemistry
- Sequence Homology, Amino Acid -
- Sharks -
- Sodium - metabolism
- Sodium-Potassium-Exchanging ATPase - chemistry