title: Structural and functional interaction sites between Na,K-ATPase and FXYD proteins.
abstract: Several members of the FXYD protein family are tissue-specific regulators of Na,K-ATPase that produce distinct effects on its apparent K(+) and Na(+) affinity. Little is known about the interaction sites between the Na,K-ATPase alpha subunit and FXYD proteins that mediate the efficient association and/or the functional effects of FXYD proteins. In this study, we have analyzed the role of the transmembrane segment TM9 of the Na,K-ATPase alpha subunit in the structural and functional interaction with FXYD2, FXYD4, and FXYD7. Mutational analysis combined with expression in Xenopus oocytes reveals that Phe(956), Glu(960), Leu(964), and Phe(967) in TM9 of the Na,K-ATPase alpha subunit represent one face interacting with the three FXYD proteins. Leu(964) and Phe(967) contribute to the efficient association of FXYD proteins with the Na,K-ATPase alpha subunit, whereas Phe(956) and Glu(960) are essential for the transmission of the functional effect of FXYD proteins on the apparent K(+) affinity of Na,K-ATPase. The relative contribution of Phe(956) and Glu(960) to the K(+) effect differs for different FXYD proteins, probably reflecting the intrinsic differences of FXYD proteins on the apparent K(+) affinity of Na,K-ATPase. In contrast to the effect on the apparent K(+) affinity, Phe(956) and Glu(960) are not involved in the effect of FXYD2 and FXYD4 on the apparent Na(+) affinity of Na,K-ATPase. The mutational analysis is in good agreement with a docking model of the Na,K-ATPase/FXYD7 complex, which also predicts the importance of Phe(956), Glu(960), Leu(964), and Phe(967) in subunit interaction. In conclusion, by using mutational analysis and modeling, we show that TM9 of the Na,K-ATPase alpha subunit exposes one face of the helix that interacts with FXYD proteins and contributes to the stable interaction with FXYD proteins, as well as mediating the effect of FXYD proteins on the apparent K(+) affinity of Na,K-ATPase.
authors.affiliation: Department of Pharmacology and Toxicology of the University, Rue du Bugnon 27, CH-1005 Lausanne, Switzerland.
authors.forename: Ciming
authors.initials: C
authors.lastname: Li
authors.forename: Aurelien
authors.initials: A
authors.lastname: Grosdidier
authors.forename: Gilles
authors.initials: G
authors.lastname: Crambert
authors.forename: Jean-Daniel
authors.initials: JD
authors.lastname: Horisberger
authors.forename: Olivier
authors.initials: O
authors.lastname: Michielin
authors.forename: Käthi
authors.initials: K
authors.lastname: Geering
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D004252:DNA Mutational Analysis; D004594:Electrophysiology; D005973:Glutamine / Q000737:chemistry; D007930:Leucine / Q000737:chemistry; D008564:Membrane Potentials; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry; D011188:Potassium / Q000737:chemistry; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013997:Time Factors; D014981:Xenopus
chemical_list: D005973:Glutamine; D010649:Phenylalanine; D000254:Sodium-Potassium-Exchanging ATPase; D007930:Leucine; D000409:Alanine; D011188:Potassium