Structural and Functional Interaction Sites between Na,K-ATPase and FXYD Proteins

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 α 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 α subunit in the structural and functional interaction with FXYD2, FXYD4, and FXYD7. Mutational analysis combined with expression in Xenopus oocytes reveals that Phe956, Glu960, Leu964, and Phe967 in TM9 of the Na,K-ATPase α subunit represent one face interacting with the three FXYD proteins. Leu964 and Phe967 contribute to the efficient association of FXYD proteins with the Na,K-ATPase α subunit, whereas Phe956 and Glu960 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 Phe956 and Glu960 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, Phe956 and Glu960 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 Phe956, Glu960, Leu964, and Phe967 in subunit interaction. In conclusion, by using mutational analysis and modeling, we show that TM9 of the Na,K-ATPase α 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.