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.
Highlights
Na,K-ATPase transports three Naϩ against two Kϩ across the plasma membrane of animal cells by using the energy of the hydrolysis of ATP
The TM9 –TM10 Region of the Na,K-ATPase ␣ Subunit Is Involved in Interaction with FXYD2—We have previously shown [11] that non-gastric H,K-ATPase does not associate with FXYD2 or FXYD4
To test whether the TM8 –TM10 region of the Na,K-ATPase ␣ subunit is involved in the interaction with FXYD2 as suggested by biochemical evidence [18], we produced a chimera containing the sequence of the Na,KATPase ␣1 subunit up to TM8 and that of the human nongastric H,K-ATPase ␣ subunit (AL1) [25] including TM9 and TM10 (Fig. 1)
Summary
It is well established that FXYD1 (phospholemman) [14], a phospholemman-like protein from shark [15], FXYD4 (corticosteroid hormone-induced factor) [11, 16], and FXYD7 [17] play a tissue-specific role in Na,K-ATPase regulation. Each of these auxiliary subunits produce a distinct functional effect on Na,K-ATPase that is adapted to the physiological needs of the tissues in which they are expressed. A recent model [19], deduced from an electron crystallographic analysis at 9.5-Å resolution of
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