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,KATPase ␣ 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 ␣ 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 ␣ 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 ␣ 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 ␣ 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.

A third Na + -binding site in the sodium pump

“…We have recently provided evidence for an interaction between the transmembrane segment of FYXD proteins and the ninth transmembrane segment of the ␣-subunit (22). A similar position of FXYD protein is supported by other approaches (31) while other locations have also been proposed recently (32).…”

“…2A) a negative slope of the K 1/2 K ext ϩ vs. Vm relation in the high negative potential (Ϫ50 to Ϫ130 mV) in the presence of extracellular sodium (Na ext ϩ ), which is due to the competitive binding of Na ext ϩ with K ext ϩ on the E2 conformation. Alanine substitution of E960, a residue predicted to mediate the interaction between the ␣-subunit and FXDY7 (22), had only a small effect, whereas the E961A mutation resulted in a large change of the voltage-dependent activation by K ext ϩ in the presence of Na ext ϩ ( Fig. 2 A) whereas there was no effect in the absence of Na ext ϩ (Fig.…”

Structural Interactions between FXYD Proteins and Na+,K+-ATPase

“…Previously, the location of the trans-membrane segment in the groove between M2, M6, and M9 was inferred on the basis of electron microscopy of renal Na ϩ ,K ϩ -ATPase (20), functional effects of mutations in M9 (22), and cross-linking of the cytoplasmic domain of the ␥ subunit to S4 of the ␣1 subunit (23). Specifically, the current experiments show that Cys-49 of CHIF is cross-linked to Cys-140 in M2 of the ␣ subunit, because either the C49F or the C140S mutation specifically interferes with the crosslink.…”

“…A denaturation study also suggested that ␥ might interact in the M8 to M10 region (21). A role for M9 of the ␣ subunit has been inferred from the effects of mutants in M9 on the stability of ␣/␤-␥, ␣/␤-CHIF, or ␣/␤-FXYD7 complexes, and the functional consequences of the mutations have been studied in Xenopus oocytes (22). Modeling of the FXYD helix was consistent with docking in the groove between M2, M6, and M9 in the ␣ subunit.…”

Mécanisme du transport des cations Na+et K+par la pompe à sodium

“…Plusieurs localisations avaient été proposées pour ce troisième site. C'est en cherchant à préciser la nature des interactions entre la sous-unité α de la Na + ,K + -ATPase et une protéine régulatrice -de la famille des protéines FXYD [6] -que nous avons découvert le rôle très important joué par un segment transmembranaire peu étudié jusque-là, M9, dans la mécanique du transport des cations. À la suite de l'observation de l'influence très importante d'une mutation (E961C) dans le segment M9 sur la Figure 1.…”