The Fourth Transmembrane Segment of the Na,K-ATPase α Subunit

Although cardiac glycosides have been used as drugs for more than 2 centuries and their primary target, the sodium pump (Na,KATPase), has already been known for 4 decades, their exact binding site is still elusive. In our efforts to define the molecular basis of digitalis glycosides binding we started from the fact that a closely related enzyme, the gastric H,K-ATPase, does not bind glycosides like ouabain. Previously, we showed that a chimera of these two enzymes, in which only the M3-M4 and M5-M6 hairpins were of Na,K-ATPase, bound ouabain with high affinity (

Section: Discussionmentioning

confidence: 53%

Reconstruction of the Complete Ouabain-binding Pocket of Na,K-ATPase in Gastric H,K-ATPase by Substitution of Only Seven Amino Acids

Qiu

Krieger

Schaftenaar

et al. 2005

“…In view of the relationship between C2 and the ␣ subunit in the three-dimensional structure (Fig. 6A) glutathionylation may nevertheless contribute to regulation, for example by influencing the PEGL motif further down transmembrane helix 4 of the ␣ subunit that is critical for Na ϩ binding (34). Other effects of glutathionylation on FXYD/␣ interaction may also contribute.…”

Section: Discussionmentioning

confidence: 99%

FXYD Proteins Reverse Inhibition of the Na+-K+ Pump Mediated by Glutathionylation of Its β1 Subunit

Bibert

Liu

Figtree

et al. 2011

114

We have reported that Na ؉ -K ؉ pump ␤ 1 subunit glutathionylation induced by oxidative signals causes pump inhibition in a previous study. In the present study, we found that ␤ 1 subunit glutathionylation and pump inhibition could be reversed by exposing myocytes to exogenous wild-type FXYD3. A cysteine-free FXYD3 derivative had no effect. Similar results were obtained with wild-type and mutant FXYD proteins expressed in oocytes. Glutathionylation of the ␤ 1 subunit was increased in myocardium from FXYD1 ؊/؊ mice. In conclusion, there is a dependence of Na

“…All residues in TM9, Ile 953 29 and Gly 40 in FXYD7, the substitution of which has previously been shown to significantly affect the association efficiency with Na,KATPase (37), were not predicted to form favorable contacts by the docking. It remains to be shown whether substitution of these glycine residues could perturb correct folding of FXYD proteins, thus limiting complex formation.…”

Section: Discussionmentioning

confidence: 99%

“…Protein Modeling-Based on our previous homology model of the Bufo Na-K-ATPase (29) in the E1 conformation, a model of the FXYD7/ Na-K-ATPase complex was built using an evolutionary algorithm. The details of the calculations will be presented separately.…”

Section: Methodsmentioning

confidence: 99%

“…We produced a model of the TM9 helix (29) and identified Ile 953 , Phe 956 , Glu 960 , Leu 964 , and Phe 967 , which could be directed to FXYD proteins and potentially could form an interaction face. We substituted these amino acids of the rat ␣1 isoform, located on the same side of the TM9 helix, either individually or in combination with alanine residues (Fig.…”

Section: Figmentioning

confidence: 99%

See 1 more Smart Citation

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

Grosdidier

Crambert

et al. 2004

Self Cite

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.