Distinct Regulatory Effects of the Na,K-ATPase γ Subunit

Abstract

The two variants of the ␥ subunit of the rat renal sodium pump, ␥ a and ␥ b , have similar effects on the Na,K-ATPase. Both increase the affinity for ATP due to a shift in the enzyme's E 1 7 E 2 conformational equilibrium toward E 1 . In addition, both increase K ؉ antagonism of cytoplasmic Na ؉ activation. To gain insight into the structural basis for these distinct effects, extramembranous N-terminal and C-terminal mutants of ␥ were expressed in rat ␣1-transfected HeLa cells. At the N terminus, the variant-distinct region was deleted (␥N⌬7) or replaced by alanine residues (␥N7A). At the C terminus, four (␥ a C⌬4) or ten (␥ a C⌬10) residues were deleted. None of these mutations abrogates the K ؉ /Na ؉ antagonism as evidenced in a similar increase in K Na seen at high (100 mM) K ؉ concentration. In contrast, the C-terminal as well as N-terminal deletions (␥N⌬7, ␥ a C⌬4, and

FXYD7, Mapping of Functional Sites Involved in Endoplasmic Reticulum Export, Association With and Regulation of Na,K-ATPase

“…The precise domain(s) or amino acids in FXYD proteins that are directly involved in the interaction with Na,K-ATPase are still unknown. Recent studies suggest that, both in FXYD2 and FXYD4, the transmembrane domain is mainly involved in the stable interaction with Na,K-ATPase (19,33) though in FXYD2 the cytoplasmic C terminus may also play a role in this process (8). In the present study, we confirm recent observations (19) that the conserved Gly 40 , which is associated with a form of renal hypomagnesemia when mutated into an arginine residue in FXYD2 (18), is involved in Na,K-ATPase-FXYD protein interaction.…”

“…Since this mutation was shown to abolish the interaction of FXYD2 with the Na,K-ATPase (19), it has been speculated that magnesium wasting associated with this pathology may be indirectly caused by a loss of Na,K-ATPase regulation by FXYD2. To learn more about the role of this conserved glycine residue, we replaced Gly 40 in FXYD7 not only by alanine (G40A) but also by tryptophan, a bulky hydrophobic amino acid (G40W), or by arginine (G40R), a positively charged residue.…”

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

“…It remains to be shown whether substitution of these glycine residues could perturb correct folding of FXYD proteins, thus limiting complex formation. Alternatively, it remains to be investigated whether there might be a correlation between the potential implication of Gly 40 in oligomer formation (38) and the association efficiency of Gly 40 mutants in FXYD7 as well as in FXYD2 (39). Finally, the residues suggested by the docking study only, such as, Tyr 149 , Ile 157 , Arg 156 , Ile 142 , and Leu 968 in the Na,K-ATPase ␣ subunit, and the predicted amino acids in FXYD7, are good candidates for further mutagenesis analysis.…”

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

“…23 is that extra-membrane regions of both CHIF and ␥ interact with both ␣ and ␤ subunits. ␥ is known to raise the apparent affinity for ATP, by stabilizing the E 1 conformation, via an interaction near the cytoplasmic C terminus (9,57). CHIF does not have such an effect (17), and there is also a detailed difference in the DST-induced cross-link in that the ␥-␣ but not CHIF-␣ cross-link is amplified in a Na ϩ -containing medium.…”