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

Abstract The two variants of the γ subunit of the rat renal sodium pump, γa and γb, have similar effects on the Na,K-ATPase. Both increase the affinity for ATP due to a shift in the enzyme's E1 ↔ E2 conformational equilibrium toward E1. 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 γ were expressed in rat α1-transfected HeLa cells. At the N terminus, the variant-distinct region was deleted (γNΔ7) or replaced by alanine residues (γN7A). At the C terminus, four (γaCΔ4) or ten (γaCΔ10) 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 (γNΔ7, γaCΔ4, and γaCΔ10) abolished the decrease in K′ATP seen with wild-type γa or γb. It is concluded that different regions of the γ chain mediate the distinct functional effects of γ, and the effects can be long-range. In the transmembrane region, the impact of G41R replacement was analyzed since this mutation is associated with autosomal dominant renal Mg2+-wasting in man (Meij, I. C., Koenderink, J. B., van Bokhoven, H., Assink, K. F. H., Groenestege, W. T., de Pont, J. J. H. H. M., Bindels, R. J. M., Monnens, L. A. H., Van den Heuvel, L. P. W. J., and Knoers, N. V. A. M. (2000) Nat. Genet. 26, 265–266). The results show that Gly-41 → Arg prevents trafficking of γ but not αβ pumps to the cell surface and abrogates functional effects of γ on αβ pumps. These findings underscore a potentially important role of γ in affecting solute transport, in this instance Mg2+ reabsorption, consequent to its primary effect on the sodium pump.