Isoform specificity of human Na+,K+-ATPase localization and aldosterone regulation in mouse kidney cells

The minimal functional unit of the Na+,K+-ATPase is a heterodimer of an alpha (α1–4) and a beta (β1–3) subunit (reviewed in Blanco & Mercer, 1998). The α subunit is the multi membrane-spanning catalytic subunit that performs ATP-driven extrusion of three Na+ in exchange for two K+. The β subunit is known to be required for structural and functional maturation of the α subunit and also slightly impacts on the K+ and Na+ activation kinetics of the mature Na+,K+-ATPase (Hasler et al. 1998). The small γ subunit or CHIF (FXYD protein family) is associated with the Na+,K+-ATPase in some kidney tubule segments and modulates pump kinetics (Crambert & Geering, 2003). The Na+,K+-ATPase composed of α1 and β1 subunits appears to be expressed ubiquitously and fulfils housekeeping functions. The α1 subunit also appears to be the major or single α subunit to be expressed along the nephron (Lucking et al. 1996), although the Ki for ouabain and the apparent Na+ affinity display axial differences (Doucet & Barlet, 1986; Barlet-Bas et al. 1990). Recent functional and localization results obtained for the γ subunits and CHIF suggest that the localized expression of these small ancillary subunits along the tubule might explain these differences (Arystarkhova et al. 1999, 2002; Therien et al. 1999; Beguin et al. 2001; Crambert & Geering, 2003). The α2 subunit is expressed in brain, skeletal muscle and heart, the α3 subunit in brain and the α4 subunit exclusively in testis. Rat α1, 2 and 3 are 87% identical and their functional differences in terms of Na+ and K+ kinetics appear relatively small (Blanco & Mercer, 1998). Functional parameters might, however, be to some extent species specific and have been determined for human Na+,K+-ATPase only in non-mammalian expression systems, i.e. in Xenopus oocytes, insect SF9 cells and yeast (Yu et al. 1997; Crambert et al. 2000; Muller-Ehmsen et al. 2001). The aldosterone-sensitive distal nephron (ASDN) is composed of the second half of the distal convoluted tubule (DCT2), the connecting tubule (CNT) and the collecting duct (CD) (Loffing et al. 2001). In this region, aldosterone action depends on transcription and translation (Summa et al. 2001) and it is the site where fine tuning of the final urinary Na+ excretion takes place (Verrey et al. 2000). The Na+ reabsorption machinery of the ASDN segment-specific epithelial cells is formed from the epithelial Na+ channel ENaC and, in DCT2, the Na+–Cl− cotransporter (NCC), which mediate luminal Na+ influx, and by the Na+,K+-ATPase that extrudes Na+ to the basolateral side and maintains the driving force for apical Na+ entry. Aldosterone controls Na+ reabsorption by regulating this transport in the short and long term (Verrey et al. 2000; Masilamani et al. 2002). Functionally, the short-term effect of aldosterone is already apparent 30 min after initiation of treatment (Verrey et al. 2000). We have recently shown in adrenalectomized rats that aldosterone increases the cell-surface expression of ENaC and of the Na+,K+-ATPase within 2 h (Loffing et al. 2001; Summa et al. 2001; Verrey et al. 2003). The effect of aldosterone on the cell-surface expression of Na+,K+-ATPase could be reproduced in vitro using the mouse cortical collecting duct-derived mpkCCDc14 cell line (Bens et al. 1999). In this model, the increase in cell-surface expression correlates with an increase in Na+ pump current. It is independent of apical Na+ influx during hormonal stimulation and depends on transcription and translation (Summa et al. 2001). Short-term stimulation of Na+,K+-ATPase by aldosterone has been previously described in Xenopus laevis A6 cells, another model for distal nephron Na+ reabsorption. However, unlike in mammalian cortical collecting duct (CCD) and mpkCCD cells, this stimulation was not mediated by an increase in the number of cell-surface pumps, meaning that the underlying mechanism is probably not the same. The aim of the present study was to test the hypothesis that the transcriptionally mediated effect of aldosterone on the cell-surface expression of Na+,K+-ATPase in mammalian CCD cells depends on isoform-specific structures of the α1 subunit. Therefore, we expressed the human Na+,K+-ATPase subunit isoforms α1, α2 and α3 in the mouse kidney cortical collecting duct cell line mpkCCDc14 and characterized their expression and their regulation by aldosterone. The results revealed an a isoform-specific distribution of the Na+,K+-ATPase and an α1 subunit-specific stimulation by aldosterone of the cell-surface Na+ pump function.