Glucocorticoids control epithelial Na+ conductance via a mechanism dependent upon serum and glucocorticoid-inducible kinse 1 (SGK1) a kinase thought to control the surface abundance of α-,β- and γ-ENaC. However, whilst dexamethasone activates SGK1 in H441 cells, this response is rapid (1 – 3 h) and transient whilst the induction of Na+ current is relatively slow (> 3 h) and sustained (Watt et al. 2010). We have therefore explored the effects of dexamethasone upon α-,β- and γ-ENaC abundance in the total and in surface protein pools; surface proteins were isolated by biotinylation (10mM sulfosuccinimidyl-2-(biotinamido)-ethyl-1,3’dithioproprionate, 1 h 4°C) / streptavidin binding. Two α-ENaC isoforms (~100 kDa and ~77 kDa) were detected in total and surface pools and dexamethasone (0.2 µM, 24 h) consistently increased the abundance of each isoform in both protein pools (Fig. 1A). Beta-ENaC was present as a single band (~100 kDa) and, whilst dexamethasone clearly increased the overall expression of this subunit, it had no effect upon surface abundance. Although two γ-ENaC isoforms (96 kDa, 76 kDa) were detected in total protein, only one band (~80 kDa) was present at the cell surface and dexamethasone had no effect upon the abundance of this surface-exposed protein (Fig. 1A). Each ENaC subunits is therefore present at the surface of glucocorticoid-deprived cells, and the fact that such cells do not display Na+ currents (Watt et al. 2010) cannot, therefore, be attributed to the absence of α-,β- and γ-ENaC from the membrane. Moreover, the induction of ENaC activity by prolonged (~24 h) dexamethasone stimulation is not associated with any change to the surface expression of β- and γ-ENaC whilst the effect on α-ENaC can be explained by an increase in the overall expression level (Fig1 A). Since glucocorticoid-induced SGK1 activity peaks after ~3 h (Watt et al. 2010), subsequent experiments compared the abundance of α-,β- and γ-ENaC at the surface of hormone-deprived cells, and cells exposed to 0.2 µM dexamethasone for 24 h or 3h. As anticipated (see above) 24 h stimulation increased the surface expression of α-ENaC with no effect upon β- or γ-ENaC (Fig. 1B). Increases in the surface abundance of α-,β- and γ-ENaC were, however, evident in cells exposed to dexamethasone for only 3 h (Fig. 1B), despite the fact that such cells do not express discernible Na+ currents (Watt et al. 2010). Whilst periods of SGK1 activation are associated with the recruitment of α-,β- and γ-ENaC to the cell surface, such co-ordinated increases in the surface expression of these channel subunits cannot explain the SGK1-dependent activation of ENaC in these cells (Watt et al. 2010).