Expression of highly selective sodium channels in alveolar type II cells is determined by culture conditions

Alveolar fluid clearance in the developing and mature lungs is believed to be mediated by some form of epithelial Na channels (ENaC). However, single- channel studies using isolated alveolar type II (ATII) cells have failed to demonstrate consistently the presence of highly selective Na+ channels tha t would be expected from ENaC expression. We postulated that in vitro cultu re conditions might be responsible for alterations in the biophysical prope rties of Na+ conductances observed in cultured ATII cells. When ATII cells were grown on glass plates submerged in media that lacked steroids, the pre dominant channel was a 21-pS nonselective cation channel (NSC) with a Na+-t o-K+ selectivity of 1; however, when grown on permeable supports in the pre sence of steroids and air interface, the predominant channel was a low-cond uctance (6.6 +/- 3.4 pS, n = 94), highly Na+-selective channel (HSC) with a P-Na/P-K >80 that is inhibited by submicromolar concentrations of amilorid e (K-0.5 = 37 nM) and is similar in biophysical properties to ENaC channels described in other epithelia. To establish the relationship of this HSC ch annel to the cloned ENaC, we employed antisense oligonucleotide methods to inhibit the individual subunit proteins of ENaC (alpha, beta, and gamma) an d used patch-clamp techniques to determine the density of this channel in a pical membrane patches of ATII cells. Overnight treatment of cells with ant isense oligonucleotides to any of the three subunits of ENaC resulted in a significant decrease in the density of HSC channels in the apical membrane cell-attached patches. Taken together, these results show that when grown o n permeable supports in the presence of steroids and air interface, the pre dominant channels expressed in ATII cells have single-channel characteristi cs resembling channels that are associated with the coexpression of the thr ee cloned ENaC subunits alpha-, beta-, and gamma -ENaC.