Antisense oligonucleotides against the alpha-subunit of ENaC decrease lungepithelial cation-channel activity

Amiloride-sensitive Na+ transport by lung epithelia plays a critical role i n maintaining alveolar Nat and water balance. It has been generally assumed that Na+ transport is mediated by the amiloride-sensitive epithelial Nat c hannel (ENaC) because molecular biology studies have confirmed the presence of ENaC subunits alpha, beta, and gamma in lung epithelia. However, the pr edominant Na+-transporting channel reported from electrophysiological studi es by most laboratories is a nonselective, high-conductance channel that is very different from the highly selective, low-conductance ENaC reported in other tissues. In our laboratory, single-channel recordings from apical me mbrane patches from rat alveolar type II (ATII) cells in primary culture re veal a nonselective cation channel with a conductance of 20.6 +/- 1.1 pS an d an Na+-to-K+ selectivity of 0.97 +/- 0.07. This channel is inhibited by s ubmicromolar concentrations of amiloride. Thus there is some question about the relationship between the gene product observed with single-channel met hods and the cloned ENaC subunits. We have employed antisense oligonucleoti de methods to block the synthesis of individual ENaC subunit proteins (alph a, beta, and gamma) and determined the effect of a reduction in the subunit expression on the density of the nonselective cation channel observed in a pical membrane patches on ATII cells. Treatment of ATII cells with antisens e oligonucleotides inhibited the production of each subunit protein; howeve r, single-channel recordings showed that only the antisense oligonucleotide targeting the a-subunit resulted in a significant decrease in the density of nonselective cation channels. Inhibition of the beta- and gamma-subunit proteins alone or together did not cause any changes in the observed channe l density. There were no changes in open probability or other channel chara cteristics. These results support the hypothesis that the alpha-subunit of ENaC alone or in combination with some protein other than the beta- or gamm a-subunit protein is the major component of lung alveolar epithelial cation channels.