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

Abstract

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 that would be expected from ENaC expression. We postulated that in vitro culture conditions might be responsible for alterations in the biophysical properties of Na+ conductances observed in cultured ATII cells. When ATII cells were grown on glass plates submerged in media that lacked steroids, the predominant channel was a 21-pS nonselective cation channel (NSC) with a Na+-to-K+ selectivity of 1; however, when grown on permeable supports in the presence of steroids and air interface, the predominant channel was a low-conductance (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 amiloride (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 channel to the cloned ENaC, we employed antisense oligonucleotide methods to inhibit the individual subunit proteins of ENaC (alpha, beta, and gamma) and used patch-clamp techniques to determine the density of this channel in apical membrane patches of ATII cells. Overnight treatment of cells with antisense 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 on permeable supports in the presence of steroids and air interface, the predominant channels expressed in ATII cells have single-channel characteristics resembling channels that are associated with the coexpression of the three cloned ENaC subunits alpha-, beta-, and gamma-ENaC.

Serial changes in nasal potential difference and lung electrical impedance tomography at high altitude

“…The exact nature of cation channels in the alveolar epithelium has been debated because of the differing biophysical properties of channels encountered in cultured cells. However, Jain and colleagues (18) demonstrated that the culture conditions under which type II pneumocytes are grown influence their biophysical properties. When ATII cells were grown on glass plates submerged in media, the predominant channel was a 21-pS nonselective cation channel.…”

Steroids and Exogenous γ-ENaC Subunit Modulate Cation Channels Formed by α-ENaC in Human B Lymphocytes

“…However, in other cell types, expression of the exogenous ␣-ENaC subunit alone can form a stretch-activated nonselective cation channel (9), and a similar nonselective cation channel has also been described in native lung epithelial alveolar type II cells (10 -12). This lung cation channel appears to be formed from ␣-ENaC alone and is equally permeable to Na ϩ and K ϩ , is sensitive to steroid hormones, and has a higher unit conductance (21 pS) than that of ␣␤␥-ENaC (4 -5 pS) (13,14). These data suggest that the ␣-ENaC subunit alone under the correct conditions is able to form a channel.…”

“…After the characterization of macroscopic currents by whole-cell recordings, single-channel analysis was also done in the cell-attached mode. For cell-attached experiments, the fraction of patches with channel activity was used to estimate the channel density as described previously (13,14).…”

Transport du Na+dans les poumons

“…Plusieurs groupes de recherche ont démontré la présence d'un canal cationique non sélectif (NSC) sensible à l'amiloride dans les cellules épithéliales pulmonaires. Les cellules alvéolaires épithé-liales expriment dans différentes proportions les canaux NSC et ENaC selon leurs conditions de culture [28]. L'identité moléculaire exacte du canal NSC n'est pas connue, mais certains résultats suggèrent qu'il serait constitué de la sous-unité ENaC mais pas des sous-unités et [28] (Tableau I).…”

“…Les cellules alvéolaires épithé-liales expriment dans différentes proportions les canaux NSC et ENaC selon leurs conditions de culture [28]. L'identité moléculaire exacte du canal NSC n'est pas connue, mais certains résultats suggèrent qu'il serait constitué de la sous-unité ENaC mais pas des sous-unités et [28] (Tableau I). Ces résultats laissent entrevoir qu'une association dans des proportions différentes des sous-unités ENaC pourrait former des canaux dotés de propriétés distinctes.…”