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Amiloride-sensitive epithelial Na؉ channels (ENaC) are responsible for trans-epithelial Na

Amiloride-sensitive Na+ transport by lung epithelia plays a critical role in maintaining alveolar Na+ and water balance. It has been generally assumed that Na+transport is mediated by the amiloride-sensitive epithelial Na+ channel (ENaC) because molecular biology studies have confirmed the presence of ENaC subunits α, β, and γ in lung epithelia. However, the predominant Na+-transporting channel reported from electrophysiological studies 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 membrane patches from rat alveolar type II (ATII) cells in primary culture reveal a nonselective cation channel with a conductance of 20.6 ± 1.1 pS and an Na+-to-K+selectivity of 0.97 ± 0.07. This channel is inhibited by submicromolar concentrations of amiloride. Thus there is some question about the relationship between the gene product observed with single-channel methods and the cloned ENaC subunits. We have employed antisense oligonucleotide methods to block the synthesis of individual ENaC subunit proteins (α, β, and γ) and determined the effect of a reduction in the subunit expression on the density of the nonselective cation channel observed in apical membrane patches on ATII cells. Treatment of ATII cells with antisense oligonucleotides inhibited the production of each subunit protein; however, single-channel recordings showed that only the antisense oligonucleotide targeting the α-subunit resulted in a significant decrease in the density of nonselective cation channels. Inhibition of the β- and γ-subunit proteins alone or together did not cause any changes in the observed channel density. There were no changes in open probability or other channel characteristics. These results support the hypothesis that the α-subunit of ENaC alone or in combination with some protein other than the β- or γ-subunit protein is the major component of lung alveolar epithelial cation channels.

Regulation of epithelial Na؉ channel (ENaC) subunit levels by protein kinase C (PKC) was investigated in A6 cells. PKC activation altered ENaC subunit levels, differentially decreasing the levels of both ␤ and ␥, but not ␣ENaC. Temporal regulation of ␤ and ␥ENaC by PKC differed; ␥ENaC decreased with a time constant of 3.7 ؎ 1.0 h, whereas ␤ENaC decreased in 13.9 ؎ 3.0 h. Activation of PKC also resulted in a decrease in trans-epithelial Na ؉ reabsorption for up to 48 h. PMA activation of PKC resulted in negative feedback inhibition of PKC protein levels beginning within 4 h. Both ␤ and ␥ENaC levels, as well as transport tended toward pretreatment values after 48 h of PMA treatment. PKC inhibitors attenuated the effects of PMA on ENaC subunit levels and Na ؉ transport. These results directly show for the first time that PKC differentially regulates ENaC subunit levels by decreasing the levels of ␤ and ␥ but not ␣ENaC protein. These results imply a PKC-dependent, long term decrease in Na ؉ reabsorption.Sodium homeostasis is essential to proper maintenance of total body water and electrolyte content, and thus, blood pressure control. The activity of luminal, epithelial Na ϩ channels (ENaC) 1 is rate-limiting for trans-epithelial Na ϩ reabsorption across the renal collecting duct and other Na ϩ reabsorbing epithelium. Thus, understanding regulation of ENaC activity is relevant to physiology as well as to treating disease with associated fluid imbalance.ENaC is a heterotetrameric channel complex composed of at least three homologous but distinct subunits: ␣, ␤, and ␥ (1). Numerous results show that expression of ENaC subunit message and protein are differentially regulated in various tissues and species (reviewed by Refs. 2-4). For example, Masilamani and colleagues (5) recently showed in rat collecting duct principal cells that ␣ENaC protein levels increase in response to aldosterone; however, we found in the amphibian A6 cell model of the collecting duct principal cell that ␣ENaC is not significantly influenced by aldosterone, but ␤ENaC protein levels are increased in response to steroid (Ref. 6; also refer to Fig. 1 of the present study). Besides aldosterone, Zentner et al. (7) recently showed in the rat parotid epithelial cell line, Pa-4, that expression of ␣ENaC mRNA and possibly protein was decreased within 6 h by protein kinase C activation.Activation of PKC decreases Na ϩ reabsorption across renal epithelium by affecting ENaC (8 -11). Studies of single channel properties show that in amphibian, rat, and rabbit distal tubule cells, ENaC activity is decreased within 5-10 min after activation of PKC (12-15). A rapid initial decrease in ENaC open probability is, in part, responsible for the early change in activity; however, it appears that PKC may also subsequently affect the number of functional channels (14, 15). Although most studies are consistent with PKC decreasing ENaC open probability initially and then subsequently reducing channel number, Els et al. (16) showed with blocker-induced noise analysis in A6...

Amiloride-sensitive epithelial sodium (Na(+)) channels (ENaC) play a crucial role in Na(+) transport and fluid reabsorption in the kidney, lung, and colon. The magnitude of ENaC-mediated Na(+) transport in epithelial cells depends on the average open probability of the channels and the number of channels on the apical surface of epithelial cells. The number of channels in the apical membrane, in turn, depends upon a balance between the rate of ENaC insertion and the rate of removal from the apical membrane. ENaC is made up of three homologous subunits, alpha, beta, and gamma. The C-terminal domain of all three subunits is intracellular and contains a proline rich motif (PPxY). Mutations or deletion of this PPxY motif in the beta and gamma subunits prevent the binding of one isoform of a specific ubiquitin ligase, neural precursor cell expressed developmentally down-regulated protein (Nedd4-2) to the channel in vitro and in transfected cell systems, thereby impeding ubiquitin conjugation of the channel subunits. Ubiquitin conjugation would seem to imply that ENaC turnover is determined by the ubiquitin-proteasome system, but when MDCK cells are transfected with ENaC, ubiquitin conjugation apparently leads to lysosomal degradation. However, in untransfected epithelial cells (A6) expressing endogenous ENaC, ENaC appears to be degraded by the ubiquitin-proteasome system. Nonetheless, in both transfected and untransfected cells, the rate of ENaC degradation is apparently controlled by the rate of Nedd4-2-mediated ENaC ubiquitination. Controlling the rate of degradation is apparently important enough to have multiple, redundant pathways to control Nedd4-2 and ENaC ubiquitination.