The Activity of the Epithelial Sodium Channel Is Regulated by Clathrin-mediated Endocytosis

Abstract

Activity of the epithelial sodium channel (ENaC) is a key determinant of sodium homeostasis and blood pressure. Liddle's syndrome, an inherited form of hypertension, is caused by mutations that delete or alter PY domains in the carboxyl termini of ␤ or ␥ ENaC subunits, leading to increased channel activity. In this study we investigated the mechanism of this effect by analysis of wild-type and mutant ENaC activity in Xenopus oocytes. By inhibiting insertion of new channels into the plasma membrane with brefeldin A, we demonstrate that the half-life of the activity of channels containing Liddle's mutations is markedly prolonged compared with wildtype channels (t1 ⁄2 of 30 h in mutant versus 3.6 in wildtype, p < 0.001). We investigated the involvement of clathrin-coated pit-mediated endocytosis by co-expressing a dominant-negative dynamin mutant with wildtype ENaC in oocytes. Expression of this specific inhibitor of endocytosis leads to a large increase in the activity of wild-type channels, demonstrating that normal turnover of this channel is through the clathrincoated pit pathway. In contrast, co-expression of Liddle's mutations and dynamin mutants leads to no further increase in channel activity, consistent with one of the effects of Liddle's mutations being the loss of endocytosis of these channels. These findings demonstrate the normal mechanism of turnover of ENaC from the cell surface and demonstrate a mechanism that can account for the increased number of channels in the plasma membrane seen in Liddle's syndrome.Expression of the amiloride-sensitive epithelial sodium channel (ENaC) 1 in the plasma membrane determines the sodium permeability of many epithelia involved in sodium reabsorption. The channel is regulated by hormones such as aldosterone, insulin, and antidiuretic hormone that increase its activity by diverse mechanisms including increase in channel synthesis, incorporation of channels from an intracellular pool to the plasma membrane, and induction of post-translational modifications of the channel proteins (1). There are fewer known mechanisms that decrease sodium permeability in cells expressing ENaC. Recently, there have been several studies that implicate the carboxyl terminus of the channel subunits in processes that normally down-regulate its activity. Mutations in the human genes of the ␤ and ␥ subunits cause a form of salt-sensitive hypertension known as Liddle's syndrome (2, 3). These mutations introduce frameshifts or premature stop codons that delete the intracellular carboxyl-terminal domains of the ␤ or ␥ subunits, or they substitute residues in a prolinerich motif present in the carboxyl termini of both subunits (4, 5). Expression of ENaC channels with truncated ␤ or ␥ subunits in Xenopus oocytes induces amiloride-sensitive whole-cell currents 3-5-fold larger in magnitude than wild-type channels (6). Two mechanisms have been proposed to explain the increase in sodium permeability induced by Liddle's mutations: an increase in the number of channels expressed at the cell surface, a...

Arachidonic Acid Regulates Surface Expression of Epithelial Sodium Channels

“…The enhanced rate of decrease of amiloride-sensitive currents in oocytes treated with BFA and ETYA, compared with oocytes treated with BFA alone, provided additional evidence that ETYA reduced functional expression of ENaC by increasing rates of channel endocytosis. Furthermore, a PY motif mutation (␤Y618A) blocked ETYA-mediated inhibition of ENaC expression and is consistent with previous studies that reported that channels with mutations in the PY motif exhibited increased cell surface expression in association with a reduction in the rate of channel endocytosis (21,28,31). Although our results suggest that arachidonic acid inhibits ENaC by reducing surface expression of channels, we cannot discard the possibility that arachidonic acid also reduced channel P o .…”

“…Stimulation of ENaC endocytosis by arachidonic acid or ETYA appears to be dependent on previously characterized internalization motifs within the C termini of ENaC subunits (21), as substitution of wild type ENaC with the PY mutant ␤Y618A blocks ETYA effects.…”

“…ENaC-mediated Na ϩ currents were then blocked by perfusion with TEV solution containing benzamil (100 M). A second assay used to examine whether ETYA enhanced rates of channel internalization was based on the interruption of delivery of newly synthesized channels to the plasma membrane by brefeldin A (BFA) (21). Thirty h following co-injection of oocytes with ␣␤␥ cRNAs, amiloride-sensitive currents were measured by TEV.…”

Genetics of arterial hypertension and hypotension

“…6). The ensuing constant inappropriate Na + reabsorption causes volume expansion, even under conditions of high salt intake, and ultimately hypertension (Synder et al 1995;Shimkets et al 1997). A considerable genetic heterogeneity between different mutations and pedigrees has been observed.…”

Maturation of the Epithelial Na+ Channel Involves Proteolytic Processing of the α- and γ-Subunits

“…In general, N-glycans on proteins prior to their delivery to the medial Golgi complex are substrates for Endo H, and N-glycans on proteins after passing this point are not substrates. However, not all N-glycans are modified during transit through the Golgi complex, and these N-glycans remain sensitive to treatment with Endo H. ENaC is apparently delivered to the cell surface via the Golgi complex because surface delivery is blocked by treatment of cells with the fungal metabolite brefeldin A that disrupts cytosolic coat assembly needed for vesicle budding throughout the Golgi complex (9,17,18). However, direct evidence for processing of N-glycans on ENaC to complex type is limited.…”