title: 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 beta or gamma 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 wild-type channels (t1/2 of 30 h in mutant versus 3.6 in wild-type, p < 0.001). We investigated the involvement of clathrin-coated pit-mediated endocytosis by co-expressing a dominant-negative dynamin mutant with wild-type 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 clathrin-coated 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.
authors.affiliation: Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06520-8026, USA.
authors.forename: R A
authors.initials: RA
authors.lastname: Shimkets
authors.forename: R P
authors.initials: RP
authors.lastname: Lifton
authors.forename: C M
authors.initials: CM
authors.lastname: Canessa
mesh_terms: D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D020126:Brefeldin A; D002966:Clathrin / Q000378:metabolism*; D003517:Cyclopentanes / Q000494:pharmacology; D004232:Diuretics / Q000494:pharmacology; D004705:Endocytosis; D053503:Epithelial Sodium Channels; D004848:Epithelium / Q000378:metabolism; D006207:Half-Life; D006973:Hypertension / Q000235:genetics; D009865:Oocytes / Q000166:cytology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D015222:Sodium Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus
chemical_list: D002966:Clathrin; D003517:Cyclopentanes; D004232:Diuretics; D053503:Epithelial Sodium Channels; D011500:Protein Synthesis Inhibitors; D015222:Sodium Channels; D020126:Brefeldin A; D000584:Amiloride