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 β or γ 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 (t½ 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.

Footnotes

  • * This work was supported by grants from the National Science Foundation (to R. S.) and the Edward Mallinckrodt, Jr. Foundation (C. C.) and by a National Institutes of Health SCOR on Hypertension.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • To whom correspondence should be addressed: Dept. of Cellular and Molecular Physiology, 333 Cedar St., New Haven, CT 06520-8026. Tel.: 203-785-5892; Fax: 203-785-4951; E-mail:cecilia_canessa{at}yale.edu.

  • Received April 25, 1997.
  • Revision received August 7, 1997.
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