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.
Highlights
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
This difference is due to a very slow rate of clathrin-mediated endocytosis of the truncated channels and was reproduced by single amino acid mutations Y618A and ␥Y628A but not by ␣Y673A. These results indicate that the tyrosine residues present in the proline-rich motifs of the  and ␥ subunits encode signals recognized by the endocytic machinery and that mutations or deletions of these signals result in accumulation of channels at the plasma membrane due to reduced retrieval
These results suggest either that truncated channels are more efficiently delivered to the plasma membrane or, alternatively, that there is a process that reduces the activity of the wild-type but not of truncated channels in the plasma membrane
Summary
Expression of the amiloride-sensitive epithelial sodium channel (ENaC) in the plasma membrane determines the sodium permeability of many epithelia involved in sodium reabsorption. We demonstrate that channels with carboxyl-terminal truncations of the  and ␥ subunits have significantly longer half-life at the plasma membrane than wild-type channels This difference is due to a very slow rate of clathrin-mediated endocytosis of the truncated channels and was reproduced by single amino acid mutations Y618A and ␥Y628A but not by ␣Y673A. These results indicate that the tyrosine residues present in the proline-rich motifs of the  and ␥ subunits encode signals recognized by the endocytic machinery and that mutations or deletions of these signals result in accumulation of channels at the plasma membrane due to reduced retrieval. Since these same residues are mutated or deleted in Liddle’s syndrome, the results explain the mechanism that increases the number of cell-surface channels in Liddle’s mutants
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