Objectives: 

Epithelial Na+ channel (ENaC) formed by alpha, beta and gamma subunits plays a key role in Na+ reabsorption across tight epithelia. Strict control of ENaC activity is essential for extracellular volume homeostasis. ENaC endocytosis is controlled by Nedd4-2 binding to PPxY motifs in the c-termini of beta and gamma subunits and subsequent ubiquitination of channel subunits. A fourth ENaC subunit, named delta, can interact with beta and gamma to form channels that are also regulated by ubiquitination. In this study we compared the trafficking of alpha/beta/gamma and delta/beta/gamma channels in Xenopus oocytes.

Materials: 

ENaC currents from different subunit combinations were quantified by two electrode voltage clamp (TEVC) in Xenopus oocytes. Fluorescence recovery after photobleaching (FRAP) was measured in a confocal (Olympus Fluoview1000) to quantify membrane insertion rates in the oocyte membrane under different conditions.

Results: 

The use of brefeldin A to block the insertion of newly synthesized channels to the membrane showed a higher endocytosis rate for alpha/beta/gamma than for delta/beta/gamma. FRAP experiments were then used to study channel membrane insertion. The results indicate that the insertion rate of alpha/beta/gamma is higher than that of delta/beta/gamma.

Conclusions: 

Our data suggests that delta modifies ENaC turnover in the membrane and thus plays a dominant role in the regulation of channel trafficking.