Effects of Aldosterone on Biosynthesis, Traffic, and Functional Expression of Epithelial Sodium Channels in A6 Cells

The collecting duct regulates Na⁺ transport by adjusting the abundance/activity of epithelial Na⁺ channels (ENaC). In this study we have investigated the synthesis, degradation, endocytosis, and activity of ENaC and the effects of aldosterone on these processes using endogenous channels expressed in the A6 cell line. Biochemical studies were performed with a newly raised set of specific antibodies against each of the three subunits of the amphibian ENaC. Our results indicate simultaneous transcription and translation of α, β, and γ subunits and enhancement of both processes by aldosterone: two- and fourfold increase, respectively. The biosynthesis of new channels can be followed by acquisition of endoglycosidase H–resistant oligosacharides in α and β subunits and, in the case of α, by the appearance of a form resistant to reducing agents. The half-life of the total pool of subunits (t_1/2 40–70 min) is longer than the fraction of channels in the apical membrane (t_1/2 12–17 min). Aldosterone induces a fourfold increase in the abundance of the three subunits in the apical membrane without significant changes in the open probability, kinetics of single channels, or in the rate of degradation of ENaC subunits. Accordingly, the aldosterone response could be accounted by an increase in the abundance of apical channels due, at least in part, to de novo synthesis of subunits.