Proteolytic processing of the epithelial sodium channel (ENaC) is thought to contribute to its activation. Intracellular proteolytic cleavage by the convertase furin at three distinct furin sites (two in α- and one in γENaC) is important for ENaC maturation along the biosynthetic pathway before the channels reaches the plasma membrane. The pivotal final step in proteolytic ENaC activation takes place at the plasma membrane where γENaC is cleaved by membrane-bound proteases and/or extracellular proteases in a region distal to the furin site. Putative cleavage sites for prostasin, plasmin, and neutrophil elastase have been described in this region. Recently, we demonstrated that mutating two phenylalanines (FF174) in the vicinity of the prostasin cleavage site prevented proteolytic activation by chymotrypsin. The aim of the present study was to identify functionally relevant cleavage sites in γENaC involved in proteolytic channel activation by trypsin.

Mutant human γENaC constructs were generated by site directed mutagenesis. Human wild-type or mutant αβγENaC were expressed in Xenopus laevis oocytes. Amiloride-sensitive whole-cell currents (δI_ami) were determined before and after superfusion of the oocytes with 2 µg/ml chymotrypsin or trypsin. Biotinylated cell-surface γENaC cleavage fragments were detected by western blot analysis using a γENaC antibody.

Mutating the putative prostasin (RKRK178), plasmin (K189), and neutrophil elastase (V182;V193) cleavage sites in γENaC did not reduce channel activation by trypsin. Adjacent to the prostasin cleavage site there are two lysine residues (K168; K170) and one arginine residue (R172) that represent putative cleavage sites for trypsin. To investigate the role of these residues in ENaC activation by trypsin, we compared the effect of trypsin on oocytes expressing wt and γ_{K168A;K170A;R172A;RKRK178AAAA;V182G;V193G;K189A} mutant ENaC. Interestingly, the γ_{K168A;K170A;R172A;RKRK178AAAA;V182G;V193G;K189A} mutation abolished the stimulatory effect by trypsin. In contrast, the stimulatory effect of chymotrypsin was preserved in oocytes expressing γ_{K168A;K170A;R172A;RKRK178AAAA;V182G;V193G;K189A} mutant channel. Furthermore, the detection of γENaC cleavage fragments at the cell surface was consistent with the current data.

In summary, we identified functionally relevant trypsin cleavage sites in the γ-subunit of human ENaC. The fact that γENaC contains several different cleavage sites in a region critical for proteolytic channel activation may provide a mechanism for differential ENaC regulation by tissue-specific proteases.