Introduction: A unique feature of the epithelial sodium channel (ENaC) is its proteolytic activation (1, 2) which involves specific cleavage sites and the release of inhibitory peptide fragments (3). Proteolytic cleavage occurs in the extracellular domains of α- and γENaC and is essential for channel activation under (patho-)physiological conditions. In humans, cleavage of δENaC may also contribute to channel activation (4). Inappropriate ENaC activation by proteases may be involved in sodium retention and the pathogenesis of arterial hypertension in the context of renal disease (e.g. in nephrotic syndrome) (5). The pivotal final step in proteolytic channel activation probably happens at the plasma membrane, where γENaC is cleaved by membrane-bound proteases and/or extracellular proteases (6). The availability of several cleavage sites within a defined region of the γ-subunit with preferences for different types of proteases may provide a basis for tissue-specific proteolytic ENaC activation. However, at present, the (patho-)physiologically relevant proteases and molecular mechanisms involved in proteolytic channel activation remain to be determined. The prototypical serine protease trypsin I can activate ENaC in vitro but is unlikely to be the physiologically relevant ENaC activating protease in vivo. In contrast, trypsin IV (mesotrypsin), a form of trypsin known to be present in several extrapancreatic epithelial cells, may be co-expressed with ENaC. Moreover, under pathophysiological conditions the cysteine protease cathepsin-S (Cat-S) may reach ENaC in the apical membrane of epithelial cells. Therefore, trypsin IV and Cat-S are candidate proteases for proteolytic ENaC activation. The aim of the present study was to determine whether trypsin IV and Cat-S (7) can indeed activate human ENaC expressed in the oocyte expression system.Methods: Site-directed mutagenesis was used to identify functionally relevant cleavage sites in the γ-subunit of the channel. Human wild-type or mutant αβγENaC was expressed in Xenopus laevis oocytes. Amiloride-sensitive whole-cell currents (ΔIami) were determined by two-electrode voltage-clamp before and after 30 min incubation of the oocytes in human trypsin IV (10 μg/ml) or Cat-S (1 µM). Biotinylated cell surface γENaC cleavage products were detected by western blot analysis using a γENaC antibody.Results: In Xenopus laevis oocytes, we monitored the proteolytic activation of ENaC currents and the appearance of γENaC cleavage products at the cell surface. We demonstrated that trypsin IV can stimulate ENaC. ENaC cleavage and activation by trypsin IV requires a critical cleavage site (K189) in the extracellular domain of the γ-subunit. To confirm that the observed ENaC activation is caused by the proteolytic activity of trypsin IV, we examined the effect of the serine protease inhibitor melagatran. Melagatran completely prevented stimulation of ENaC currents by trypsin IV. In contrast, activation of ENaC by trypsin IV was not prevented by the polypeptide inhibitor SBTI (soybean trypsin inhibitor) confirming that trypsin IV, unlike trypsin, is resistant to SBTI. Furthermore, we demonstrated that Cat-S stimulates ΔIami in ENaC expressing oocytes. ENaC stimulation by Cat-S was associated with the appearance of a γENaC cleavage fragment at the plasma membrane indicating proteolytic channel activation. Interestingly, mutating two valine residues (V182;V193) in the critical region of γENaC prevented proteolytic activation of ENaC by Cat-S.Conclusion: Our results show, for the first time, an activation of ENaC by trypsin IV and by Cat-S, a member of the family of cysteine proteases. Interestingly, both proteases use specific cleavage sites to achieve channel activation. Hence, preferential cleavage sites may provide a mechanism for differential ENaC regulation by tissue-specific proteases under (patho-)physiological conditions.