Proteolytic activation of the epithelial sodium channel (ENaC) involves cleavage of its γ subunit in a critical region targeted by several proteases. Our aim was to identify cleavage sites in this region that are functionally important for activation of human ENaC by plasmin and chymotrypsin. Sequence alignment revealed a putative plasmin cleavage site in human γENaC (K189) that corresponds to a plasmin cleavage site (K194) in mouse γENaC. We mutated this site to alanine (K189A) and expressed human wild-type (wt) αβγENaC and αβγK189AENaC in Xenopus laevis oocytes. The γK189A mutation reduced but did not abolish activation of ENaC whole cell currents by plasmin. Mutating a putative prostasin site (γRKRK178AAAA) had no effect on the stimulatory response to plasmin. In contrast, a double mutation (γRKRK178AAAA;K189A) prevented the stimulatory effect of plasmin. We conclude that in addition to the preferential plasmin cleavage site K189, the putative prostasin cleavage site RKRK178 may serve as an alternative site for proteolytic channel activation by plasmin. Interestingly, the double mutation delayed but did not abolish ENaC activation by chymotrypsin. The time-dependent appearance of cleavage products at the cell surface nicely correlated with the stimulatory effect of chymotrypsin on ENaC currents in oocytes expressing wt or double mutant ENaC. Delayed proteolytic activation of the double mutant channel with a stepwise recruitment of so-called near-silent channels was confirmed in single-channel recordings from outside-out patches. Mutating two phenylalanines (FF174) in the vicinity of the prostasin cleavage site prevented proteolytic activation by chymotrypsin. This indicates that chymotrypsin preferentially cleaves at FF174. The close proximity of FF174 to the prostasin site may explain why mutating the prostasin site impedes channel activation by chymotrypsin. In conclusion, this study supports the concept that different proteases have distinct preferences for certain cleavage sites in γENaC, which may be relevant for tissue-specific proteolytic ENaC activation.

Proteolytic processing of the amiloride-sensitive epithelial sodium channel (ENaC) by serine proteases is known to be important for channel activation. Inappropriate ENaC activation by proteases may contribute to the pathophysiology of cystic fibrosis and could be involved in sodium retention and the pathogenesis of arterial hypertension in the context of renal disease. We hypothesized that in addition to serine proteases, cathepsin proteases may activate ENaC. Cathepsin proteases belong to the group of cysteine proteases and play a pathophysiological role in inflammatory diseases. Under pathophysiological conditions, cathepsin-S (Cat-S) may reach ENaC in the apical membrane of epithelial cells. The aim of this study was to investigate the effect of purified Cat-S on human ENaC heterologously expressed in Xenopus laevis oocytes and on ENaC-mediated sodium transport in cultured M-1 mouse renal collecting duct cells. We demonstrated that Cat-S activates amiloride-sensitive whole-cell currents in ENaC-expressing oocytes. The stimulatory effect of Cat-S was preserved at pH 5. ENaC stimulation by Cat-S was associated with the appearance of a γENaC cleavage fragment at the plasma membrane indicating proteolytic channel activation. Mutating two valine residues (V182 and V193) in the critical region of γENaC prevented proteolytic activation of ENaC by Cat-S. Pre-incubation of the oocytes with the Cat-S inhibitor morpholinurea-leucine-homophenylalanine-vinylsulfone-phenyl (LHVS) prevented the stimulatory effect of Cat-S on ENaC. In contrast, LHVS had no effect on ENaC activation by the prototypical serine proteases trypsin and chymotrypsin. Cat-S also stimulated ENaC in differentiated renal epithelial cells. These findings demonstrate that the cysteine protease Cat-S can activate ENaC which may be relevant under pathophysiological conditions.

Proteolytic channel activation is a unique feature of the epithelial sodium channel (ENaC) but is not yet fully understood. The serine protease trypsin is known to activate ENaC in vitro but may not be relevant in vivo. In this study, we wanted to investigate whether the trypsin‐like serine protease trypsin IV, known to be expressed in several epithelial tissues, can activate human ENaC. Moreover, using site‐directed mutagenesis we wanted to identify functionally relevant cleavage sites in the γ‐subunit of the channel. In the Xenopus laevis oocyte expression system, we monitored the proteolytic activation of ENaC currents and the appearance of γ‐ENaC cleavage products at the cell surface. We demonstrated that trypsin IV can stimulate ENaC and that this activation involves a critical cleavage site (K189) in the extracellular domain of the γ‐subunit. Moreover, we showed that channel activation by trypsin was prevented by mutating three putative trypsin cleavage sites (K168; K170; R172) in addition to mutating previously described prostasin (RKRK178), plasmin (K189), and neutrophil elastase (V182;V193) sites. In conclusion, we demonstrated for the first time that trypsin IV can activate ENaC and that trypsin and trypsin IV use specific cleavage sites to achieve channel activation. Preferential cleavage sites may provide a mechanism for differential ENaC regulation by tissue‐specific proteases. Grant Funding Source: Supported by grants of the Interdisziplinäres Zentrum für Klinische Forschung (IZKF) (S.H., M.K.)