Activation of the epithelial sodium channel by plasmin and cathepsin-S.

Proteolytic channel activation is a unique feature of the epithelial sodium channel (ENaC). Cleavage at specific sites in the extracellular domains of α-, δ-, and γENaC is essential for channel activation [1; 2]. However, the (patho-)physiologically relevant proteases and molecular mechanisms involved in proteolytic channel activation remain to be determined. Inappropriate ENaC activation by proteases may be involved in sodium retention and the pathogenesis of arterial hypertension in the context of renal disease. Recently, we and others reported that the serine protease plasmin can proteolytically activate ENaC which may contribute to renal sodium retention in nephrotic syndrome [3; 4]. In mouse γENaC a putative plasmin cleavage site (K194) has been reported [4]. For human ENaC, the relevant cleavage site(s) for plasmin remain to be determined. 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. Our aims were (a) to identify functionally relevant plasmin cleavage sites in human γENaC [5] and (b) to investigate the effect of Cat-S on human ENaC [6]. Mutant human γENaC constructs were generated by site-directed mutagenesis. Human wild-type or mutant αβγENaC was expressed in Xenopus laevis oocytes. Amiloride-sensitive whole-cell currents (ΔI_aml) were determined by two-electrode voltage-clamp before and after 30 min incubation of the oocytes in human plasmin (10 μg/ml), chymotrypsin (2 μg/ml) or Cat-S (1 μM). Biotinylated cell surface γENaC cleavage products were detected by western blot analysis using a γENaC antibody. (a) 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 αβγENaC and αβγK189AENaC in 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 wild-type or double mutant ENaC. This indicates a causal link between proteolytic cleavage and channel activation. 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. Interestingly, mutating two phenylalanines (γFF174) adjacent to the prostasin cleavage site pre vented proteolytic activation by chymotrypsin. This indicates that the two phenylalanines constitute a preferential cleavage site for ENaC activation by chymotrypsin. (b) We demonstrated that Cat-S activates ΔI_aml 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. Mutat ing two valine residues (V182 and V193) in the critical region of γENaC prevented proteolytic activation of ENaC by Cat-S. The stimulatory effect of Cat-S on ENaC activity and the con comitant appearance of a γENaC cleavage product at the cell surface were prevented by the Cat-S inhibitor LHVS (morpholinurea-leucine-homophenylalanine-vinylsulfone-phenyl). In contrast, LHVS had no effect on ENaC activation by the prototypical serine proteases trypsin and chymotrypsin. To our knowledge, this is the first report that the cysteine protease Cat-S can activate ENaC which may be relevant under patho physiological conditions. In this study, we identified cleavage sites in a region critical for proteolytic channel activation in the γ-subunit of human ENaC with functional importance for its proteolytic activation by plasmin, chymotrypsin and Cat-S. The fact that γENaC contains several different cleavage sites in this region is likely to have physiological implications. Indeed, it may provide a mechanism for differential ENaC regulation by tissue-specific proteases. [ABSTRACT FROM AUTHOR]