Plasmin and chymotrypsin have distinct preferences for channel activating cleavage sites in the γ subunit of the human epithelial sodium channel

Abstract

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.

Association of Plasminuria with Overhydration in Patients with CKD

“…Importantly, we showed that plasmin concentrations in the range of those observed in urine samples of patients with CKD were sufficient to activate ENaC currents in vitro. In previously published studies, higher plasmin concentrations have been used to show its activating effect on ENaC (9,10,24,25). However, the exposure time to plasmin in those studies was relatively short (5-30 minutes), whereas in this study, plasmin at comparatively low concentrations showed a stimulating effect on ENaC after 4 hours of exposure.…”

“…Oocytes were collected from Xenopus laevis with the approval of the animal welfare officer for the University of Erlangen-Nürnberg as described (7,9,10). Defolliculated stages 5 and 6 oocytes were injected with complementary RNA encoding human a-, b-, and g-ENaC (0.2 ng complementary RNA per subunit of ENaC).…”

“…Defolliculated stages 5 and 6 oocytes were injected with complementary RNA encoding human a-, b-, and g-ENaC (0.2 ng complementary RNA per subunit of ENaC). ENaC-mediated whole-cell currents were measured using the two-electrode voltage clamp technique as previously described (7,9,10).…”

“…Studies with protein-rich urine from both nephrotic rats and patients have shown a stimulation of ENaC currents in vitro that was attributed to occurrence of the serine protease plasmin in the urine (4,5). It is an emerging concept that serine proteases contribute to ENaC regulation by cleaving specific sites in the extracellular domains of the a-and g-subunits (6)(7)(8)(9)(10), resulting in the release of inhibitory peptides and activation of the channel (11)(12)(13). Proteolytic activation of ENaC by serine proteases as shown in native renal tissue (14) is enhanced by low-salt diet or aldosterone infusion (15) and might involve membrane-anchored prostasin (16) and/or tissue (urinary) kallikreins (17).…”

Proteolytic Regulation of Epithelial Sodium Channels by Urokinase Plasminogen Activator

“…Indeed, ENaC activation by plasmin has been recently demonstrated (33,34). Both uPA (abbokinase) and tPA (alteplase) are extensively used for fibrinolytic therapy for asthma, pleural effusion, and other respiratory diseases.…”