Proteolytic Activation of the Human Epithelial Sodium Channel by Trypsin IV and Trypsin I Involves Distinct Cleavage Sites

Abstract

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.)

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).…”

Extracellular protease trypsin activates amiloride‐insensitive sodium channels in human leukemia cells

“…Additionally, trypsin at low concentrations was reported to stimulate membrane‐resident ENaCs in Xenopus oocytes via G‐protein–coupled receptors distinct from PARs . In contrast, direct action of trypsin involving proteolytic cleavage of α‐ENaC and γ‐ENaC subunits was observed on ENaCs expressed in 3T3 cells or X. oocytes . Consistently, extracellular proteases were concluded to stimulate ENaC channels in native renal mouse tubules and cultured M‐1 cortical collecting duct cells .…”