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

Silke Haerteis,Annabel Krappitz,Matteus Krappitz,Jane E Murphy,Marko Bertog,Bettina Krueger,Regina Nacken,Hyunjae Chung,Morley D Hollenberg,Wolfgang Knecht,Nigel W Bunnett,Christoph Korbmacher

doi:10.1074/jbc.m113.538470

Abstract

Proteolytic activation is a unique feature of the epithelial sodium channel (ENaC). However, the underlying molecular mechanisms and the physiologically relevant proteases remain to be identified. The serine protease trypsin I can activate ENaC in vitro but is unlikely to be the physiologically relevant activating protease in ENaC-expressing tissues in vivo. Herein, we investigated whether human trypsin IV, a form of trypsin that is co-expressed in several extrapancreatic epithelial cells with ENaC, can activate human ENaC. In Xenopus laevis oocytes, we monitored proteolytic activation of ENaC currents and the appearance of γENaC cleavage products at the cell surface. We demonstrated that trypsin IV and trypsin I can stimulate ENaC heterologously expressed in oocytes. ENaC cleavage and activation by trypsin IV but not by trypsin I required a critical cleavage site (Lys-189) in the extracellular domain of the γ-subunit. In contrast, channel activation by trypsin I was prevented by mutating three putative cleavage sites (Lys-168, Lys-170, and Arg-172) in addition to mutating previously described prostasin (RKRK(178)), plasmin (Lys-189), and neutrophil elastase (Val-182 and Val-193) sites. Moreover, we found that trypsin IV is expressed in human renal epithelial cells and can increase ENaC-mediated sodium transport in cultured human airway epithelial cells. Thus, trypsin IV may regulate ENaC function in epithelial tissues. Our results show, for the first time, that trypsin IV can stimulate ENaC and that trypsin IV and trypsin I activate ENaC by cleavage at distinct sites. The presence of distinct cleavage sites may be important for ENaC regulation by tissue-specific proteases.

Highlights

Proteolysis is required for ENaC activity, but the proteases activating ENaC in epithelial tissues are unknown
The Serine Protease Inhibitor Melagatran Prevents Trypsin IV and Trypsin I Activation of ENaC—To confirm that the observed ENaC activation is caused by the proteolytic activity of trypsin IV and not by a contaminant, we examined the effect of the serine protease inhibitor melagatran, which inhibits both trypsin IV and trypsin I [23]. ⌬Iami was measured before and after 30-min incubation of oocytes in solutions containing trypsin I (2 ␮g/ml), trypsin IV (10 ␮g/ml), melagatran (10 ␮M), trypsin I plus melagatran, or trypsin IV plus melagatran
We report that trypsin IV stimulates ENaC-mediated wholecell currents in oocytes expressing human ENaC

Summary

Introduction

Proteolysis is required for ENaC activity, but the proteases activating ENaC in epithelial tissues are unknown. Results: Human trypsin IV and trypsin I activate ENaC by cleavage at distinct sites in the channel’s ␥-subunit. Conclusion: Cleavage at distinct sites may provide a mechanism for differential ENaC regulation by tissue-specific proteases. Proteolytic activation is a unique feature of the epithelial sodium channel (ENaC). The serine protease trypsin I can activate ENaC in vitro but is unlikely to be the physiologically relevant activating protease in ENaC-expressing tissues in vivo. We investigated whether human trypsin IV, a form of trypsin that is co-expressed in several extrapancreatic epithelial cells with ENaC, can activate human ENaC. ENaC cleavage and activation by trypsin IV but not by trypsin I required a critical cleavage site (Lys-189) in the extracellular domain of the ␥-subunit. We found that trypsin IV is expressed in human renal epithelial cells and can increase ENaC-mediated sodium transport in cultured human airway

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Conclusion