Proteolytic activation of the human epithelial sodium channel by trypsin IV and trypsin I involves distinct cleavage sites.
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.
From the Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Waldstrasse 6, 91054 Erlangen, Germany.
Silke
S
Haerteis
From the Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Waldstrasse 6, 91054 Erlangen, Germany.
Annabel
A
Krappitz
From the Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Waldstrasse 6, 91054 Erlangen, Germany.
Matteus
M
Krappitz
the UCSF Center for the Neurobiology of Digestive Diseases, Department of Surgery, University of California, San Francisco, California.
Jane E
JE
Murphy
From the Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Waldstrasse 6, 91054 Erlangen, Germany.
Marko
M
Bertog
From the Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Waldstrasse 6, 91054 Erlangen, Germany.
Bettina
B
Krueger
From the Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Waldstrasse 6, 91054 Erlangen, Germany.
Regina
R
Nacken
the Department of Physiology and Pharmacology, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
Hyunjae
H
Chung
the Department of Physiology and Pharmacology, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
Morley D
MD
Hollenberg
Bioscience, CVGI iMed, AstraZeneca Research and Development, 43181 Mölndal, Sweden.
Wolfgang
W
Knecht
the Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, Victoria 3052, Australia, and the Department of Pharmacology, University of Melbourne, Melbourne, Victoria 3010, Australia.
Nigel W
NW
Bunnett
From the Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Waldstrasse 6, 91054 Erlangen, Germany, christoph.korbmacher@fau.de.
Christoph
C
Korbmacher
D000595:Amino Acid Sequence; D000818:Animals; D001384:Azetidines / Q000494:pharmacology; D001596:Benzylamines / Q000494:pharmacology; D001665:Binding Sites; D004847:Epithelial Cells / Q000378:metabolism; D053503:Epithelial Sodium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D017434:Protein Structure, Tertiary; D059748:Proteolysis / Q000187:drug effects; D014357:Trypsin / Q000378:metabolism*; D014982:Xenopus laevis / Q000235:genetics
D001384:Azetidines; D001596:Benzylamines; D053503:Epithelial Sodium Channels; C109573:melagatran; D014357:Trypsin
Epithelial Sodium Channel (ENaC); Ion Channel; Oocyte; Protease; Proteolytic Channel Activation; Serine Protease; Trypsin; Trypsin IV; Two-electrode Voltage Clamp