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Proteolytic activation of the human epithelial sodium channel by trypsin IV and trypsin I involves distinct cleavage sites

Haerteis, Silke ; Krappitz, Annabel ; Krappitz, Matteus ; Murphy, Jane E ; Bertog, Marko ; Krueger, Bettina ; Nacken, Regina ; Chung, Hyunjae ; Hollenberg, Morley D and Knecht, Wolfgang LU , et al. (2014) In The Journal of biological chemistry 289(27). p.19067-19078
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... (More)

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.

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type
Contribution to journal
publication status
published
subject
keywords
Amino Acid Sequence, Animals, Azetidines/pharmacology, Benzylamines/pharmacology, Binding Sites, Epithelial Cells/metabolism, Epithelial Sodium Channels/chemistry, Extracellular Space/metabolism, Humans, Kidney/metabolism, Molecular Sequence Data, Mutation, Oocytes/metabolism, Protein Structure, Tertiary, Proteolysis/drug effects, Trypsin/metabolism, Xenopus laevis/genetics
in
The Journal of biological chemistry
volume
289
issue
27
pages
19067 - 19078
publisher
American Society for Biochemistry and Molecular Biology
external identifiers
  • scopus:84903823511
  • pmid:24841206
ISSN
1083-351X
DOI
10.1074/jbc.M113.538470
language
English
LU publication?
no
id
78ca25ad-119a-437e-a80c-ca84e0eb2a98
date added to LUP
2020-07-17 14:06:39
date last changed
2024-05-15 16:11:39
@article{78ca25ad-119a-437e-a80c-ca84e0eb2a98,
  abstract     = {{<p>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. </p>}},
  author       = {{Haerteis, Silke and Krappitz, Annabel and Krappitz, Matteus and Murphy, Jane E and Bertog, Marko and Krueger, Bettina and Nacken, Regina and Chung, Hyunjae and Hollenberg, Morley D and Knecht, Wolfgang and Bunnett, Nigel W and Korbmacher, Christoph}},
  issn         = {{1083-351X}},
  keywords     = {{Amino Acid Sequence; Animals; Azetidines/pharmacology; Benzylamines/pharmacology; Binding Sites; Epithelial Cells/metabolism; Epithelial Sodium Channels/chemistry; Extracellular Space/metabolism; Humans; Kidney/metabolism; Molecular Sequence Data; Mutation; Oocytes/metabolism; Protein Structure, Tertiary; Proteolysis/drug effects; Trypsin/metabolism; Xenopus laevis/genetics}},
  language     = {{eng}},
  month        = {{07}},
  number       = {{27}},
  pages        = {{19067--19078}},
  publisher    = {{American Society for Biochemistry and Molecular Biology}},
  series       = {{The Journal of biological chemistry}},
  title        = {{Proteolytic activation of the human epithelial sodium channel by trypsin IV and trypsin I involves distinct cleavage sites}},
  url          = {{http://dx.doi.org/10.1074/jbc.M113.538470}},
  doi          = {{10.1074/jbc.M113.538470}},
  volume       = {{289}},
  year         = {{2014}},
}