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The lectin‐like domain of TNF directly increases ENaC activity
Author(s) -
Czikora Istvan,
Alli Abdel A,
Bao HuiFang,
White Richard,
Shabbir Waheed,
LemmensGruber Rosa,
Tzotzos Susan,
Fischer Bernhard,
Matthay Michael A,
Eaton Douglas C,
Lucas Rudolf
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.913.40
Subject(s) - epithelial sodium channel , internalization , peptide , amiloride , chemistry , microbiology and biotechnology , lectin , protein subunit , extracellular , biophysics , biochemistry , receptor , biology , sodium , organic chemistry , gene
Rationale Aims of this study were to investigate whether the TIP peptide, mimicking the alveolar liquid clearance (ALC)‐promoting lectin‐like domain of TNF, is able to activate amiloride‐sensitive Na + transport in H441 cells and to directly interact with specific ENaC domains. Methods Peptide effects on Na + ‐uptake in cells pretreated or not with N‐glycosidase F , were assessed using perforated patch or single channel measurements. Peptide‐ENaC interactions were assessed upon incubating either a) biotinylated TIP or mutant TIP peptide‐coupled or b) GST‐ENaC‐α domain‐coupled beads with lysates from ENaC‐α overexpressing H441 cells or peptides, respectively. Results TIP, but not mutant TIP peptide activates amiloride‐sensitive Na + currents and single ENaC channels in glycosylated, but not de‐glycosyated H441 cells. Binding to human ENaC‐α subunit occurs within the regulatory carboxy‐terminal, but not the extracellular loop or N‐terminal domains of the recombinant protein. Conclusions These data suggest for the first time that the lectin‐like domain of TNF, upon internalization, directly activates ENaC, by binding to the carboxy‐terminal domain of the channel's α‐subunit. This provides a rationale for the observed therapeutically promising ALC‐promoting activities of the peptide in vivo .