The lectin‐like domain of TNF directly increases ENaC activity

Abstract

RationaleAims 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.MethodsPeptide 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.ResultsTIP, 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.ConclusionsThese 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.