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The Epsilon Subunit of Epithelial Sodium Channels in Xenopus laevis Prevents Channel Activation by Proteolytic Cleavage
Author(s) -
Wichmann Lukas,
Vowinkel Kirsty,
Althaus Mike
Publication year - 2017
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.31.1_supplement.856.4
Subject(s) - epithelial sodium channel , xenopus , protein subunit , amiloride , chemistry , microbiology and biotechnology , sodium channel , extracellular , patch clamp , ion channel , acid sensing ion channel , cleavage (geology) , furin , voltage clamp , biophysics , biochemistry , biology , sodium , membrane potential , enzyme , receptor , gene , paleontology , fracture (geology) , organic chemistry
Epithelial Sodium Channels (ENaC) are sodium‐selective ion channels which facilitate the absorption of Na + ions across epithelial tissues. The heterotrimeric channel is composed of three homologous subunits (α, β, γ) residing in the apical membrane of epithelial cells. Proteolytic cleavage of the α‐ and γ‐subunits is a major regulatory mechanism determining ENaC open probability. In Xenopus laevis, an additional epsilon (ɛ) subunit has been identified which can form functional ENaCs by substituting for the α‐subunit. Since channel properties as well as regulatory characteristics of ENaC containing the ɛ‐subunit are poorly understood, we aimed to investigate the regulation of ɛβγ‐ and αβγ‐ENaC by proteolytic processing. Heterologously expressed ɛβγ‐ and αβγ‐ENaCs in Xenopus laevis oocytes were functionally characterized using the two‐electrode voltage‐clamp technique. Oocytes expressing ɛβγ‐ENaC had larger amiloride‐sensitive transmembrane currents than oocytes expressing ENaC containing the α‐subunit. In contrast to αβγ‐ENaC, currents mediated by ɛβγ‐ENaC readily decreased in the presence of high extracellular Na + concentrations due to enhanced sodium self‐inhibition. The employment of MTSET‐sensitive β S540C‐ ENaC mutants as well as cell‐attached patch‐clamp recordings indicated that the presence of the ɛ‐ENaC subunit produced channels with a higher open probability than those containing the α‐subunit. Proteolytic processing of ENaC is a multi‐step mechanism that increases channel open probability by releasing inhibitory peptides from the α‐ and γ‐ENaC subunits. Proteolytic maturation is initiated through intracellular cleavage of both ENaC subunits by furin. At the cell surface the channels are further processed through cleavage of γ‐ENaC by extracellular proteases. Extracellular chymotrypsin increased currents mediated by αβγ‐ENaC but had no effect on ɛβγ‐ENaC. Channels containing the Xenopus ɛβ‐ENaC subunits together with human γ‐ENaC were also unaffected by chymotrypsin. Incubation of oocytes with a furin‐inhibitor potentiated the effect of chymotrypsin on αβγ‐ENaC expressing oocytes whereas it had no effect on ɛβγ‐ENaC mediated currents. Analysis of ENaC cleavage fragments by immunodetection of N‐terminally (HA) and C‐ terminally (V5) epitope‐tagged α‐, ɛ‐ and γ‐ENaC subunits suggested that there is no intracellular proteolytic processing of the ɛ‐ENaC subunit. Furthermore, the conserved inhibitory peptide sequence identified in α‐ENaC was absent in ɛ‐ENaC. In conclusion, these results demonstrate that incorporation of the ɛ‐ENaC subunit changes ENaC regulation by proteolytic processing and renders the channel resistant to cleavage by extracellular proteases. Hence, the subunit composition of ENaC might play a major role in the susceptibility of ENaC and transepithelial Na + absorption to proteolytic regulation.