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Titratable residues in the extracellular domain of human gamma‐ENaC contribute to H+ regulation of channel activity
Author(s) -
Collier Daniel M,
Snyder Peter M
Publication year - 2011
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.25.1_supplement.860.4
Subject(s) - epithelial sodium channel , extracellular , xenopus , amiloride , chemistry , protein subunit , mutant , microbiology and biotechnology , biophysics , biochemistry , sodium , biology , gene , organic chemistry
The extracellular domain of the epithelial sodium channel (ENaC) plays a critical role in tuning channel activity in response to dynamic extracellular conditions. We have previously demonstrated that H + have dual effects on ENaC activity. H + increase ENaC activity by decreasing Na + self‐inhibition. H + also inhibit ENaC activity by increasing Cl − inhibition. In the current work, we investigated the mechanisms by which H + stimulate ENaC. We took advantage of our previous finding that rat ENaC has a reduced response to pH compared to human ENaC. By expressing combinations of human and rat subunits, we localized the difference to the γ subunit. Our strategy was to mutate H + titratable residues in the extracellular domain of human γENaC that are not conserved in rat γENaC. We expressed the mutant γENaC cDNAs (with wild type α‐ and βENaC) in Xenopus oocytes and tested the effect of pH changes on amiloride‐sensitive Na + current (by TEVC at −60 mV). We identified a group of residues in the extracellular domain of γENaC (D164, Q165, D166, E292, E315, E335, H439, and E455) that, when individually mutated to Ala, decreased H + activation of ENaC. Furthermore, combining single mutations resulted in an additive decrease in the pH response. The data demonstrate that multiple residues in human γENaC are required for regulation by pH.