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Conserved charged residues at the surface and interface of epithelial sodium channel subunits – roles in cell surface expression and the sodium self‐inhibition response
Author(s) -
Edelheit Oded,
BenShahar Refael,
Dascal Nathan,
Hanukoglu Aaron,
Hanukoglu Israel
Publication year - 2014
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12765
Subject(s) - epithelial sodium channel , protein subunit , chemistry , xenopus , mutant , transmembrane domain , biophysics , transmembrane protein , sodium channel , sodium , microbiology and biotechnology , biochemistry , biology , amino acid , gene , receptor , organic chemistry
The epithelial sodium channel ( E Na C ) is composed of three homologous subunits that form a triangular pyramid‐shaped funnel, anchored in the membrane with a stem of six transmembrane domains. We examined the structure–function relationships of 17 conserved charged residues on the surface of the ectodomain of human γ‐ EN a C subunit by alanine mutagenesis and co‐expression with α‐ and β‐ EN a C subunits in X enopus oocytes. The results showed that N a + conductance of cells expressing these mutants can be accounted for by two parameters: (a) the EN a C density on the cell surface as measured by the fluorescence of an α‐ E na C –yellow fluorescent protein hybrid and (b) the sodium self‐inhibition ( SSI ) response that reflects the open probability of the channel ( P o ). Overall, the activity of all 17 mutants was correlated with surface levels of EN a C . There was no significant correlation between these parameters measured for α‐ and γ‐ EN a C subunit mutants at nine homologous positions. Thus, the functions of most of the homologous surface residues examined differ between the two subunits. Only four mutants (K328, D510, R514 and E518) significantly reduced the SSI response. The α‐ EN a C homologs of three of these (R350, E530 and E538) also severely affected the SSI response. The c ASIC 1 homologs of these (K247, E417, Q421) are located at the interface between subunits, on or about the ion pathway at the rotational symmetry axis in the center of the trimer. Thus, it is likely that these residues are involved in conformational changes that lead to channel constriction and the SSI response upon Na + ion flooding.