Mutations in a membrane spanning domain of the Epithlial Na+ Channel (ENaC) identify residues that are critical to the channel pore

A. L. Bradford, B. K. Berdiev, D. J. Benos

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One feature of the epithelial amiloride-sensitive Na+ channel (ENaC) that is critical for channel function is the pore-forming region. By mutating amino acids in the putative membrane spanning domains (M1 and M2) of the channel subunits (α, β, and γ), the residues that contribute to the conductivity and/or selectivity of the pore can be deduced. We mutated specific negatively charged residues of the human α subunit that are predicted by α helical wheel analysis to occur at the pore-facing side of M1 or M2 to arginines. Appropriate cRNA's were coinjected into oocytes with wild type (wt) β and γ hENaC. Two-electrode voltage clamp recordings revealed that an E108R M1 mutation had no effect on whole-cell amiloride-sensitive Na+ current compared to wt αβγhENaC. Conversely, a D575R M2 mutant and a combination mutant E108R+E568R+E571R both showed decreased amiloride-sensitive whole-cell current. We have also prepared membrane vesicles from wt and mutant ENaC infected oocytes and incorporated them into planar bilayer membranes for single channel recording. Consistent with the oocyte whole-cell data, the normalized current of the αB108Rβγ mutant was 0.15 and that of the wt channel was 0.13. I-V curves from both channels in asymmetric solutions (100 mM KClcia/100 mM NaCltrans) were identical. In contrast, the single channel conductance of αD575R (no β and γ) in the bilayer was 27 pS compared to a wt conductance or 35 pS, also consistent with the data obtained under whole-cell conditions. This mutation did not affect channel PNa+/PK+ selectivity. These results indicate that negative residues in the M2 region of the α subunit of hENaC contribute to cation conduction through the channel.

Original languageEnglish
Pages (from-to)A982
JournalFASEB Journal
Issue number5
Publication statusPublished - Mar 20 1998

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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