A Region Directly Following the Second Transmembrane Domain in γENaC Is Required for Normal Channel Gating

We used a yeast one-hybrid complementation screen to identify regions within the cytosolic tails of the mouse α,β, and γ epithelial Na⁺ channel (ENaC) important to protein-protein and/or protein-lipid interactions at the plasma membrane. The cytosolic COOH terminus of αENaC contained a strongly interactive domain just distal to the second transmembrane region (TM2) between Met⁶¹⁰ and Val⁶³². Likewise, γENaC contained such a domain just distal to TM2 spanning Gln ⁵⁷³-Pro⁶⁰⁰. Interactive domains were also localized within Met¹-Gln⁵⁴ and the last 17 residues of α- and βENaC, respectively. Confocal images of Chinese hamster ovary cells transfected with enhanced green fluorescent fusion proteins of the cytosolic tails of mENaC subunits were consistent with results in yeast. Fusion proteins of the NH₂ terminus of αENaC and the COOH termini of all three subunits co-localized with a plasma membrane marker. The functional importance of the membrane interactive domain in the COOH terminus of γENaC was established with whole-cell patch clamp experiments of wild type (α, β, and γ) and mutant (α, β, and γ _ΔQ573-P600) mENaC reconstituted in Chinese hamster ovary cells. Mutant channels had about 13% of the activity of wild type channels with 0.33 ± 0.14 versus 2.5 ± 0.80 nA of amiloridesensitive inward current at -80 mV. Single channel analysis of recombinant channels demonstrated that mutant channels had a decrease in P_o with 0.16 ± 0.03 versus 0.67 ± 0.07 for wild type. Mutant γENaC associated normally with the other two subunits in co-immunoprecipitation studies and localized to the plasma membrane in membrane labeling experiments and when visualized with evanescent-field fluorescence microscopy. Similar to deletion of Gln ⁵⁷³-Pro⁶⁰⁰, deletion of Gin⁵⁷³-AXg ⁵⁸³ but not Thr⁵⁸⁴-Pro⁶⁰⁰ decreased ENaC activity. The current results demonstrate that residues within Gln ⁵⁷³-Arg⁵⁸³ of γENaC are necessary for normal channel gating.