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

• Published in: The Journal of biological chemistry Vol. 278; no. 42; pp. 41367 - 41379
• Main Authors: Booth, Rachell E., Tong, Qiusheng, Medina, Jorge, Snyder, Peter M., Patel, Pravina, Stockand, James D.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 17.10.2003
• Subjects: Amino Acid Motifs; Animals; Biotinylation; Blotting, Western; Cell Membrane - metabolism; CHO Cells; Cricetinae; Cytosol - metabolism; Electrophysiology; Epithelial Sodium Channels; Gene Deletion; Mice; Microscopy, Confocal; Microscopy, Fluorescence; Mutation; Patch-Clamp Techniques; Plasmids - metabolism; Precipitin Tests; Protein Binding; Protein Structure, Tertiary; Recombinant Fusion Proteins - metabolism; Sodium Channels - chemistry; Sodium Channels - metabolism; Sodium Channels - physiology; Structure-Activity Relationship; Temperature; Transfection; Two-Hybrid System Techniques
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M305400200

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 Gln573–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 γΔQ573-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 Po 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 Gln573–Pro600, deletion of Gln573–Arg583 but not Thr584–Pro600 decreased ENaC activity. The current results demonstrate that residues within Gln573–Arg583 of γENaC are necessary for normal channel gating.