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A region directly following the second transmembrane domain in gamma ENaC is required for normal channel gating
Journal article   Open access   Peer reviewed

A region directly following the second transmembrane domain in gamma ENaC is required for normal channel gating

Rachell E Booth, Qiusheng Tong, Jorge Medina, Peter M Snyder, Pravina Patel and James D Stockand
The Journal of biological chemistry, Vol.278(42), pp.41367-41379
10/17/2003
DOI: 10.1074/jbc.M305400200
PMID: 12897071
url
https://doi.org/10.1074/jbc.M305400200View
Published (Version of record) Open Access

Abstract

We used a yeast one-hybrid complementation screen to identify regions within the cytosolic tails of the mouse alpha, beta, and gamma epithelial Na+ channel (ENaC) important to protein-protein and/or protein-lipid interactions at the plasma membrane. The cytosolic COOH terminus of alphaENaC contained a strongly interactive domain just distal to the second transmembrane region (TM2) between Met610 and Val632. Likewise, gammaENaC 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 alpha- and betaENaC, 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 alphaENaC 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 gammaENaC was established with whole-cell patch clamp experiments of wild type (alpha, beta, and gamma) and mutant (alpha, beta, and gammadeltaQ573-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 gammaENaC 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 gammaENaC are necessary for normal channel gating.
 Temperature Electrophysiology Sodium Channels - physiology Structure-Activity Relationship Recombinant Fusion Proteins - metabolism Epithelial Sodium Channels Transfection Gene Deletion Sodium Channels - metabolism Cell Membrane - metabolism CHO Cells Protein Structure, Tertiary Cricetinae Biotinylation Plasmids - metabolism Blotting, Western Precipitin Tests Amino Acid Motifs Microscopy, Confocal Two-Hybrid System Techniques Patch-Clamp Techniques Animals Sodium Channels - chemistry Protein Binding Cytosol - metabolism Mice Mutation Microscopy, Fluorescence

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