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

Rachell E. Booth, Qiusheng Tong, Jorge Medina, Peter M. Snyder, Pravina Patel, James D Stockand

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

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 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 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.

Original languageEnglish (US)
Pages (from-to)41367-41379
Number of pages13
JournalJournal of Biological Chemistry
Volume278
Issue number42
DOIs
StatePublished - Oct 17 2003

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Epithelial Sodium Channels
Cell membranes
Cell Membrane
Cricetulus
Proteins
Yeast
Tail
Ovary
Fusion reactions
Yeasts
Cells
Evanescent fields
Membranes
Fluorescence microscopy
Clamping devices
Fluorescence Microscopy
Immunoprecipitation
Labeling
Experiments
Lipids

ASJC Scopus subject areas

  • Biochemistry

Cite this

A Region Directly Following the Second Transmembrane Domain in γENaC Is Required for Normal Channel Gating. / Booth, Rachell E.; Tong, Qiusheng; Medina, Jorge; Snyder, Peter M.; Patel, Pravina; Stockand, James D.

In: Journal of Biological Chemistry, Vol. 278, No. 42, 17.10.2003, p. 41367-41379.

Research output: Contribution to journalArticle

Booth, Rachell E. ; Tong, Qiusheng ; Medina, Jorge ; Snyder, Peter M. ; Patel, Pravina ; Stockand, James D. / A Region Directly Following the Second Transmembrane Domain in γENaC Is Required for Normal Channel Gating. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 42. pp. 41367-41379.
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