title: Multiple epithelial Na+ channel domains participate in subunit assembly.
abstract: Epithelial sodium channels (ENaCs) are composed of three structurally related subunits that form a tetrameric channel. The Xenopus laevis oocyte expression system was used to identify regions within the ENaC alpha-subunit that confer a dominant negative phenotype on functional expression of alphabetagamma-ENaC to define domains that have a role in subunit-subunit interactions. Coexpression of full-length mouse alphabetagamma-ENaC with either 1) the alpha-subunit first membrane-spanning domain and short downstream hydrophobic domain (alpha-M1H1); 2) alpha-M1H1 and its downstream hydrophilic extracellular loop (alpha-M1H1-ECL); 3) the membrane-spanning domain of a control type 2 transmembrane protein (glutamyl transpeptidase; gamma-GT) fused to the alpha-ECL (gamma-GT-alpha-ECL); 4) the extracellular domain of a control type 1 transmembrane protein (Tac) fused to the alpha-subunit second membrane-spanning domain and short upstream hydrophobic domain (Tac-alpha-H2M2); or 5) the alpha-subunit cytoplasmic COOH terminus (alpha-Ct) significantly reduced amiloride-sensitive Na+ currents in X. laevis oocytes. Functional expression of Na+ channels was not inhibited when full-length alphabetagamma-ENaC was coexpressed with either 1) the alpha-ECL lacking a signal-anchor sequence, 2) alpha-M1H1 and alpha-Ct expressed as a fusion protein, 3) full-length gamma-GT, or 4) full-length Tac. Furthermore, the expression of ROMK channels was not inhibited when full-length ROMK was coexpressed with either alpha-M1H1-ECL or alpha-Ct. Full-length FLAG-tagged alpha-, beta-, or gamma-ENaC coimmunoprecipitated with myc-tagged alpha-M1H1-ECL, whereas wild-type gamma-GT did not. These data suggest that multiple sites within the alpha-subunit participate in subunit-subunit interactions that are required for proper assembly of the heterooligomeric ENaC complex.
authors.affiliation: Department of Medicine, University of Pittsburgh, 15261, USA.
authors.forename: James B
authors.initials: JB
authors.lastname: Bruns
authors.forename: Baofeng
authors.initials: B
authors.lastname: Hu
authors.forename: Yoon J
authors.initials: YJ
authors.lastname: Ahn
authors.forename: Shaohu
authors.initials: S
authors.lastname: Sheng
authors.forename: Rebecca P
authors.initials: RP
authors.lastname: Hughey
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Kleyman
mesh_terms: D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004232:Diuretics / Q000494:pharmacology; D053503:Epithelial Sodium Channels; D005260:Female; D005799:Genes, Dominant; D051379:Mice; D009865:Oocytes; D010641:Phenotype; D020033:Protein Isoforms / Q000737:chemistry / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D017434:Protein Structure, Tertiary / Q000502:physiology; D015222:Sodium Channels / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014982:Xenopus laevis
chemical_list: D004232:Diuretics; D053503:Epithelial Sodium Channels; D020033:Protein Isoforms; D015222:Sodium Channels; D000584:Amiloride