title: Epithelial sodium channel pore region. structure and role in gating.
abstract: Epithelial sodium channels (ENaC) have a crucial role in the regulation of extracellular fluid volume and blood pressure. To study the structure of the pore region of ENaC, the susceptibility of introduced cysteine residues to sulfhydryl-reactive methanethiosulfonate derivatives ((2-aminoethyl)methanethiosulfonate hydrobromide (MTSEA) and [(2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET)) and to Cd(2+) was determined. Selected mutants within the amino-terminal portion (alphaVal(569)-alphaTrp(582)) of the pore region responded to MTSEA, MTSET, or Cd(2+) with stimulation or inhibition of whole cell Na(+) current. The reactive residues were not contiguous but were separated by 2-3 residues where substituted cysteine residues did not respond to the reagents and line one face of an alpha-helix. The activation of alphaS580Cbetagamma mENaC by MTSET was associated with a large increase in channel open probability. Within the carboxyl-terminal portion (alphaSer(583)-alphaSer(592)) of the pore region, only one mutation (alphaS583C) conferred a rapid, nearly complete block by MTSEA, MTSET, and Cd(2+), whereas several other mutant channels were partially blocked by MTSEA or Cd(2+) but not by MTSET. Our data suggest that the outer pore of ENaC is formed by an alpha-helix, followed by an extended region that forms a selectivity filter. Furthermore, our data suggest that the pore region participates in ENaC gating.
authors.affiliation: Department of Medicine, Physiology and Pathology, School of Medicine, University of Pennsylvania and Veterans Affairs Medical Center, Philadelphia, Pennsylvania 19104, USA.
authors.forename: S
authors.initials: S
authors.lastname: Sheng
authors.forename: J
authors.initials: J
authors.lastname: Li
authors.forename: K A
authors.initials: KA
authors.lastname: McNulty
authors.forename: T
authors.initials: T
authors.lastname: Kieber-Emmons
authors.forename: T R
authors.initials: TR
authors.lastname: Kleyman
mesh_terms: D000584:Amiloride / Q000494:pharmacology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D003545:Cysteine; D053503:Epithelial Sodium Channels; D005260:Female; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015222:Sodium Channels / Q000737:chemistry* / Q000502:physiology*; D014982:Xenopus laevis
chemical_list: D053503:Epithelial Sodium Channels; D011994:Recombinant Proteins; D015222:Sodium Channels; D000584:Amiloride; D003545:Cysteine