title: Side chain orientation of residues lining the selectivity filter of epithelial Na+ channels.
abstract: Epithelial Na(+) channels (ENaCs) selectively conduct Na(+) and Li(+) but exclude K(+). A three-residue tract ((G/S)XS) present within all three subunits has been identified as a key structure forming a putative selectivity filter. We investigated the side chain orientation of residues within this tract by analyzing accessibility of the introduced sulfhydryl groups to thiophilic Cd(2+). Xenopus oocytes were used to express wild-type or mutant mouse alphabetagammaENaCs. The blocking effect of external Cd(2+) was examined by comparing amiloride-sensitive Na(+) currents measured by two-electrode voltage clamp in the absence and presence of Cd(2+) in the bath solution. The currents in mutant channels containing a single Cys substitution at the first or third position within the (G/S)XS tract (alphaG587C, alphaS589C, betaG529C, betaS531C, gammaS546C, and gammaS548C) were blocked by Cd(2+) with varying inhibitory constants (0.06-13 mm), whereas the currents in control channels were largely insensitive to Cd(2+) at concentrations up to 10 mm. The Cd(2+) blocking effects were fast, with time constants in the range of seconds, and were only partially reversible. The blocked currents were restored by 10 mm dithiothreitol. Mutant channels containing alanine or serine substitutions at these sites within the alpha subunit were only poorly and reversibly blocked by 10 mm Cd(2+). These results indicate that the introduced sulfhydryl groups face the conduction pore and suggest that serine hydroxyl groups within the selectivity filter in wild-type ENaCs face the conduction pore and may contribute to cation selectivity by participating in coordination of permeating cations.
authors.affiliation: Renal-Electrolyte Division, Department of Medicine, School of Medicine, University of Pittsburgh, PEnnsylvania 15261, USA. shaohu@pitt.edu
authors.forename: Shaohu
authors.initials: S
authors.lastname: Sheng
authors.forename: Clint J
authors.initials: CJ
authors.lastname: Perry
authors.forename: Ossama B
authors.initials: OB
authors.lastname: Kashlan
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Kleyman
mesh_terms: D000409:Alanine / Q000737:chemistry; D000584:Amiloride / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002104:Cadmium / Q000737:chemistry*; D002412:Cations; D003545:Cysteine / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D053503:Epithelial Sodium Channels; D051379:Mice; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018075:RNA, Complementary / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D015222:Sodium Channels / Q000737:chemistry*; D013997:Time Factors; D014981:Xenopus
chemical_list: D002412:Cations; D053503:Epithelial Sodium Channels; D018075:RNA, Complementary; D015222:Sodium Channels; D002104:Cadmium; D012694:Serine; D000584:Amiloride; D003545:Cysteine; D000409:Alanine; D004229:Dithiothreitol