title: Mechanistic signs of double-barreled structure in a fluoride ion channel.
abstract: The Fluc family of F(-) ion channels protects prokaryotes and lower eukaryotes from the toxicity of environmental F(-). In bacteria, these channels are built as dual-topology dimers whereby the two subunits assemble in antiparallel transmembrane orientation. Recent crystal structures suggested that Fluc channels contain two separate ion-conduction pathways, each with two F(-) binding sites, but no functional correlates of this unusual architecture have been reported. Experiments here fill this gap by examining the consequences of mutating two conserved F(-)-coordinating phenylalanine residues. Substitution of each phenylalanine specifically extinguishes its associated F(-) binding site in crystal structures and concomitantly inhibits F(-) permeation. Functional analysis of concatemeric channels, which permit mutagenic manipulation of individual pores, show that each pore can be separately inactivated without blocking F(-) conduction through its symmetry-related twin. The results strongly support dual-pathway architecture of Fluc channels.
authors.affiliation: Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, United States.
authors.forename: Nicholas B
authors.initials: NB
authors.lastname: Last
authors.affiliation: Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, United States.
authors.forename: Ludmila
authors.initials: L
authors.lastname: Kolmakova-Partensky
authors.affiliation: Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, United States.
authors.forename: Tania
authors.initials: T
authors.lastname: Shane
authors.affiliation: Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, United States.
authors.forename: Christopher
authors.identifier: 0000-0002-0273-8653
authors.initials: C
authors.lastname: Miller
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000201:enzymology; D005459:Fluorides / Q000378:metabolism*; D007473:Ion Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010539:Permeability; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation
chemical_list: D007473:Ion Channels; D050505:Mutant Proteins; D010649:Phenylalanine; D005459:Fluorides
keywords: E. coli; biophysics; fluoride ion channel; membrane protein architecture; permeation; structural biology