title: Two-sided block of a dual-topology F- channel.
abstract: The Fluc family is a set of small membrane proteins forming F(-)-specific electrodiffusive ion channels that rescue microorganisms from F(-) toxicity during exposure to weakly acidic environments. The functional channel is built as a dual-topology homodimer with twofold symmetry parallel to the membrane plane. Fluc channels are blocked by nanomolar-affinity fibronectin-domain monobodies originally selected from phage-display libraries. The unusual symmetrical antiparallel dimeric architecture of Flucs demands that the two chemically equivalent monobody-binding epitopes reside on opposite ends of the channel, a double-sided blocking situation that has never before presented itself in ion channel biophysics. However, it is not known if both sites can be simultaneously occupied, and if so, whether monobodies bind independently or cooperatively to their transmembrane epitopes. Here, we use direct monobody-binding assays and single-channel recordings of a Fluc channel homolog to reveal a novel trimolecular blocking behavior that reveals a doubly occupied blocked state. Kinetic analysis of single-channel recordings made with monobody on both sides of the membrane shows substantial negative cooperativity between the two blocking sites.
authors.affiliation: Department of Biochemistry and Howard Hughes Medical Institute, Brandeis University, Waltham, MA 02454.
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Turman
authors.affiliation: Department of Biochemistry and Howard Hughes Medical Institute, Brandeis University, Waltham, MA 02454.
authors.forename: Jacob T
authors.initials: JT
authors.lastname: Nathanson
authors.affiliation: Department of Biochemistry and Howard Hughes Medical Institute, Brandeis University, Waltham, MA 02454.
authors.forename: Randy B
authors.initials: RB
authors.lastname: Stockbridge
authors.affiliation: Department of Biochemistry and.
authors.forename: Timothy O
authors.initials: TO
authors.lastname: Street
authors.affiliation: Department of Biochemistry and Howard Hughes Medical Institute, Brandeis University, Waltham, MA 02454 cmiller@brandeis.edu.
authors.forename: Christopher
authors.initials: C
authors.lastname: Miller
mesh_terms: D016880:Anisotropy; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D000939:Epitopes / Q000737:chemistry; D005454:Fluorescence Polarization; D005461:Fluorine / Q000737:chemistry*; D007473:Ion Channels / Q000737:chemistry*; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008962:Models, Theoretical; D009154:Mutation; D009282:Naphthalenesulfonates / Q000737:chemistry; D011485:Protein Binding; D055672:Static Electricity
chemical_list: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D000939:Epitopes; D007473:Ion Channels; D008051:Lipid Bilayers; D009282:Naphthalenesulfonates; D005461:Fluorine; C063126:5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid; D003545:Cysteine
keywords: block; cooperativity; ion channel; monobody