title: Phenylalanine in the pore of the Erwinia ligand-gated ion channel modulates picrotoxinin potency but not receptor function.
abstract: The Erwinia ligand-gated ion channel (ELIC) is a bacterial homologue of eukaryotic Cys-loop ligand-gated ion channels. This protein has the potential to be a useful model for Cys-loop receptors but is unusual in that it has an aromatic residue (Phe) facing into the pore, leading to some predictions that this protein is incapable of ion flux. Subsequent studies have shown this is not the case, so here we probe the role of this residue by examining the function of the ELIC in cases in which the Phe has been substituted with a range of alternative amino acids, expressed in Xenopus oocytes and functionally examined. Most of the mutations have little effect on the GABA EC50, but the potency of the weak pore-blocking antagonist picrotoxinin at F16'A-, F16'D-, F16'S-, and F16'T-containing receptors was increased to levels comparable with those of Cys-loop receptors, suggesting that this antagonist can enter the pore only when residue 16' is small. T6'S has no effect on picrotoxinin potency when expressed alone but abolishes the increased potency when combined with F16'S, indicating that the inhibitor binds at position 6', as in Cys-loop receptors, if it can enter the pore. Overall, the data support the proposal that the ELIC pore is a good model for Cys-loop receptor pores if the role of F16' is taken into consideration.
authors.affiliation: Department of Biochemistry, University of Cambridge , Cambridge CB2 1QW, U.K.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Thompson
authors.forename: Mona
authors.initials: M
authors.lastname: Alqazzaz
authors.forename: Kerry L
authors.initials: KL
authors.lastname: Price
authors.forename: David A
authors.initials: DA
authors.lastname: Weston
authors.forename: Sarah C R
authors.initials: SC
authors.lastname: Lummis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive / Q000187:drug effects; D058466:Cysteine Loop Ligand-Gated Ion Channel Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004885:Erwinia / Q000235:genetics / Q000378:metabolism*; D005260:Female; D058787:GABA-A Receptor Antagonists / Q000378:metabolism / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D009865:Oocytes / Q000378:metabolism / Q000502:physiology; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010852:Picrotoxin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D054830:Sesterterpenes; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D058466:Cysteine Loop Ligand-Gated Ion Channel Receptors; D058787:GABA-A Receptor Antagonists; D010649:Phenylalanine; D010852:Picrotoxin; D054830:Sesterterpenes; D005680:gamma-Aminobutyric Acid; C022961:picrotoxinin