title: Mutations in the extracellular domains of glutamate-gated chloride channel alpha3 and beta subunits from ivermectin-resistant Cooperia oncophora affect agonist sensitivity.
abstract: Two full-length glutamate-gated chloride channel (GluCl) cDNAs, encoding GluClalpha3 and GluClbeta subunits, were cloned from ivermectin-susceptible (IVS) and -resistant (IVR) Cooperia oncophora adult worms. The IVS and IVR GluClalpha3 subunits differ at three amino acid positions, while the IVS and IVR GluClbeta subunits differ at two amino acid positions. The aim of this study was to determine whether mutations in the IVR subunits affect agonist sensitivity. The subunits were expressed singly and in combination in Xenopus laevis oocytes. Electrophysiological whole-cell voltage-clamp recordings showed that mutations in the IVR GluClalpha3 caused a modest but significant threefold loss of sensitivity to glutamate, the natural ligand for GluCl receptors. As well, a significant decrease in sensitivity to the anthelmintics ivermectin and moxidectin was observed in the IVR GluClalpha3 receptor. Mutations in the IVR GluClbeta subunit abolished glutamate sensitivity. Co-expressing the IVS GluClalpha3 and GluClbeta subunits resulted in heteromeric channels that were more sensitive to glutamate than the respective homomeric channels, demonstrating co-assembly of the subunits. In contrast, the heteromeric IVR channels were less sensitive to glutamate than the homomeric IVR GluClalpha3 channels. The heteromeric IVS channels were significantly more sensitive to glutamate than the heteromeric IVR channels. Of the three amino acids distinguishing the IVS and IVR GluClalpha3 subunits, only one of them, L256F, accounted for the differences in response between the IVS and IVR GluClalpha3 homomeric channels.
authors.affiliation: Institute of Parasitology, McGill University, 21-111 Lakeshore Road, Ste-Anne-de-Bellevue, Quebec, Canada H9X 3V9.
authors.forename: Annete I
authors.initials: AI
authors.lastname: Njue
authors.forename: Jon
authors.initials: J
authors.lastname: Hayashi
authors.forename: Lyle
authors.initials: L
authors.lastname: Kinne
authors.forename: Xiao-Peng
authors.initials: XP
authors.lastname: Feng
authors.forename: Roger K
authors.initials: RK
authors.lastname: Prichard
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000969:Antinematodal Agents / Q000494:pharmacology; D002478:Cells, Cultured; D065101:Chloride Channel Agonists; D018118:Chloride Channels / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance / Q000235:genetics*; D018698:Glutamic Acid / Q000493:pharmacokinetics; D007559:Ivermectin / Q000494:pharmacology*; D008024:Ligands; D018942:Macrolides / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary / Q000235:genetics; D021122:Protein Subunits / Q000819:agonists / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014251:Trichostrongyloidea / Q000235:genetics* / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D000969:Antinematodal Agents; D065101:Chloride Channel Agonists; D018118:Chloride Channels; D008024:Ligands; D018942:Macrolides; D021122:Protein Subunits; C109777:glutamate-gated chloride channels; D018698:Glutamic Acid; D007559:Ivermectin; C027837:moxidectin