Genetic and biochemical evidence for a novel avermectin-sensitive chloride channel in Caenorhabditis elegans -: Isolation and characterization

Avermectins are a class of macrocyclic lactones that is widely used in crop protection and to treat helminth infections in man and animals. Two complementary DNAs (GluCl alpha and GluCl beta) encoding chloride channels that are gated by avermectin and glutamate, respectively, were isolated from Caenorhabditis elegans. To study the role of these subunits in conferring avermectin sensitivity we isolated a mutant C. elegans strain with a Tc1 transposable element insertion that functionally inactivated the GluCl alpha gene (GluCl alpha::Tc1). GluCl alpha::Tc1 animals exhibit a normal phenotype including typical avermectin sensitivity, Xenopus oocytes expressing GluCl alpha::Tc1 strain mRNA elicited reduced amplitude avermectin and glutamate-dependent chloride currents. Avermectin binding assays in GluCl alpha::Tc1 strain membranes showed the presence of high affinity binding sites, with a reduced B-max. These experiments suggest that GluCl alpha is a target for avermectin and that additional glutamate-gated and avermectin-sensitive chloride channel subunits exist in C. elegans. We isolated a cDNA (GluCl alpha 2) encoding a chloride channel that shares 75% amino acid identity with GluCl alpha. This subunit forms homomeric channels that are gated irreversibly by avermectin and reversibly by glutamate, GluCl alpha 2 coassembles with GluCl beta to form heteromeric channels that are gated by both ligands. The presence of subunits related to GluCl alpha may explain the low level and rarity of target site involvement in resistance to the avermectin class of compounds.