Glutamate-gated chloride channels are ligand-gated ion channels found only in invertebrates. They are the targets of the macrocyclic lactone family of anthelmintics and insecticides, of which ivermectin is probably the best known. The
avr-14 gene of C. elegans encodes two GluCl subunits, alpha 3A and alpha 3B, and mutations in this gene contribute to avermectin resistance: an amino-acid substitution (L256F) has also found in the alpha;3B subunit of ivermectin-resistant Cooperia oncophora, a parasite of cattle. We have been comparing the pharmacology of GluClα3B from C. elegans and Haemonchus contortus, a close-related parasite of sheep and goats. When expressed in Xenopus oocytes, the H. contortus receptor is much more sensitive to L-glutamate (the natural ligand), with an EC50 of 27.5 ± 1.1mM compared to that of 2.23 ± 1.2 mM for the C. elegans channel. In addition, L-glutamate activation of the channel exhibits positive co-operativity (nH = 1.89 ± 0.35) which is not observed for the C. elegans channel. Ivermectin opens the HcGluClα3B channels slowly and irreversibly, at concentrations down to 0.1nM, with an EC50 of ~12nM. When we introduced the L256F mutation into the HcGluCl alpha 3B channel, the EC50 for L-glutamate was increased to 92.2 mM and the positive co-operativity was lost. Ivermectin binding to the channel was assessed by expressing wild-type and mutated HcGluClα3B in COS-7 and measuring the specific binding of radiolabelled ivermectin to membrane preparations. The kD for ivermectin binding was not significantly different between wild-type (2.64nM) and L256F mutant (2.96nM) receptors, but again the Hill number was reduced (from 3.2 to 1.8), indicating a reduction in co-operativity in the binding to the mutant channel. These data indicate that the H. contortus GluCl alpha 3B channel has a higher affinity for both L-glutamate and ivermectin than its C. elegans counterpart and that it is possible that the L256F polymorphism may contribute to ivermectin resistance, possibly by affecting the interactions between individual subunits to prevent co-operative binding of agonists. Preliminary attempts to detect this polymorphism in ivermectin-resistant isolates of H. contortus have been unsuccessful.