Effects of glutamate and ivermectin on single glutamate-gated chloride channels of the parasitic nematode H. contortus

Ivermectin (IVM) is a widely-used anthelmintic that works by binding to and activating glutamate-gated chloride channel receptors (GluClRs) in nematodes. The resulting chloride flux inhibits the pharyngeal muscle cells and motor neurons of nematodes, causing death by paralysis or starvation. IVM resistance is an emerging problem in many pest species, necessitating the development of novel drugs. However, drug optimisation requires a quantitative understanding of GluClR activation and modulation mechanisms. Here we investigated the biophysical properties of homomeric α (avr-14b) GluClRs from the parasitic nematode, H. contortus, in the presence of glutamate and IVM. The receptor proved to be highly responsive to low nanomolar concentrations of both compounds. Analysis of single receptor activations demonstrated that the GluClR oscillates between multiple functional states upon the binding of either ligand. The G36’A mutation in the third transmembrane domain, which was previously thought to hinder access of IVM to its binding site, was found to decrease the duration of active periods and increase receptor desensitisation. On an ensemble macropatch level the mutation gave rise to enhanced current decay and desensitisation rates. Because these responses were common to both glutamate and IVM, and were observed under conditions where agonist binding sites were likely saturated, we infer that G36’A affects the intrinsic properties of the receptor with no specific effect on IVM binding mechanisms. These unexpected results provide new insights into the activation and modulatory mechanisms of the H. contortus GluClRs and provide a mechanistic framework upon which the actions of drugs can be reliably interpreted.Author summary: IVM is a gold standard anti-parasitic drug that is used extensively to control invertebrate parasites pest species. The drug targets the glutamate-gated chloride channel receptor (GluClR) found on neurons and muscle cells of these organisms, causing paralysis and death. However, IVM resistance is becoming a serious problem in human and animal health, as well as human food production. We provide the first comprehensive investigation of the functional properties of the GluClR of H. contortus, which is a major parasite in grazing animals, such as sheep and goats. We compared glutamate and IVM induced activity of the wild-type and a mutant GluClR, G36’A, that markedly reduces IVM sensitivity in wild populations of pests. Our data demonstrate that the mutation reduces IVM sensitivity by altering the functional properties of the GluClR rather than specifically affecting the binding of IVM, even though the mutation occurs at the IVM binding site. This study provides a mechanistic framework upon which the actions of new candidate anthelmintic drugs can be interpreted.