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

Abstract

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.

A Whole Genome Re-Sequencing Based GWA Analysis Reveals Candidate Genes Associated with Ivermectin Resistance in Haemonchus contortus

“…The end result is a flaccid paralysis or starvation, resulting in the death of the worm [73]. Mutations in these channels have been reported to confer IVM resistance in C. elegans [15] and pests [69,[74][75][76]. Although, we did not find any other of the previously proposed candidate genes in our set of selected candidates, we cannot discount the previous candidates too in other populations of H. contortus.…”

“…One of our candidate genes encodes glutamate-gated chloride channel (HCON_00148840, glc-3), and it has been analyzed as IVM-resistance-associated candidate previously [68][69][70]. The glutamate-gated chloride channel is the direct target site of IVM, where IVM binds irreversibly [71,72].…”

SAHA (Vorinostat) Corrects Inhibitory Synaptic Deficits Caused by Missense Epilepsy Mutations to the GABAA Receptor γ2 Subunit

“…However, the α1β2γ2 K289M GABA A Rs deactivated more rapidly than α1β2γ2 GABA A Rs, with mean time constants of 13.7 ± 0.7 ms ( n = 5) and 22.9 ± ms ( n = 12), respectively (Figures 10E,G ). As macropatch deactivation times correspond to single receptor mean activation periods (Scott et al, 2015 ; Atif et al, 2017 ; Dixon et al, 2015a ), single receptor recordings were also analysed for mean active duration and intra-activation open probability (P O ). The mean active duration of individual receptors was significantly reduced from 193 ± 18 ms ( n = 8) for α1β2γ2 GABA A Rs to 119 ± 13 ms ( n = 8, p = 0.0067) for α1β2γ2 K289M GABA A Rs (Figure 10H ).…”

Mutations in GluCl associated with field ivermectin-resistant head lice from Senegal

“…The mutation in this residue was also associated with elevated levels of abamectin resistance in wild isolates of T. urticae [24,26] . Even if this residue occurs at the ivermectin-binding site, the mutation in this site was found to reduce ivermectin sensitivity by altering the functional properties of the GluCl rather than specifically affect the binding of ivermectin [33] . A sSimilar mechanism can be suggested for the A251V mutation.…”