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.

Glutamate-gated chloride channel receptors (GluClRs) mediate inhibitory neurotransmission at invertebrate synapses and are primary targets of parasites that impact drastically on agriculture and human health. Ivermectin (IVM) is a broad-spectrum pesticide that binds and potentiates GluClR activity. Resistance to IVM is a major economic and health concern, but the molecular and synaptic mechanisms of resistance are ill-defined. Here we focus on GluClRs of the agricultural endoparasite, Haemonchus contortus. We demonstrate that IVM potentiates inhibitory input by inducing a tonic current that plateaus over 15 minutes and by enhancing post-synaptic current peak amplitude and decay times. We further demonstrate that IVM greatly enhances the active durations of single receptors. These effects are greatly attenuated when endogenous IVM-insensitive subunits are incorporated into GluClRs, suggesting a mechanism of IVM resistance that does not affect glutamate sensitivity. We discovered functional groups of IVM that contribute to tuning its potency at different isoforms and show that the dominant mode of access of IVM is via the cell membrane to the receptor.

Background and Purpose Between half to 1 million people die annually from malaria. Anopheles gambiae mosquitoes are major malaria vectors. Unfortunately, resistance has emerged to the agents currently used to control A. gambiae, creating a demand for novel control measures. The pentameric glutamate‐gated chloride channel (GluCl) expressed in the muscle and nerve cells of these organisms are a potentially important biological target for malaria control. The pharmacological properties of Anophiline GluCl receptors are, however, largely unknown. Accordingly, we compared the efficacy of four insecticides (lindane, fipronil, picrotoxin, and ivermectin) on two A. gambiae GluCl receptor splice variants with the aim of providing a molecular basis for designing novel anti‐malaria treatments. Experimental Approach The A. gambiae GluCl receptor b1 and c splice variants were expressed homomerically in Xenopus laevis oocytes and studied with electrophysiological techniques, using two‐electrode voltage‐clamp. Key Results The b1 and c GluCl receptors were activated with similar potencies by glutamate and ivermectin. Fipronil was more potent than picrotoxin and lindane at inhibiting glutamate‐ and ivermectin‐gated currents. Importantly, b1 GluCl receptors exhibited reduced sensitivity to picrotoxin and lindane. They also recovered from these effects to a greater extent than c GluCl receptors Conclusions and Implications The two splice variant subunits exhibited differential sensitivities to multiple, structurally divergent insecticides, without accompanying changes in the sensitivity to the endogenous neurotransmitter, glutamate, implying that drug resistance may be caused by alterations in relative subunit expression levels, without affecting physiological function. Our results strongly suggest that it should be feasible to develop novel subunit‐specific pharmacological agents.

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