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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.
Highlights
Pentameric ligand gated ion channels are membrane-bound receptors that facilitate the diffusion of ions across cell membranes in response to the binding of agonists
A detailed study of the biophysical properties of GluClR activation has not been undertaken, even though GluClRs constitute a major group of Pentameric ligand gated ion channels (pLGICs), many organisms that express them are serious parasitic pests, or vectors for disease transmission and they are a major target for anthelminthic drugs
In this study we investigated the biophysical properties of the homomeric α GluClR from H. contortus as: 1) H. contortus is a major parasitic pest of domestic ruminant animals, 2) IVM is used widely to control H. contortus, 3) IVM resistance has emerged as a major problem in this species [43], and 4) GluClRs comprising or containing the α subunit are most likely the major biological IVM target in this species [4, 25]
Summary
Pentameric ligand gated ion channels (pLGICs) are membrane-bound receptors that facilitate the diffusion of ions across cell membranes in response to the binding of agonists. The glutamate-gated chloride channel receptor (GluClR), first identified in arthropods, such as insects and crustaceans [1,2,3], is an anion-selective pLGIC found at neuronal and neuromuscular inhibitory synapses [4]. O. volvulus and W. bancrofti are nematodes that cause river blindness (onchocerciasis) and elephantiasis (lymphatic filariasis), respectively, in humans. Another nematode, H. contortus [17] is a serious pathogen in ruminant agricultural animals such as cattle, sheep and goats. The flatworm blood fluke, S. mansoni, inflicts schistosomiasis (associated with serious systemic morbidities) on hundreds of millions of people in underdeveloped communities [23]
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