Abstract

The broad-spectrum anthelmintic drug ivermectin (IVM) activates and stabilizes an open-channel conformation of invertebrate chloride-selective glutamate receptors (GluClRs), thereby causing a continuous inflow of chloride ions and sustained membrane hyperpolarization. These effects suppress nervous impulses and vital physiological processes in parasitic nematodes. The GluClRs are pentamers. Homopentameric receptors assembled from the Caenorhabditis elegans (C. elegans) GluClα (GLC-1) subunit can inherently respond to IVM but not to glutamate (the neurotransmitter). In contrast, heteromeric GluClα/β (GLC-1/GLC-2) assemblies respond to both ligands, independently of each other. Glutamate and IVM bind at the interface between adjacent subunits, far away from each other; glutamate in the extracellular ligand-binding domain, and IVM in the ion-channel pore periphery. To understand the importance of putative intersubunit contacts located outside the glutamate and IVM binding sites, we introduced mutations at intersubunit interfaces, between these two binding-site types. Then, we determined the effect of these mutations on the activation of the heteromeric mutant receptors by glutamate and IVM. Amongst these mutations, we characterized an α-subunit point mutation located close to the putative IVM-binding pocket, in the extracellular end of the first transmembrane helix (M1). This mutation (αF276A) moderately reduced the sensitivity of the heteromeric GluClαF276A/βWT receptor to glutamate, and slightly decreased the receptor subunits’ cooperativity in response to glutamate. In contrast, the αF276A mutation drastically reduced the sensitivity of the receptor to IVM and significantly increased the receptor subunits’ cooperativity in response to IVM. We suggest that this mutation reduces the efficacy of channel gating, and impairs the integrity of the IVM-binding pocket, likely by disrupting important interactions between the tip of M1 and the M2-M3 loop of an adjacent subunit. We hypothesize that this physical contact between M1 and the M2-M3 loop tunes the relative orientation of the ion-channel transmembrane helices M1, M2 and M3 to optimize pore opening. Interestingly, pre-exposure of the GluClαF276A/βWT mutant receptor to subthreshold IVM concentration recovered the receptor sensitivity to glutamate. We infer that IVM likely retained its positive modulation activity by constraining the transmembrane helices in a preopen orientation sensitive to glutamate, with no need for the aforementioned disrupted interactions between M1 and the M2-M3 loop.

Highlights

  • Chloride-selective glutamate receptors (GluClRs) are pentameric glutamate (Glu)-gated chloride channels unique to invertebrates (Wolstenholme, 2012)

  • All the GluCl receptor assemblies mentioned below originate from the C. elegans GluClα (GLC-1) and GluClβ (GLC-2) subunits; so, the species name is avoided hereafter

  • We have recently shown that CHO cells transfected with the wild type GluClα subunit alone display very weak responses to 10 mM Glu but robust responses to 500 nM IVM (Degani-Katzav et al, 2016)

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Summary

Introduction

Chloride-selective glutamate receptors (GluClRs) are pentameric glutamate (Glu)-gated chloride channels unique to invertebrates (Wolstenholme, 2012). GluClRs are specific targets for ivermectin (IVM), a macrocyclic lactone widely used as an anthelmintic drug to treat filarial diseases like onchocerciasis (river blindness), which is caused by Onchocerca volvulus, and elephantiasis (lymphatic filariasis) that is caused by Wuchereria bancrofti, Brugia malayi, Brugia timori and Brugia pahangithat. These diseases afflict hundreds of millions of people worldwide, mainly in equatorial Africa (Crump and Omura, 2011; Campbell, 2012). This long-lasting effect eventually leads to suppression of locomotion (Cook et al, 2006); inhibition of the pharyngeal muscle activity, which interrupts with feeding behavior (Geary et al, 1993; Brownlee et al, 1997; Dent et al, 2000); and interruption of secretion processes in the parasite that are crucial for evading the host immune system (Moreno et al, 2010; reviewed in Geary and Moreno, 2012; Wolstenholme, 2012; Wolstenholme et al, 2016)

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