Probing Allosteric Relationships Between the Neurotransmitter-Binding Site and the Binding Pocket of the Anthelmintic Drug Ivermectin in Glutamate-Gated Chloride Channels

Abstract

Ivermectin (IVM) is a broad-spectrum anthelmintic drug used to treat human parasitic diseases like onchocerciasis (river blindness) and elephantiasis (lymphatic filariasis). IVM acts on invertebrate Cys-loop receptors (GluClRs) that naturally bind the neurotransmitter glutamate (Glu) to open an intrinsic chloride-selective channel. Native heteropentameric GluClα/βRs are irreversibly stabilized in an open-channel conformation by IVM. Hence, IVM causes sustained inflow of chloride ions and hyperpolarization that suppress nervous impulses in nematodes. Consequently, vital physiological processes in the nematode are abolished. Upon expression in Xenopus oocytes, homomeric GluClαRs are activated (being opened) by IVM but not by Glu, whereas homomeric GluClβRs are activated by Glu but not by IVM (Cully et al., Nature, 1994). A heteromeric GluClα/βR carrying a mutation in the β-subunit that inhibits Glu binding is readily activated by IVM (Li et al., FEBS Lett, 2002). These differential responses suggest that the drug and the neurotransmitter binding sites are independent of each other. Yet, a recent X-ray crystal structure shows that IVM-bound homomeric GluClαR can accommodate Glu (Hibbs and Gouaux, Nature, 2011), in line with a study showing that robust activation of homomeric GluClαRs by IVM enables further (slight) activation by Glu (Etter et al., JBC, 1996). Here, we explored whether the structurally separated IVM and Glu binding sites are functionally coupled in heteromeric GluClα/βRs. Mutations introduced between the Glu and IVM binding pockets or inside the IVM binding pocket were examined for their influence on agonistic efficacies. We found mutations that improve IVM efficacy without changing Glu efficacy, and mutations that either reduce or increase the agonistic efficacies of IVM and Glu concomitantly. Our results strongly indicate that the IVM and Glu binding sites in heteromeric GluClRs are functionally coupled.