Abstract

Ivermectin is an anthelmintic drug that works by activating glutamate-gated chloride channel receptors (GluClRs) in nematode parasites. GluClRs belong to the Cys-loop receptor family that also includes glycine receptor (GlyR) chloride channels. GluClRs and A288G mutant GlyRs are both activated by low nanomolar ivermectin concentrations. The crystal structure of the Caenorhabditis elegans α GluClR complexed with ivermectin has recently been published. Here, we probed ivermectin sensitivity determinants on the α1 GlyR using site-directed mutagenesis and electrophysiology. Based on a mutagenesis screen of transmembrane residues, we identified Ala288 and Pro230 as crucial sensitivity determinants. A comparison of the actions of selamectin and ivermectin suggested the benzofuran C05-OH was required for high efficacy. When taken together with docking simulations, these results supported a GlyR ivermectin binding orientation similar to that seen in the GluClR crystal structure. However, whereas the crystal structure shows that ivermectin interacts with the α GluClR via H-bonds with Leu218, Ser260, and Thr285 (α GluClR numbering), our data indicate that H-bonds with residues homologous to Ser260 and Thr285 are not important for high ivermectin sensitivity or direct agonist efficacy in A288G α1 GlyRs or three other GluClRs. Our data also suggest that van der Waals interactions between the ivermectin disaccharide and GlyR M2-M3 loop residues are unimportant for high ivermectin sensitivity. Thus, although our results corroborate the ivermectin binding orientation as revealed by the crystal structure, they demonstrate that some of the binding interactions revealed by this structure do not pertain to other highly ivermectin-sensitive Cys-loop receptors.

Highlights

  • The ivermectin-binding site on the glutamate-gated chloride channel was recently resolved by crystallography

  • Whereas the crystal structure shows that ivermectin interacts with the ␣ GluClR via H-bonds with Leu218, Ser260, and Thr285 (␣ GluClR numbering), our data indicate that H-bonds with residues homologous to Ser260 and Thr285 are not important for high ivermectin sensitivity or direct agonist efficacy in A288G ␣1 glycine receptor (GlyR) or three other GluClRs

  • Disruption of Ivermectin Efficacy via Mutations at the LBDTMD Interface—Because the conservative Y279F mutation in the ␣1 GlyR M2–M3 loop dramatically reduces ivermectin sensitivity [19], we investigated whether residues at the ligand-binding domain (LBD)-transmembrane domain (TMD) interfacial region may contribute to ivermectin-binding or -gating mechanisms

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Summary

Introduction

The ivermectin-binding site on the glutamate-gated chloride channel was recently resolved by crystallography. Results: Ivermectin binds in a similar orientation to the structurally related glycine receptor, two H-bonds apparent in the crystal structure proved unimportant for binding to glycine receptors. Conclusion: Ivermectin-binding mechanisms vary among Cys-loop receptors. Ivermectin is an anthelmintic drug that works by activating glutamate-gated chloride channel receptors (GluClRs) in nematode parasites. We probed ivermectin sensitivity determinants on the ␣1 GlyR using site-directed mutagenesis and electrophysiology. When taken together with docking simulations, these results supported a GlyR ivermectin binding orientation similar to that seen in the GluClR crystal structure. Our results corroborate the ivermectin binding orientation as revealed by the crystal structure, they demonstrate that some of the binding interactions revealed by this structure do not pertain to other highly ivermectin-sensitive Cys-loop receptors

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