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Molecular determinants of ivermectin sensitivity at the glycine receptor chloride channel.

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 ? GluClR complexed with ivermectin has recently been published. Here, we probed ivermectin sensitivity determinants on the ?1 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 ? 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 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.

SUBMITTER: Lynagh T

PROVIDER: S-EPMC3243547 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Molecular determinants of ivermectin sensitivity at the glycine receptor chloride channel.

Lynagh Timothy T Webb Timothy I TI Dixon Christine L CL Cromer Brett A BA Lynch Joseph W JW

The Journal of biological chemistry 20111027 51

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 determ ...[more]

PMID: 22033924

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Project description:Ivermectin is an anthelmintic drug that works by inhibiting neuronal activity and muscular contractility in arthropods and nematodes. It works by activating glutamate-gated chloride channels (GluClRs) at nanomolar concentrations. These receptors, found exclusively in invertebrates, belong to the pentameric Cys-loop receptor family of ligand-gated ion channels (LGICs). Higher (micromolar) concentrations of ivermectin also activate or modulate vertebrate Cys-loop receptors, including the excitatory nicotinic and the inhibitory GABA type-A and glycine receptors (GlyRs). An X-ray crystal structure of ivermectin complexed with the C. elegans ? GluClR demonstrated that ivermectin binds to the transmembrane domain in a cleft at the interface of adjacent subunits. It also identified three hydrogen bonds thought to attach ivermectin to its site. Site-directed mutagenesis and voltage-clamp electrophysiology have also been employed to probe the binding site for ivermectin in ?1 GlyRs. These have raised doubts as to whether the hydrogen bonds are essential for high ivermectin potency. Due to its lipophilic nature, it is likely that ivermectin accumulates in the membrane and binds reversibly (i.e., weakly) to its site. Several lines of evidence suggest that ivermectin opens the channel pore via a structural change distinct from that induced by the neurotransmitter agonist. Conformational changes occurring at locations distant from the pore can be probed using voltage-clamp fluorometry (VCF), a technique which involves quantitating agonist-induced fluorescence changes from environmentally sensitive fluorophores covalently attached to receptor domains of interest. This technique has demonstrated that ivermectin induces a global conformational change that propagates from the transmembrane domain to the neurotransmitter binding site, thus suggesting a mechanism by which ivermectin potentiates neurotransmitter-gated currents. Together, this information provides new insights into the mechanisms of action of this important drug.

Project description:Anthelmintic resistance is a major problem in livestock farming, especially of small ruminants, but our understanding of it has been limited by the difficulty in carrying out functional genetic studies on parasitic nematodes. An important nematode infecting sheep and goats is Haemonchus contortus; in many parts of the world this species is resistant to almost all the currently available drugs, including ivermectin. It is extremely polymorphic and to date it has proved impossible to relate any sequence polymorphisms to its ivermectin resistance status. Expression of candidate drug-resistance genes in Caenorhabditis elegans could provide a convenient means to study the effects of polymorphisms found in resistant parasites, but may be complicated by differences between the gene families of target and model organisms. We tested this using the glutamate-gated chloride channel (GluCl) gene family, which forms the ivermectin drug target and are candidate resistance genes. We expressed GluCl subunits from C. elegans and H. contortus in a highly resistant triple mutant C. elegans strain (DA1316) under the control of the avr-14 promoter; expression of GFP behind this promoter recapitulated the pattern previously reported for avr-14. Expression of ivermectin-sensitive subunits from both species restored drug sensitivity to transgenic worms, though some quantitative differences were noted between lines. Expression of an ivermectin-insensitive subunit, Hco-GLC-2, had no effect on drug sensitivity. Expression of a previously uncharacterised parasite-specific subunit, Hco-GLC-6, caused the transgenic worms to become ivermectin sensitive, suggesting that this subunit also encodes a GluCl that responds to the drug. These results demonstrate that both orthologous and paralogous subunits from C. elegans and H. contortus are able to rescue the ivermectin sensitivity of mutant C. elegans, though some quantitative differences were observed between transgenic lines in some assays. C. elegans is a suitable system for studying parasitic nematode genes that may be involved in drug resistance.

Dataset Information

Molecular determinants of ivermectin sensitivity at the glycine receptor chloride channel.

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Molecular determinants of ivermectin sensitivity at the glycine receptor chloride channel.

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