Glutamate-gated chloride channels and the mode of action of the abamectin/milbemycin anthelmintics

Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK.
Parasitology (Impact Factor: 2.56). 02/2005; 131 Suppl(Suppl.):S85-95. DOI: 10.1017/S0031182005008218
Source: PubMed


The macrocyclic lactones are the biggest selling and arguably most effective anthelmintics currently available. They are good substrates for the P-glycoproteins, which might explain their selective toxicity for parasites over their vertebrate hosts. Changes in the expression of these pumps have been implicated in resistance to the macrocyclic lactones, but it is clear that they exert their anthelmintic effects by binding to glutamate-gated chloride channels expressed on nematode neurones and pharyngeal muscle cells. This effect is quite distinct from the channel opening induced by glutamate, the endogenous transmitter acting at these receptors, which produces rapidly opening and desensitising channels. Ivermectin-activated channels open very slowly but essentially irreversibly, leading to a very long-lasting hyperpolarisation or depolarisation of the neurone or muscle cell and therefore blocking further function. Molecular and genetic studies have shown that there are multiple GluCl isoforms in both free-living and parasitic nematodes: the exact genetic make-up and functions of the GluCl may vary between species. The known expression patterns of the GluCl explain most of the observed biological effects of treatment with the macrocyclic lactones, though the reason for the long-lasting inhibition of larval production in filarial species is still poorly understood.

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Available from: Adrian J Wolstenholme,
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    • "Its biological target is the GluClR, a pLGIC that is highly expressed in nerve and muscle cells of nematodes and arthropods, but absent in vertebrate species. Low nanomolar concentrations of ivermectin irreversibly activate a chloride influx through these GluClRs that electrically silences nerve and muscle activity, leading to death by flaccid paralysis or starvation (Wolstenholme and Rogers, 2005). Importantly, ivermectin and related compounds also bind to and either directly activate or positively modulate several vertebrate pLGICs including GlyRs (Shan et al., 2001; Lynagh and Lynch, 2010; Lynagh et al., 2011), GABA A Rs (Sigel and Baur, 1987; Krüsek and Zemková, 1994; Adelsberger et al., 2000), and α7 nAChRs (Krause et al., 1998), albeit at relatively low potencies. "
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    ABSTRACT: GABAA receptors (GABAARs) are the major inhibitory neurotransmitter receptors in the brain and are therapeutic targets for many indications including sedation, anesthesia and anxiolysis. There is, however, considerable scope for the development of new therapeutics with improved beneficial effects and reduced side-effect profiles. The anthelminthic drug, ivermectin, activates the GABAAR although its binding site is not known. The molecular site of action of ivermectin has, however, been defined by crystallography in the homologous glutamate-gated chloride channel. Resolving the molecular mechanisms of ivermectin binding to α1β2γ2L GABAARs may provide insights into the design of improved therapeutics. Given that ivermectin binds to subunit interfaces, we sought to define (1) which subunit interface sites it binds to, (2) whether these sites are equivalent in terms of ivermectin sensitivity or efficacy, and (3) how many must be occupied for maximal efficacy. Our approach involved precluding ivermectin from binding to particular interfaces by introducing bulky M3 domain 36'F sidechains to the "+" side of those interfaces. We thereby demonstrated that ivermectin produces irreversible channel activation only when it binds to the single γ2L-β2 interface site. When it binds to α1-β2 sites it elicits potentiation of GABA-gated currents but has no irreversible activating effect. Ivermectin cannot bind to the β2-α1 interface site due to its endogenous bulky 36' methionine. Replacing this with an alanine creates a functional site at this interface, but surprisingly it is inhibitory. Molecular docking simulations reveal that the γ2L-β2 interface forms more contacts with ivermectin than the other interfaces, possibly explaining why ivermectin appears to bind irreversibly at this interface. This study demonstrates unexpectedly stark pharmacological differences among GABAAR ivermectin binding sites.
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    • "In gastro-intestinal parasites and insects, the best-studied members of this family are the glutamate-gated chloride channels (GluCls), which are widely expressed in the nervous system and on pharyngeal muscle (Wolstenholme, 2012). The MLs cause an essentially irreversible activation of these receptors, which has several deleterious consequences for the parasite, including paralysis together with inhibition of feeding and of egg-production (Wolstenholme and Rogers, 2005). In D. immitis, infertility caused by MLs has been attributed to effects on the male parasites (Lok et al. 1995). "
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    • "Insecticides that act on GluCls include abamectin, ivermectin, fipronil and the indole diterpenoid compound nodulisporic acid. A particular mutation in the α-subunit of GluCls causes the substitution of one amino acid, resulting in reduced sensitivity of the mutant channel to insecticide and thereby causing insecticide resistance [52]. A total of 7 GluCls sequences were identified from the CSM transcriptome (Table S8), but most of insects, such as D. melanogaster, T. castaneum and A. mellifera, only have one glutamate-gated chloride channel subunit. "
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