Adrian J. Wolstenholme

Nematode glutamate-gated chloride channels are targets of the macrocyclic lactones, the most important group of anthelmintics available. In Xenopus laevis oocytes, channels formed by the GluClα3B subunit from the parasite Haemonchus contortus were more sensitive to l-glutamate (EC₅₀ = 27.6 ± 2.7 μM) than those formed by the homologous subunit from Caenorhabditis elegans (EC₅₀ = 2.2 ± 0.12 mM). Ibotenate was a partial agonist (EC₅₀ = 87.7 ± 3.5 μM). The H. contortus channels responded to low concentrations of ivermectin (estimated EC₅₀ =∼0.1 ± 1.0 nM), opening slowly and irreversibly in a highly cooperative manner: the rate of channel opening was concentration-dependent. Responses to glutamate and ivermectin were inhibited by picrotoxinin and fipronil. Mutating an N-terminal domain amino acid, leucine 256, to phenylalanine increased the EC₅₀ for l-glutamate to 92.2 ± 3.5 μM, and reduced the Hill number from 1.89 ± 0.35 to 1.09 ± 0.16. It increased the K_d for radiolabeled ivermectin binding from 0.35 ± 0.1 to 2.26 ± 0.78 nM. Two other mutations (E114G and V235A) had no effect on l-glutamate activation or ivermectin binding: one (T300S) produced no detectable channel activity, but ivermectin binding was similar to wild-type. The substitution of any aromatic amino acid for Leu256 had similar effects in the radioligand binding assay. Molecular modeling studies suggested that the GluCl subunits have a fold similar to that of other Cys-loop ligand-gated ion channels and that amino acid 256 was unlikely to play a direct role in ligand binding but may be involved in mediating the allosteric properties of the receptor.

SUMMARY: Benzimidazoles (BZ) are widely used to treat parasitic nematode infections of humans and animals, but resistance is widespread in veterinary parasites. Several polymorphisms in β-tubulin genes have been associated with BZ-resistance. In the present study, we investigated β-tubulin isotype 1 sequences of 18 Haemonchus contortus isolates with varying levels of resistance to thiabendazole. The only polymorphism whose frequency was significantly increased in the resistant isolates was TTC to TAC at codon 200. Real-time PCR (using DNA from 100 third-stage larvae, L3s) and pyrosequencing (from DNA from 1000–10 000 L3s) were used to measure allele frequencies at codon 200 of these isolates, producing similar results; drug sensitivity decreased with increasing TAC frequency. Pyrosequencing was also used to measure allele frequencies at positions 167 and 198. We showed that such measurements are sufficient to assess the BZ-resistance status of most H. contortus isolates. The concordance between real-time PCR and pyrosequencing results carried out in different laboratories indicated that these tools are suitable for the routine diagnosis of BZ-resistance in H. contortus. The molecular methods were more sensitive than the ‘egg hatch test’, and less time-consuming than current in vivo- or in vitro-anthelmintic resistance detection methods. Thus, they provide a realistic option for routine molecular resistance testing on farms.

Nematode cys-loop ligand gated ion channels (CLGIC) mediate neurotransmission and are important targets for anthelmintics in parasitic nematodes. The CLGIC superfamily in nematodes includes ion channels gated by acetylcholine, γ-amino butyric acid (GABA), glutamate, glycine and 5-HT. The macrocyclic lactones and the nicotinic agonists are important groups of anthelmintics that target the glutamate gated chloride channels and the nicotinic acetylcholine receptors, respectively. The model organism Caenorhabditis elegans has the most diverse families of cys-loop LGIC known in any organism. Many parasitic nematodes have homologues of C. elegans receptors but to date no genome wide investigations have been done. The genome sequencing projects of Brugia malayi (clade III) and Trichinella spiralis (clade I) have allowed us to characterise the CLGIC families in these species. Although the main groups of CLGICs targeted by anthelmintics are represented in both the nematode genomes investigated here, the CLGIC family is much smaller in B. malayi and T. spiralis, suggesting that care must be taken when using C. elegans as a model organism for distantly related nematodes.

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.

Glutamate‐gated chloride channels (GluCl) are related to γ‐aminobutyric acid‐A (GABA_A) receptors and are the target sites for the avermectin/milbemycin (A/M) anthelmintics, drugs that cause paralysis of the somatic and pharyngeal muscles in nematodes. We have previously identified four GluCl subunits, HcGluClα, HcGluClβ, HcGluClα3A, and HcGluClα3B from the sheep parasite Haemonchus contortus. We raised specific antisera against all of these subunits and used them in immunofluorescence experiments on adult parasites. All of the subunits were expressed in the motor nervous system, especially motor neuron commissures. Double‐immunostaining experiments suggested that HcGluClα and HcGluClβ were expressed on the same commissures; these were also stained with an anti‐GABA antibody, suggesting that they may be inhibitory motor neurons. The HcGluClβ subunit was also detected in lateral and sublateral nerve cords. The HcGluClα3A and ‐B subunits, products of an alternatively spliced gene, were expressed in different neurons. We found HcGluClαA in a pair of sensory, possibly amphid, neurons in the head, in addition to the motor neuron commissures. HcGluClα3B was detected in three cell bodies, probably of pharyngeal neurons, and to ventral and lateral cords. These results indicate that the GluCl are widely distributed in the H. contortus nervous system and suggest that they have critical roles controlling locomotion, pharyngeal function, and possibly sensory processing in parasitic nematodes. They also provide an explanation for the observed effects of the A/M anthelmintics. J. Comp. Neurol. 462:213–222, 2003.

We report the cloning and expression of a novel Caenorhabditis elegans polypeptide, GLC‐3, with high sequence identity to previously cloned L‐glutamate‐gated chloride channel subunits from nematodes and insects. Expression of glc‐3 cRNA in Xenopus oocytes resulted in the formation of homo‐oligomeric L‐glutamate‐gated chloride channels with robust and rapidly desensitizing currents, an EC₅₀ of 1.9±0.03 mM and a Hill coefficient of 1.5±0.1. GABA, glycine, histamine and NMDA all failed to activate the GLC‐3 homo‐oligomer at concentrations of 1 mM. The anthelminthic, ivermectin, directly and irreversibly activated the L‐glutamate‐gated channel with an EC₅₀ of 0.4±0.02 μM. The GLC‐3 channels were selective for chloride ions, as shown by the shift in the reversal potential for L‐glutamate‐gated currents after the reduction of external Cl⁻ from 107.6 to 62.5 mM. Picrotoxinin failed to inhibit L‐glutamate agonist responses at concentrations up to 1 mM. The polycyclic dinitrile, 3,3‐bis‐trifluoromethyl‐bicyclo[2,2,1]heptane‐2,2‐dicarbonitrile (BIDN), completely blocked L‐glutamate‐induced chloride currents recorded from oocytes expressing GLC‐3 with an IC₅₀ of 0.2±0.07 μM. The phenylpyrazole insecticide, fipronil, reversibly inhibited L‐glutamate‐gated currents recorded from the GLC‐3 receptor with an IC₅₀ of 11.5±0.11 μM. In this study, we detail the unusual antagonist pharmacology of a new GluCl subunit from C. elegans. Unlike all other native and recombinant nematode GluCl reported to date, the GLC‐3 receptor is insensitive to picrotoxinin, but is sensitive to two other channel blockers, BIDN and fipronil. Further study of this receptor may provide insights into the molecular basis of non‐competitive antagonism by these compounds. British Journal of Pharmacology (2001) 132, 1247–1254; doi:10.1038/sj.bjp.0703937

Nematode membrane preparations contain high amounts of low-affinity specific L-glutamate binding sites. The numbers of these sites were increased in 2 isolates, one field-derived and the other laboratory-derived, of ivermectin-resistant Haemonchus contortus and a field isolate of ivermectin-resistant Telodorsagia circumcincta, when compared to control, drug- sensitive isolates. Specific [³H]ivermectin binding to these membrane preparations showed no differences between ivermectin-sensitive and resistant isolates and the number of ivermectin binding sites was approximately 100-fold less than the number of L-glutamate binding sites. Kinetic analysis of L-glutamate binding suggested the presence of at least 2 classes of binding site. L-Glutamate binding was blocked by ibotenic acid, kynurenic acid and β-hydroxyaspartate, but not by ivermectin, argiopine, kainate, quisqualate or NMDA. Competition assays with ibotenic acid suggested that there were 2 distinct populations of glutamate binding sites and that the site with the lower affinity for ibotenate was upregulated in the ivermectin-resistant nematodes. In the field isolate of resistant H. contortus we found no coding changes in the cDNAs encoding glutamate-gated chloride channel subunits HG2, HG3 and HG4, nor were any changes in channel expression detected using subunit-specific antibodies. The low-affinity binding site is unlikely to be associated with the ivermectin receptor in these nematodes.