Sean G. Forrester

A glutamate-gated chloride channel subunit from Haemonchus contortus:: Expression in a mammalian cell line, ligand binding, and modulation of anthelmintic binding by glutamate

Glutamate-gated chloride channels (GluCls) are inhibitory ion channels that are sensitive to the antiparasitic drugs ivermectin (IVM) and moxidectin (MOX). We have transiently transfected COS-7 cells with a subunit of a GluCl (HcGluCla) from the parasitic nematode Haemonchus contortus. This subunit bound [3H]-IVM and [3H]-MOX with K(d) values of 0.11+/-0.021 and 0.18+/-0.02nM, respectively. Displacement analysis revealed that IVM and MOX bind to the same site on HcGluCla and that this site is likely distinct from the glutamate binding site. Glutamate was found to be an allosteric modulator of [3H]-MOX and [3H]-IVM binding and increased the affinity of [3H]-MOX for HcGluCla by more than 50% and that of [3H]-IVM by more than 7-fold. These results point to both similarities and differences in the interactions of IVM and MOX with the GluCl. Aspartate, which is structurally similar to glutamate, had little or no effect on [3H]-IVM and [3H]-MOX binding, suggesting that this ligand does not induce the conformational change necessary to potentiate macrocyclic lactone binding. These results also indicate that it may be possible to enhance the efficacy of macrocyclic lactone anthelmintics by administering these compounds with ligands acting allosterically to enhance their binding.

Haemonchus contortus: HcGluCla expressed in Xenopus oocytes forms a glutamate-gated ion channel that is activated by ibotenate and the antiparasitic drug ivermectin

Ion channels are targets for many drugs including insecticides and anthelminthic agents such as ivermectin (IVM) and moxidectin (MOX). IVM has been shown to activate glutamate-gated chloride channels (GluCls) from the free-living nematode, Caenorhabditis elegans. Haemonchus contortus is a parasitic nematode that is also extremely sensitive to IVM. The high sensitivity of H. contortus to IVM is probably the result of the fact that, like C. elegans, H. contortus also express GluCls. To investigate the potential physiological response to IVM in H. contortus we have expressed a GluCl from this parasite (H. contortus glutamate-gated chloride channel, HcGluCla) in Xenopus oocytes. HcGluCla expressed in oocytes formed a homomeric channel that responded to glutamate and ibotenate as well as the anthelmintics IVM and MOX. The response to glutamate and ibotenate was fast acting and reversible whereas the response to IVM and MOX was a slower activating channel that was essentially irreversible. These results suggest that IVM toxicity in H. contortus is the result of its irreversible activation of GluCls.

Characterization of a novel tyramine-gated chloride channel from Haemonchus contortus.

Tyramine (TA), a trace amine, is becoming accepted as a main stream neurotransmitter in invertebrates. Recent evidence indicates that part of the function of TA in nematodes involves a novel receptor (Cel-LGC-55) from the ligand-gated chloride channel class of ionotropic receptors. However, the role of TA or its receptors in the biology of nematode parasites is limited. Haemonchus contortus is a deadly parasitic worm which causes significant economic burden in the production of small ruminants in many parts of the world. In this study, we have cloned and characterized a novel LGCC from H. contortus which we have named Hco-LGC-55. This receptor subunit is a clear orthologue of Cel-LGC-55 and is able to form a homomeric chloride channel that is gated by tyramine, dopamine and octopamine. Semi-quantitative reverse transcription-polymerase chain reaction (sqRT-PCR) shows that this subunit is expressed in all life-cycle stages of the worm, but appears to have reduced mRNA expression in the adult male.

Association of ion-channel genotype and macrocyclic lactone sensitivity traits in Haemonchus contortus.

Resistance to macrocyclic lactones in the strongylid parasite of sheep, Haemonchus contortus, is associated with specific alleles of several genes, including those encoding ligand-gated chloride-channels. While previous functional studies of the ion-channels encoded by these resistant alleles have revealed alterations in ligand binding and response to the anthelmintics, we still do not know how these alleles are contributing to resistance in vivo. To understand the phenomenon of anthelmintic resistance in detail we need to link changes in the genes of individual parasites with their ability to resist the effects of anthelmintic exposure. We have determined the genotype of individual adult and larval H. contortus with respect to the glc-5 and lgc-37 genes linked with macrocyclic lactone resistance. In these same individuals, we measured feeding and movement, two characteristics targeted by the drug, which are believed to contribute to parasite killing. Both genes are linked with altered behavior in the absence of drug, providing evidence that genetic resistance may be associated with alterations in parasite biology. In the presence of macrocyclic lactones, both genes are associated with a degree of protection against drug action. Whether this protection may be effective under the application of anthelmintic as part of normal farm practice and whether this could explain the evolution of resistance remain unclear.

Pharmacological characterization of the Haemonchus contortus GABA-gated chloride channel, Hco-UNC-49: modulation by macrocyclic lactone anthelmintics and a receptor for piperazine.

Invertebrate ligand-gated chloride channels are well recognized as important targets for several insecticides and anthelmintics. Hco-UNC-49 is a GABA-gated chloride channel from the parasitic nematode Haemonchus contortus and is an orthologue to the neuromuscular receptor (Cel-UNC-49) from the free-living nematode Caenorhabditis elegans. While the receptors from the two nematodes are similar in sequence, they exhibit different sensitivities to GABA which may reflect differences in in vivo function. The aim of the current study was to further characterize the pharmacology of the Hco-UNC-49 receptor by examining its sensitivity to various insecticides and anthelmintics using two-electrode voltage clamp. Specifically, the insecticides fipronil and picrotoxin appear to inhibit the channel in a similar manner. The IC(50) of picrotoxin on the homomeric channel was 3.65 ± 0.64 μM and for the heteromeric channel was 134.56 ± 44.12 μM. On the other hand, dieldrin, a well-known insect GABA receptor blocker, had little effect on the UNC-49 channel. The anthelmintics ivermectin and moxidectin both moderately potentiated the activation of Hco-UNC-49 by GABA, while piperazine was able to directly activate both the Hco-UNC-49 homomeric and heteromeric channels with EC(50) values of 6.23 ± 0.45 mM and 5.09 ± 0.32 mM, respectively. This piperazine current was reversibly blocked by picrotoxin which demonstrates that the anthelmintic specifically targets Hco-UNC-49. These results demonstrate that Hco-UNC-49 exhibits binding sites for several molecules including piperazine and macrocyclic lactone anthelmintics. In addition, this is the first report of the heterologous expression and subsequent characterization of a receptor for piperazine.

An UNC‐49 GABA receptor subunit from the parasitic nematode Haemonchus contortus is associated with enhanced GABA sensitivity in nematode heteromeric channels

J. Neurochem. (2010) 113, 1113–1122.

Characterization of cys-loop receptor genes involved in inhibitory amine neurotransmission in parasitic and free living nematodes

We have isolated two genes, Hco-lgc-53 and Hco-mod-1, from the parasitic nematode Haemonchus contortus, which are orthologs of previously characterized genes that encode dopamine and serotonin-gated chloride channels, respectively, in Caenorhabditis elegans. A search of transcriptome data for the filarial nematode parasites Loa loa, Brugia malayi, and Wucheria bancrofti revealed predicted coding sequences for orthologs of acetylcholine, serotonin and dopamine-gated chloride channels, which correspond to the C. elegans clades acc-1, mod-1 and ggr-3, respectively. Genome data for the more distantly related nematode parasite, Trichinella spiralis, contain genes predicted to encode members of the acc-1 clade only, but all three clades were absent from the trematode Schistosoma mansoni. Analysis of the ratio of non-synonymous to synonymous substitutions (ω) for receptor subunit sequences revealed strong selective constraint over the entire protein, consistent with the known highly conserved 3D structure of cys-loop receptors. This constraint was significantly greater for binding loop residues that are predicted to contact bound ligand and residues of the transmembrane domains. The substitution rate for ligand binding residues was significantly higher for branches leading to the acc-1 and mod-1 clades, where the convergent evolution for binding acetylcholine and serotonin, respectively, is thought to have occurred. Homology models of both Hco-MOD-1 and Hco-LGC-53 channels revealed the presence of binding structures typical of the cys-loop receptor family, including the presence of an aromatic box that is important for the formation of the binding pocket. Both receptors contain a tryptophan in loop C that appears to be a key residue important for the binding of amines to ligand-gated chloride channels. As additional ligand-gated chloride-channel sequences become available for a wider range of species the combination of molecular modeling and analysis of sequence evolution should provide an effective tool to understand the wide diversity of neurotransmitters that bind to this unique group of receptors.

Nematode cys-loop GABA receptors: biological function, pharmacology and sites of action for anthelmintics

Parasitic nematode infection of humans and livestock is a major problem globally. Attempts to control nematode populations have led to the development of several classes of anthelmintic, which target cys-loop ligand-gated ion channels. Unlike the vertebrate nervous system, the nematode nervous system possesses a large and diversified array of ligand-gated chloride channels that comprise key components of the inhibitory neurotransmission system. In particular, cys-loop GABA receptors have evolved to play many fundamental roles in nematode behaviour such as locomotion. Analysis of the genomes of several free-living and parasitic nematodes suggests that there are several groups of cys-loop GABA receptor subunits that, for the most part, are conserved among nematodes. Despite many similarities with vertebrate cys-loop GABA receptors, those in nematodes are quite distinct in sequence similarity, subunit composition and biological function. With rising anthelmintic resistance in many nematode populations worldwide, GABA receptors should become an area of increased scientific investigation in the development of the next generation of anthelmintics.

Localisation of serotonin and dopamine in Haemonchus contortus.

Serotonin and dopamine play important roles in the biology of nematodes where they exert their effect on feeding, locomotion and reproductive behavior. Haemonchus contortus, a parasitic nematode which infects small ruminants, is responsible for considerable economic losses in agriculture. In the current study we have mapped the localisation of these two neurotransmitters in this parasite using immuno-staining. Serotonin localised in amphidial and pharyngeal neurons in both adult female and male worms. Serotonin was also found in ray sensory neurons as well as in a few ventral cord motor neurons exclusively in adult males. Surprisingly, dopamine was only detected in the neuronal commissures linking the lateral and sub-lateral nerve cords in both sexes. We also studied the effect of these two molecules on female adult worms in vitro. Serotonin mainly inhibited movement whereas dopamine had a profound paralytic effect on the mid-body of the worms.

Recent Duplication and Functional Divergence in Parasitic Nematode Levamisole-Sensitive Acetylcholine Receptors.

Helminth parasites rely on fast-synaptic transmission in their neuromusculature to experience the outside world and respond to it. Acetylcholine plays a pivotal role in this and its receptors are targeted by a wide variety of both natural and synthetic compounds used in human health and for the control of parasitic disease. The model, Caenorhabditis elegans is characterized by a large number of acetylcholine receptor subunit genes, a feature shared across the nematodes. This dynamic family is characterized by both gene duplication and loss between species. The pentameric levamisole-sensitive acetylcholine receptor has been characterized from C. elegans, comprised of five different subunits. More recently, cognate receptors have been reconstituted from multiple parasitic nematodes that are found to vary in subunit composition. In order to understand the implications of receptor composition change and the origins of potentially novel drug targets, we investigated a specific example of subunit duplication based on analysis of genome data for 25 species from the 50 helminth genome initiative. We found multiple independent duplications of the unc-29, acetylcholine receptor subunit, where codon substitution rate analysis identified positive, directional selection acting on amino acid positions associated with subunit assembly. Characterization of four gene copies from a model parasitic nematode, Haemonchus contortus, demonstrated that each copy has acquired unique functional characteristics based on phenotype rescue of transgenic C. elegans and electrophysiology of receptors reconstituted in Xenopus oocytes. We found evidence that a specific incompatibility has evolved for two subunits co-expressed in muscle. We demonstrated that functional divergence of acetylcholine receptors, driven by directional selection, can occur more rapidly than previously thought and may be mediated by alteration of receptor assembly. This phenomenon is common among the clade V parasitic nematodes and this work provides a foundation for understanding the broader context of changing anthelmintic drug targets across the parasitic nematodes.