The Haemonchus contortus LGC-39 subunit is a novel subtype of an acetylcholine-gated chloride channel.

The nematode genome exhibits a vast array of Cys-loop receptors that are activated by a diverse set of neurotransmitters and anthelmintic drugs such as ivermectin and levamisole. While many Cys-loop receptors have been functionally and pharmacologically characterized, there remains a large subset of orphan receptors where the agonist remains unknown. We have identified an orphan Cys-loop receptor, LGC-39, from the parasitic nematode Haemonchus contortus that is a novel type of cholinergic-sensitive ligand-gated chloride channel. This receptor groups outside of the acetylcholine-gated chloride channel family, in the previously named GGR-1 (GABA/Glycine Receptor-1) group of Cys-loop receptors. We found that LGC-39 forms a functional homomeric receptor when expressed in Xenopus laevis oocytes and is activated by several cholinergic ligands including acetylcholine, methacholine and surprisingly, atropine with an EC50 for atropine on the low µM range. A homology model was generated which revealed some key features of the LGC-39 ligand-binding pocket that may explain some of the elements important for atropine recognition of the LGC-39 receptor. Overall these results suggest that the GGR-1 family (now called LGC-57) of Cys-loop receptors includes novel acetylcholine-gated chloride channel subtypes and may represent important future drug targets.

Characterization of adjacent charged residues near the agonist binding site of the nematode UNC-49 GABA receptor.

The UNC-49 receptor is a Cys-loop GABA receptor that is unique to the nematode phylum. The receptor differs from mammalian GABA receptors both in amino acid sequence and pharmacology which highlights its potential as a novel anthelmintic target. Sequence differences within and near the various ligand-binding loops of the nematode receptor suggest that there could be structural differences compared to mammalian receptors that result in different pharmacological and functional features. Here we investigated three residues in the UNC-49 receptor from the parasitic nematode Haemonchus contortus: K181, E183, and T230. Analysis of these residues was conducted via site-directed mutagenesis, electrophysiology, MD simulations, and mutant cycling analysis. In the UNC-49 receptor, E183 lies in close proximity to K181 where together they appear to play a role in GABA sensitivity and pharmacology, possibly interacting via an ionic bond. While the introduction of single alanine residues at each position separately had a negative impact on GABA EC50, the double alanine mutant (K181A/E183A) exhibited wildtype-level GABA EC50 and some differences in pharmacology. Overall, this study has revealed a potentially novel role for these two residues in nematode UNC-49 GABA receptors that could aid in understanding their function.

Isolation and characterization of a novel member of the ACC ligand-gated chloride channel family, Hco-LCG-46, from the parasitic nematode Haemonchus contortus.

The ACC-1 family of cys-loop receptors are ligand-gated chloride channels sensitive to acetylcholine (ACh), and are only present in invertebrates. Studies of this family of inhibitory receptors has provided insight into how they bind and respond to ACh in a manner vastly different from nicotinic acetylcholine receptors and appear to be present in tissues that are relevant to anthelmintic action. Here, we have identified two members of the ACC-1 family from the parasitic nematode Haemonchus contortus, Hco-LGC-46 and Hco-ACC-4. Hco-LGC-46 is an ACC subunit that has never been previously expressed and pharmacologically characterized. We found that Hco-LGC-46 when expressed in Xenopus laevis oocytes forms a functional homomeric channel that is responsive to the cholinergic agonists ACh and methylcholine. hco-lgc-46 expressed in a C. elegans lgc-46 null strain (ok2900) suppressed hypersensitivity to aldicarb in a manner similar to cel-lgc-46. It was also found that Hco-LGC-46 assembles with Hco-ACC-1 and produces a receptor that is over 5-fold more sensitive to ACh and responds to the cholinergic agonists methycholine and carbachol. In contrast, the co-expression of Hco-LGC-46 with Hco-ACC-4 resulted in non-functional channels in oocytes. Hco-ACC-4 also appears to form heteromeric channels with a previously characterized subunit, Hco-ACC-2. Co-expression of Hco-ACC-4 with Hco-ACC-2 resulted in a functional heteromeric channel with an EC50 value similar to that of the Hco-ACC-2 homomeric channel. However, the maximum currents generated in the ACC-4/ACC-2 channel were significantly (p < 0.005) lower than those from the ACC-2 homomeric channel. Overall, this is the first report confirming that lgc-46 encodes an acetylcholine-gated chloride channel which when co-expressed with acc-4 results in reduced receptor function or trafficking in oocytes.

Investigating the function and possible biological role of an acetylcholine-gated chloride channel subunit (ACC-1) from the parasitic nematode Haemonchus contortus.

The cys-loop superfamily of ligand-gated ion channels are well recognized as important drug targets for many invertebrate specific compounds. With the rise in resistance seen worldwide to existing anthelmintics, novel drug targets must be identified so new treatments can be developed. The acetylcholine-gated chloride channel (ACC) family is a unique family of cholinergic receptors that have been shown, using Caenorhabditis elegans as a model, to have potential as anti-parasitic drug targets. However, there is little known about the function of these receptors in parasitic nematodes. Here, we have identified an acc gene (hco-acc-1) from the sheep parasitic nematode Haemonchus contortus. While similar in sequence to the previously characterized C. elegans ACC-1 receptor, Hco-ACC-1 does not form a functional homomeric channel in Xenopus oocytes. Instead, co-expression of Hco-ACC-1 with a previously characterized subunit Hco-ACC-2 produced a functional heteromeric channel which was 3x more sensitive to acetylcholine compared to the Hco-ACC-2 homomeric channel. We have also found that Hco-ACC-1 can be functionally expressed in C. elegans. Overexpression of both cel-acc-1 and hco-acc-1 in both C. elegans N2 and acc-1 null mutants decreased the time for worms to initiate reversal avoidance to octanol. Moreover, antibodies were generated against the Hco-ACC-1 protein for use in immunolocalization studies. Hco-ACC-1 consistently localized to the anterior half of the pharynx, specifically in pharyngeal muscle tissue in H. contortus. On the other hand, expression of Hco-ACC-1 in C. elegans was restricted to neuronal tissue. Overall, this research has provided new insight into the potential role of ACC receptors in parasitic nematodes.

A mutational and molecular dynamics study of the cys-loop GABA receptor Hco-UNC-49 from Haemonchus contortus: Agonist recognition in the nematode GABA receptor family.

The UNC-49 receptor is a unique nematode γ-aminobutyric acid (GABA)-gated chloride channel that may prove to be a novel target for the development of nematocides. Here we have characterized various charged amino acid residues in and near the agonist binding site of the UNC-49 receptor from the parasitic nematode Haemonchus contorts. Utilizing the Caenorhabditis elegans GluCl crystal structure as a template, a model was generated and various charged residues [D83 (loop D), E131 (loop A), H137 (pre-loop E), R159 (Loop E), E185 (Loop B) and R241 (Loop C)] were investigated based on their location and conservation. These residues may contribute to structure, function, and molecular interactions with agonists. It was found that all residues chosen were important for receptor function to varying degrees. Results of the mutational analysis and molecular simulations suggest that R159 may be interacting with D83 by an ionic interaction that may be crucial for general GABA receptor function. We have used the results from this study as well as knowledge of residues involved in GABA receptor binding to identify sequence patterns that may assist in understanding the function of lesser known GABA receptor subunits from parasitic nematodes.

Molecular and pharmacological characterization of an acetylcholine-gated chloride channel (ACC-2) from the parasitic nematode Haemonchus contortus.

Nematode cys-loop ligand-gated ion channels (LGICs) have been shown to be attractive targets for the development of novel anti-parasitic drugs. The ACC-1 family of receptors are a unique group of acetylcholine-gated chloride channels present only in invertebrates, and sequence analysis suggests that they contain a novel binding site for acetylcholine. We have isolated a novel member of this family, Hco-ACC-2, from the parasitic nematode Haemonchus contortus and using site-directed mutagenesis, electrophysiology and molecular modelling examined how two aromatic amino acids in the binding site contributed to agonist recognition. It was found that instead of a tryptophan residue in binding loop B, which essential for ligand binding in mammalian nAChRs, there is a phenylalanine (F200) in Hco-ACC-2. Amino acid changes at F200 to either a tyrosine or tryptophan were fairly well tolerated, where a F200Y mutation resulted in a channel hypersensitive to ACh and nicotine as well as other cholinergic agonists such as carbachol and methacholine. In addition, both pyrantel and levamisole were partial agonists at the wild-type receptor and like the other agonists showed an increase in sensitivity at F200Y. On the other hand, in Hco-ACC-2 there is a tryptophan residue at position 248 in loop C that appears to be essential for receptor function, as mutations to either phenylalanine or tyrosine resulted in a marked decrease in agonist sensitivity. Moreover, mutations that swapped the residues F200 and W248 (ie. F200W/W248F) produced non-functional receptors. Overall, Hco-ACC-2 appears to have a novel cholinergic binding site that could have implications for the design of specific anthelmintics that target this family of receptors in parasitic nematodes.

Molecular Characterization of Binding Loop E in the Nematode Cys-Loop GABA Receptor.

Nematodes exhibit a vast array of cys-loop ligand-gated ion channels with unique pharmacologic characteristics. However, many of the structural components that govern the binding of various ligands are unknown. The nematode cys-loop GABA receptor uncoordinated 49 (UNC-49) is an important receptor found at neuromuscular junctions that plays an important role in the sinusoidal movement of worms. The unique pharmacologic features of this receptor suggest that there are structural differences in the agonist binding site when compared with mammalian receptors. In this study, we examined each amino acid in one of the main agonist binding loops (loop E) via the substituted cysteine accessibility method (SCAM) and analyzed the interaction of various residues by molecular dynamic simulations. We found that of the 18 loop E mutants analyzed, H142C, R147C, and S157C had significant changes in GABA EC50 and were accessible to modification by a methanethiosulfonate reagent (MTSET) resulting in a change in I GABA In addition, the residue H142, which is unique to nematode UNC-49 GABA receptors, appears to play a negative role in GABA sensitivity as its mutation to cysteine increased sensitivity to GABA and caused the UNC-49 receptor partial agonist 5-aminovaleric acid (DAVA) to behave as a full agonist. Overall, this study has revealed potential differences in the agonist binding pocket between nematode UNC-49 and mammalian GABA receptors that could be exploited in the design of novel anthelmintics.

Evaluating the longevity of surgically extracted Xenopus laevis oocytes for the study of nematode ligand-gated ion channels.

Xenopus laevis oocytes have been extensively used as a heterologous expression system for the study of ion channels. While used successfully worldwide as tool for expressing and characterizing ion channels from a wide range of species, the limited longevity of oocytes once removed from the animal can pose significant challenges. In this study, we evaluate a simple and useful method that extends the longevity of Xenopus oocytes after removal from the animal and quantitatively assessed the reliability of the electrophysiological date obtained. The receptor used for this study was the UNC-49 receptor originally isolated from the sheep parasite, Haemonchus contortus. Overall, we found that immediate storage of the ovary in supplemented ND96 storage buffer at 4 °C could extend their use for up to 17 days with almost 80% providing reliable electrophysiological data. This means that a single extraction can provide at least 3 weeks of experiments. In addition, we examined 24-day-old oocytes (week 4) extracted from a single frog and also obtained reliable data using the same approach. However, 50% of these oocytes were usable for full dose-response experiments. Overall, we did find that this method has the potential to significantly extend the use of single oocyte extractions for two-electrode voltage clamp electrophysiology.

Efficient synthesis of the GABAA receptor agonist trans-4-aminocrotonic acid (TACA).

Investigations into the pharmacology of different types of cys-loop GABA receptor have relied for years on the chemical modification of GABA-like compounds. The GABA metabolite GABOB is an attractive molecule to modify due to its convenient chemical structure. In the process of developing new GABA-mimic compounds from GABOB as a starting compound three small molecule GABA derivatives were synthesized using a variety of chemical transformations. Amongst these, a new and reliable method to synthesize TACA (trans-4-aminocrotonic acid) is reported.

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.

A molecular characterization of the agonist binding site of a nematode cys-loop GABA receptor.

BACKGROUND AND PURPOSE: Cys-loop GABA receptors represent important targets for human chemotherapeutics and insecticides and are potential targets for novel anthelmintics (nematicides). However, compared with insect and mammalian receptors, little is known regarding the pharmacological characteristics of nematode Cys-loop GABA receptors. Here we have investigated the agonist binding site of the Cys-loop GABA receptor UNC-49 (Hco-UNC-49) from the parasitic nematode Haemonchus contortus. EXPERIMENTAL APPROACH: We used two-electrode voltage-clamp electrophysiology to measure channel activation by classical GABA receptor agonists on Hco-UNC-49 expressed in Xenopus laevis oocytes, along with site-directed mutagenesis and in silico homology modelling. KEY RESULTS: The sulphonated molecules P4S and taurine had no effect on Hco-UNC-49. Other classical Cys-loop GABAA receptor agonists tested on the Hco-UNC-49B/C heteromeric channel had a rank order efficacy of GABA > trans-4-aminocrotonic acid > isoguvacine > imidazole-4-acetic acid (IMA) > (R)-(-)-4-amino-3-hydroxybutyric acid [R(-)-GABOB] > (S)-(+)-4-amino-3-hydroxybutyric acid [S(+)-GABOB] > guanidinoacetic acid > isonipecotic acid > 5-aminovaleric acid (DAVA) (partial agonist) > ß-alanine (partial agonist). In silico ligand docking revealed some variation in binding between agonists. Mutagenesis of a key serine residue in binding loop C to threonine had minimal effects on GABA and IMA but significantly increased the maximal response to DAVA and decreased twofold the EC50 for R(-)- and S(+)-GABOB. CONCLUSIONS AND IMPLICATIONS: The pharmacological profile of Hco-UNC-49 differed from that of vertebrate Cys-loop GABA receptors and insect resistance to dieldrin receptors, suggesting differences in the agonist binding pocket. These findings could be exploited to develop new drugs that specifically target GABA receptors of parasitic nematodes.

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.

Hco-LGC-38 is novel nematode cys-loop GABA receptor subunit.

We have identified and characterized a novel cys-loop GABA receptor subunit (Hco-LGC-38) from the parasitic nematode Haemonchus contortus. This subunit is present in parasitic and free-living nematodes and shares similarity to both the UNC-49 group of GABA receptor subunits from nematodes and the resistant to dieldrin (RDL) receptors of insects. Expression of the Hco-lgc-38 gene in Xenopus oocytes and subsequent electrophysiological analysis has revealed that the gene encodes a homomeric channel sensitive to GABA (EC(50) 19 µM) and the GABA analogue muscimol. The sensitivity of the Hco-LGC-38 channel to GABA is similar to reported values for the Drosophila RDL receptor whereas its lower sensitivity to muscimol is similar to nematode GABA receptors. Hco-LGC-38 is also highly sensitive to the channel blocker picrotoxin and moderately sensitive to fipronil and dieldrin. Homology modeling of Hco-LGC-38 and subsequent docking of GABA and muscimol into the binding site has uncovered several types of potential interactions with binding-site residues and overall appears to share similarity with models of other invertebrate GABA 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.

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.

The Haemonchus contortus UNC-49B subunit possesses the residues required for GABA sensitivity in homomeric and heteromeric channels.

Hco-UNC-49 is a GABA receptor from the parasitic nematode Haemonchus contortus that has a relatively low overall sequence similarity to vertebrate GABA receptors but is very similar to the UNC-49 receptor found in the free living nematode Caenorhabditis elegans. While the nematode receptors do share >80% sequence similarity they exhibit different sensitivities to GABA. In addition, the UNC-49C subunit appears to be a positive modulator of GABA sensitivity in the H. contortus heteromeric channel, but is a negative modulator in the C. elegans heteromeric channel. The cause(s) of these differences is currently unknown since the structural elements essential for GABA sensitivity in nematode receptors have been largely unexplored. Thus, the overall aim of this study was to investigate the residues that are important for UNC-49 receptor sensitivity through the use of homology modeling, site-directed mutagenesis, and two-electrode voltage clamp. This study revealed that Met(170) in Loop B of the GABA binding-site may partially account for the observed differences in GABA receptor sensitivity between the nematode species. Residues in Loops A-D that have been reported to form the GABA binding pocket in mammalian receptors, including those forming the conserved 'aromatic box', also appear to play analogous roles in Hco-UNC-49. In addition, the two mutations that produced the most significant reduction in GABA sensitivity were R66S and Y166S. Homology modeling indicates that these two residues share a hydrogen bond and are positioned close to the carboxyl end of the GABA molecule. However, of residues examined in this study, only those on the Hco-UNC-49B subunit and not its subunit partner, Hco-UNC-49C, appear important for GABA sensitivity. Overall, results from this study suggest that the binding site of the UNC-49 heteromeric GABA receptor exhibits some differences compared to classical vertebrate GABA(A) receptors.

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.

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.

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

We have identified two genes from the parasitic nematode Haemonchus contortus, Hco-unc-49B and Hco-unc-49C that encode two GABA-gated chloride channel subunits. Electrophysiological analysis revealed that this channel has properties similar to those of the UNC-49 channel from the free-living nematode Caenorhabditis elegans. For example, the Hco-UNC-49B subunit forms a functional homomeric channel that responds to GABA and is highly sensitive to picrotoxin. Hco-UNC-49C alone does not respond to GABA but can assemble with Hco-UNC-49B to form a heteromeric channel with a lower sensitivity to picrotoxin. However, we did find that the Hco-UNC-49B/C heteromeric channel is significantly more responsive to agonists compared to the Hco-UNC-49B homomeric channel, which is the opposite trend to what has been found previously for the C. elegans channel. To investigate the subunit requirements for high agonist sensitivity, we generated cross-assembled channels by co-expressing the H. contortus subunits with UNC-49 subunits from C. elegans (Cel-UNC-49). Co-expressing Cel-UNC-49B with Hco-UNC-49C produced a heteromeric channel with a reduced sensitivity to GABA compared to that of the Cel-UNC-49B homomeric channel. In contrast, co-expressing Hco-UNC-49B with Cel-UNC-49C produced a heteromeric channel that, like the Hco-UNC-49B/C heteromeric channel, exhibits an increased sensitivity to GABA. These results suggest that the Hco-UNC-49B subunit is the key determinant for the high agonist sensitivity of heteromeric channels.