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.

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.

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.

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.