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Int J Parasitol Drugs Drug Resist
2018 Dec 01;83:526-533. doi: 10.1016/j.ijpddr.2018.10.010.
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Investigating the function and possible biological role of an acetylcholine-gated chloride channel subunit (ACC-1) from the parasitic nematode Haemonchus contortus.
Callanan MK
,
Habibi SA
,
Law WJ
,
Nazareth K
,
Komuniecki RL
,
Forrester SG
.
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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 muscletissue 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.
Fig. 1. Isolation of hco-acc-1 and protein sequence analysis. A. Protein sequence alignment of H. contortus and C. elegans ACC-1 with the nAChR. Stars indicate regions of amino acid identity. Dashes represent no alignment between sequences while colons indicate similar amino acids. The 6 ligand binding loops (Loops A-F), 4 transmembrane regions (M1-M4) and the cys-loop are indicated. B. Homology model of a hypothetical receptor showing a dimer of Hco-ACC-1 and Hco-ACC-2 that was used for ligand-docking. C. End point PCR analysis of the hco-acc-1 cDNA in the adult male and L3 larval stages of H. contortus. PCR reactions were simultaneously performed on the housekeeping gene β-tubulin. Replicate experiments showed a similar trend. No PCR products were detected in negative controls (including negative RT control). D. Phylogenetic analysis of the ACC family from various nematodes. Cel - Caenorhabditis elegans Hco – Haemonchus contortus; Tci - Teladorsagia circumcincta; Ace - Ancylostoma ceylanicum; Sra - Strongyloides ratti; Tca - Toxocara canis. Hco-ACC-1 is indicated by arrow.
Fig. 2. Hco-ACC-1 and Hco-ACC-2 form a functional heteromeric receptor. A. Representative electrophysiological traces of the acetylcholine responses to oocytes expressing Hco-ACC-1 alone, Hco-ACC-2 alone and Hco-ACC-1 + 2. B. Does response curves comparing the sensitivities of the Hco-ACC-2 channel and the Hco-ACC-1/2 channel to acetylcholine and carbachol. Each data point is a mean ± SEM with n ≥ 4. C. Current/Voltage relationship of the Hco-ACC-1/2 channel comparing full Cl− ND96 (103.6 mM) partial Cl− ND96 (62.5 mM). D. Ligand docking of acetylcholine to the Hco-ACC1/2 receptor shown the distance of the ligand to W225. E. Ligand docking of carbachol to the Hco-ACC-1/2 receptor shown the distance of the ligand to W225.
Fig. 3. Immunolocalization of Hco-ACC-1 in adult female H. contortus worms. A cartoon schematic of the anterior end of the female H. contortus adult nematode is shown at the top. Green denotes detected signal in the anterior half of the pharynx seen during immunlocalization experiments. A. 25x magnification of the anterior end of H. contortus PF23 strain. B. 40X magnification stack of 25 of 5 μm confocal slices of H. contortus PF23 focused on the anterior half of the pharyngeal muscle tissue. C. 25x magnification of a female adult H. contortus MOF23 parasite focusing on the anterior end of the worm. D. 25x magnification of isolated pharynx from a female H. contortus MOF23 adult worm. E. 25x magnification light micrograph of D. F. Pre-immune serum negative control of H. contortus PF23, 25x magnification focusing on anterior end of the worm. G. Peptide-absorbed control of H. contortus MOF23 at 10x magnification focusing on the anterior end of the worm. Lines denote 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 4. Mean time to reverse of C. elegans in the presence of 30% octanol. N2, wild type; ACC-1 vc40177, Cel-ACC-1 knockout strain. PACC-1::ACC-1 FL(+) – overexpression of Cel-ACC-1 in N2 WT. PACC-1::Hco-ACC-1::GFP expression of Hco-ACC-1 in the wild type N2 C. elegans strain and the ACC-1 vc40177 (Cel-ACC-1 knockout) strains. Stars denote significant difference (P < 0.0005) compared to N2.
Fig. 5. Confocal image of N2 Caenorhabditis elegans worms expressing hco-acc-1 under control of the cel-acc-1 wild type promoter. Images A and B both show the expression of the construct in neurons near the posterior (terminal bulb) of the pharynx. Line represents 100 μm.
Figs1. Confocal images of the tail region of N2 C. elegans worms expressing hco-acc-1 under control of the cel-acc-1 wild type promoter.
Abdelmassih,
Evaluating the longevity of surgically extracted Xenopus laevis oocytes for the study of nematode ligand-gated ion channels.
2017, Pubmed,
Xenbase
Abdelmassih,
Evaluating the longevity of surgically extracted Xenopus laevis oocytes for the study of nematode ligand-gated ion channels.
2017,
Pubmed
,
Xenbase
Accardi,
Nematode cys-loop GABA receptors: biological function, pharmacology and sites of action for anthelmintics.
2012,
Pubmed
Akk,
Activation of muscle nicotinic acetylcholine receptor channels by nicotinic and muscarinic agonists.
1999,
Pubmed
Bamber,
The Caenorhabditis elegans unc-49 locus encodes multiple subunits of a heteromultimeric GABA receptor.
1999,
Pubmed
,
Xenbase
Beech,
Characterization of cys-loop receptor genes involved in inhibitory amine neurotransmission in parasitic and free living nematodes.
2013,
Pubmed
Beene,
Cation-pi interactions in ligand recognition by serotonergic (5-HT3A) and nicotinic acetylcholine receptors: the anomalous binding properties of nicotine.
2002,
Pubmed
,
Xenbase
Besier,
The Pathophysiology, Ecology and Epidemiology of Haemonchus contortus Infection in Small Ruminants.
2016,
Pubmed
Blanchard,
Deciphering the molecular determinants of cholinergic anthelmintic sensitivity in nematodes: When novel functional validation approaches highlight major differences between the model Caenorhabditis elegans and parasitic species.
2018,
Pubmed
,
Xenbase
Boulin,
Functional reconstitution of Haemonchus contortus acetylcholine receptors in Xenopus oocytes provides mechanistic insights into levamisole resistance.
2011,
Pubmed
,
Xenbase
Celie,
Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures.
2004,
Pubmed
Chao,
Feeding status and serotonin rapidly and reversibly modulate a Caenorhabditis elegans chemosensory circuit.
2004,
Pubmed
Cully,
Cloning of an avermectin-sensitive glutamate-gated chloride channel from Caenorhabditis elegans.
1994,
Pubmed
,
Xenbase
Dent,
The genetics of ivermectin resistance in Caenorhabditis elegans.
2000,
Pubmed
Dent,
avr-15 encodes a chloride channel subunit that mediates inhibitory glutamatergic neurotransmission and ivermectin sensitivity in Caenorhabditis elegans.
1997,
Pubmed
Duguet,
Recent Duplication and Functional Divergence in Parasitic Nematode Levamisole-Sensitive Acetylcholine Receptors.
2016,
Pubmed
,
Xenbase
Forrester,
Haemonchus contortus: HcGluCla expressed in Xenopus oocytes forms a glutamate-gated ion channel that is activated by ibotenate and the antiparasitic drug ivermectin.
2003,
Pubmed
,
Xenbase
Frohman,
Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer.
1988,
Pubmed
Glendinning,
Glutamate-gated chloride channels of Haemonchus contortus restore drug sensitivity to ivermectin resistant Caenorhabditis elegans.
2011,
Pubmed
Habibi,
Molecular and pharmacological characterization of an acetylcholine-gated chloride channel (ACC-2) from the parasitic nematode Haemonchus contortus.
2018,
Pubmed
Hernando,
Caenorhabditis elegans neuromuscular junction: GABA receptors and ivermectin action.
2014,
Pubmed
Hobert,
PCR fusion-based approach to create reporter gene constructs for expression analysis in transgenic C. elegans.
2002,
Pubmed
Irwin,
ZINC: a free tool to discover chemistry for biology.
2012,
Pubmed
Jensen,
Mutational studies using a cation-conducting GABAA receptor reveal the selectivity determinants of the Cys-loop family of ligand-gated ion channels.
2005,
Pubmed
Komuniecki,
Context-dependent modulation reconfigures interactive sensory-mediated microcircuits in Caenorhabditis elegans.
2014,
Pubmed
Komuniecki,
Monoamines activate neuropeptide signaling cascades to modulate nociception in C. elegans: a useful model for the modulation of chronic pain?
2012,
Pubmed
Kusano,
Cholinergic and catecholaminergic receptors in the Xenopus oocyte membrane.
1982,
Pubmed
,
Xenbase
Laing,
The genome and transcriptome of Haemonchus contortus, a key model parasite for drug and vaccine discovery.
2013,
Pubmed
Laughton,
Reporter gene constructs suggest that the Caenorhabditis elegans avermectin receptor beta-subunit is expressed solely in the pharynx.
1997,
Pubmed
McCavera,
An ivermectin-sensitive glutamate-gated chloride channel from the parasitic nematode Haemonchus contortus.
2009,
Pubmed
,
Xenbase
Mello,
DNA transformation.
1995,
Pubmed
Morris,
AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility.
2009,
Pubmed
Mu,
Different binding orientations for the same agonist at homologous receptors: a lock and key or a simple wedge?
2003,
Pubmed
Pereira,
A cellular and regulatory map of the cholinergic nervous system of C. elegans.
2015,
Pubmed
Pettersen,
UCSF Chimera--a visualization system for exploratory research and analysis.
2004,
Pubmed
Portillo,
Distribution of glutamate-gated chloride channel subunits in the parasitic nematode Haemonchus contortus.
2003,
Pubmed
Putrenko,
A family of acetylcholine-gated chloride channel subunits in Caenorhabditis elegans.
2005,
Pubmed
Rao,
A dopamine-gated ion channel (HcGGR3*) from Haemonchus contortus is expressed in the cervical papillae and is associated with macrocyclic lactone resistance.
2009,
Pubmed
,
Xenbase
Raza,
Larval development assays reveal the presence of sub-populations showing high- and low-level resistance in a monepantel (Zolvix®)-resistant isolate of Haemonchus contortus.
2016,
Pubmed
Sali,
Comparative protein modelling by satisfaction of spatial restraints.
1993,
Pubmed
Siddiqui,
An UNC-49 GABA receptor subunit from the parasitic nematode Haemonchus contortus is associated with enhanced GABA sensitivity in nematode heteromeric channels.
2010,
Pubmed
,
Xenbase
Trott,
AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading.
2010,
Pubmed
Urdaneta-Marquez,
A dyf-7 haplotype causes sensory neuron defects and is associated with macrocyclic lactone resistance worldwide in the nematode parasite Haemonchus contortus.
2014,
Pubmed
Van Doren,
Trans-spliced leader RNA exists as small nuclear ribonucleoprotein particles in Caenorhabditis elegans.
1988,
Pubmed
Weston,
Virulence in Trypanosoma cruzi infection correlates with the expression of a distinct family of sialidase superfamily genes.
1999,
Pubmed
Wever,
The Validation of Nematode-Specific Acetylcholine-Gated Chloride Channels as Potential Anthelmintic Drug Targets.
2015,
Pubmed
Williamson,
The cys-loop ligand-gated ion channel gene family of Brugia malayi and Trichinella spiralis: a comparison with Caenorhabditis elegans.
2007,
Pubmed
Wolstenholme,
Glutamate-gated chloride channels.
2012,
Pubmed
Zhang,
Structural determinants for antagonist pharmacology that distinguish the rho1 GABAC receptor from GABAA receptors.
2008,
Pubmed
,
Xenbase
