Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Front Mol Neurosci
2020 Jan 01;13:601102. doi: 10.3389/fnmol.2020.601102.
Show Gene links
Show Anatomy links
A Functional Comparison of Homopentameric Nicotinic Acetylcholine Receptors (ACR-16) Receptors From Necator americanus and Ancylostoma ceylanicum.
Kaji MD
,
Geary TG
,
Beech RN
.
???displayArticle.abstract???
Effective control of hookworm infections in humans and animals relies on using a small group of anthelmintics. Many of these drugs target cholinergic ligand-gated ion channels, yet the direct activity of anthelmintics has only been studied in a subset of these receptors, primarily in the non-parasitic nematode, Caenorhabditis elegans. Here we report the characterization of a homopentameric ionotropic acetylcholine receptor (AChR), ACR-16, from Necator americanus and Ancylostoma ceylanicum, the first known characterization of human hookworm ion channels. We used two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes to determine the pharmacodynamics of cholinergics and anthelmintics on ACR-16 from both species of hookworm. The A. ceylanicum receptor (Ace-ACR-16) was more sensitive to acetylcholine (EC50 = 20.64 ± 0.32 μM) and nicotine (EC50 = 24.37 ± 2.89 μM) than the N. americanus receptor (Nam-ACR-16) (acetylcholine EC50 = 170.1 ± 19.23 μM; nicotine EC50 = 597.9 ± 59.12 μM), at which nicotine was a weak partial agonist (% maximal acetylcholine response = 30.4 ± 7.4%). Both receptors were inhibited by 500 μM levamisole (Ace-ACR-16 = 65.1 ± 14.3% inhibition, Nam-ACR-16 = 79.5 ± 7.7% inhibition), and responded to pyrantel, but only Ace-ACR-16 responded to oxantel. We used in silico homology modeling to investigate potential structural differences that account for the differences in agonist binding and identified a loop E isoleucine 130 of Nam-ACR-16 as possibly playing a role in oxantel insensitivity. These data indicate that key functional differences exist among ACR-16 receptors from closely related species and suggest mechanisms for differential drug sensitivity.
FIGURE 1. (A) Sequence alignment of ACR-16 from Ancylostoma ceylanicum (accession # MT163735), Necator americanus (accession # MT163736) Ancylostoma caninum (accession # QEM53385.1), Caenorhabditis elegans (accession # CCD64102.1), Haemonchus contortus (accession # AZS27833.1), Ascaris suum (accession # KP756901), Parascaris equorum (accession # AZS27834.1), and the human α7 acetylcholine receptor subunit (accession # P36544.5). Amino acids are shaded by consensus sequence similarity; black is most, and white is least similar. The ECD ligand binding loops A-E are denoted in red, the characteristics cys-loop is indicated in blue, the cation selectivity motif is shown in purple and the transmembrane domains are in green. Red star indicates site of Ile130 of Nam-ACR-16 (B)% Identity matrix of the polypeptide sequences for comparison.
FIGURE 2. Functional expression of ACR-16 receptors in response to acetylcholine. (A) Amplitude of current response to 1 mM acetylcholine in the presence or absence of the accessory protein RIC-3 48 h after injection. Each point represents recordings from individual oocytes. n > 7; p < 0.05. (B) Ace-ACR-16 response profile to increasing concentrations of acetylcholine. (C) Reproducibility of Nam-ACR-16 and (D) Ace-ACR-16 current response profile to repeated concentrations of acetylcholine. Nam-ACR-16 displays reduced current responses to repeated exposures to acetylcholine.
FIGURE 3. (A) The effect of time between subsequent applications of 1 mM acetylcholine on the magnitude of current elicited from Nam-ACR-16. Oocytes given 2 min recovery time from an initial acetylcholine exposure produced significantly larger currents than any other timepoint. Data at each time point were derived from experiments conducted on oocytes from at least two different frogs. n > 6, *p = 0.0004, **p < 0.0001. (B) Representative tracing of current responses from oocytes expressing Nam-ACR-16 induced by varying time between exposures to acetylcholine.
FIGURE 4. (A) Concentration-response curves for acetylcholine on Ace- and Nam-ACR-16. Individual oocytes were exposed to increasing concentrations of acetylcholine and all responses were standardized to the maximal current achieved within each oocyte. n > 6 (B) Concentration-response curves for nicotine on Ace- and Nam-ACR-16. Individual oocytes were exposed to repeated maximal concentrations of acetylcholine to determine stability of response, and to serve as a maximal effect reference to standardize all nicotine current responses to. Nicotine acts as a full agonist on Ace-ACR-16 but as a weak partial agonist of Nam-ACR-16 n > 6.
FIGURE 5. (A) BAPTA-AM was used to examine the role of activation of intrinsic Ca2+ sensing Cl– channels induced by Ca2+ influx from Ace-ACR-16 activation. BAPTA-AM treatment had no effect on the ability of acetylcholine to activate Ace-ACR-16 in oocytes. n > 5; Vc = –60 mV (B) Current-voltage relationship of ACR-16 induced by 100 μM acetylcholine in ND96 (reversal potential = –8.5 mV), ND96 with BAPTA-treatment (reversal potential = 13.6 mV), 96 mM sodium gluconate (reversal potential = 15.9 mV), 1.8 mM CaCl2 (reversal potential = 35.8 mV). BAPTA-associated inhibition of Ca2+ gated Cl– channels altered the conductance from activating ACR-16. Oocytes were exposed to 100 μM acetylcholine at holding potentials beginning at –75 mV and increasing by 25 mV to +50 mV. (C) 100 μM acetylcholine produced no current responses in 96 mM glucosamine HCl solution. Channel activity in response to acetylcholine was restored when this solution was replaced with ND96.
FIGURE 6. (A) Ace-ACR-16 (left) and Nam-ACR-16 (right) agonist response profiles to classical cholinergics and anthelmintics. All current responses are standardized relative to that of a maximal 1 mM acetylcholine response; n = 5 (B) inhibition of EC50 acetylcholine-induced responses in Ace- and Nam-ACR-16 by levamisole and the AChR antagonist mecamylamine; n ≥ 4.
FIGURE 7. Homology models of the Ace-ACR-16 and Nam-ACR-16, docking: acetylcholine (A,B), nicotine (C,D), pyrantel (E,F), and oxantel (G,H). The principal (+) subunit contributing to key residues of Loops A, B and C, is colored green and the subunit contributing the complementary (-) face of the binding pocket contributing Loops D, E and F is colored in purple ribbon. Carbon atoms of docked agonists are colored in orange ball and stick. Red and blue molecules show oxygen and nitrogen atoms, respectively.
Abongwa,
Pharmacological profile of Ascaris suum ACR-16, a new homomeric nicotinic acetylcholine receptor widely distributed in Ascaris tissues.
2016, Pubmed,
Xenbase
Abongwa,
Pharmacological profile of Ascaris suum ACR-16, a new homomeric nicotinic acetylcholine receptor widely distributed in Ascaris tissues.
2016,
Pubmed
,
Xenbase
Atchison,
Comparative neuromuscular blocking actions of levamisole and pyrantel-type anthelmintics on rat and gastrointestinal nematode somatic muscle.
1992,
Pubmed
Ballivet,
Nicotinic acetylcholine receptors in the nematode Caenorhabditis elegans.
1996,
Pubmed
,
Xenbase
Barda,
Efficacy and tolerability of moxidectin alone and in co-administration with albendazole and tribendimidine versus albendazole plus oxantel pamoate against Trichuris trichiura infections: a randomised, non-inferiority, single-blind trial.
2018,
Pubmed
Bartos,
Glutamine 57 at the complementary binding site face is a key determinant of morantel selectivity for {alpha}7 nicotinic receptors.
2009,
Pubmed
Bartos,
Molecular determinants of pyrantel selectivity in nicotinic receptors.
2006,
Pubmed
Bartsch,
The Global Economic and Health Burden of Human Hookworm Infection.
2016,
Pubmed
Behnke,
Sensitivity to ivermectin and pyrantel of Ancylostoma ceylanicum and Necator americanus.
1993,
Pubmed
Bennett,
Xenopus laevis RIC-3 enhances the functional expression of the C. elegans homomeric nicotinic receptor, ACR-16, in Xenopus oocytes.
2012,
Pubmed
,
Xenbase
Blum,
Nicotinic pharmacophore: the pyridine N of nicotine and carbonyl of acetylcholine hydrogen bond across a subunit interface to a backbone NH.
2010,
Pubmed
,
Xenbase
Blum,
Global "worming": Climate change and its projected general impact on human helminth infections.
2018,
Pubmed
Botero,
Comparative study of pyrantel pamoate, bephenium hydroxynaphthoate, and tetrachloroethylene in the treatment of Necator americanus infections.
1973,
Pubmed
Boulin,
Eight genes are required for functional reconstitution of the Caenorhabditis elegans levamisole-sensitive acetylcholine receptor.
2008,
Pubmed
,
Xenbase
Boulin,
Functional reconstitution of Haemonchus contortus acetylcholine receptors in Xenopus oocytes provides mechanistic insights into levamisole resistance.
2011,
Pubmed
,
Xenbase
Buxton,
Investigation of acetylcholine receptor diversity in a nematode parasite leads to characterization of tribendimidine- and derquantel-sensitive nAChRs.
2014,
Pubmed
,
Xenbase
Celie,
Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures.
2004,
Pubmed
Charvet,
Nicotine-sensitive acetylcholine receptors are relevant pharmacological targets for the control of multidrug resistant parasitic nematodes.
2018,
Pubmed
,
Xenbase
Choudhary,
Pharmacological characterization of a homomeric nicotinic acetylcholine receptor formed by Ancylostoma caninum ACR-16.
2019,
Pubmed
,
Xenbase
Courtot,
Functional Characterization of a Novel Class of Morantel-Sensitive Acetylcholine Receptors in Nematodes.
2015,
Pubmed
,
Xenbase
Cully,
Solubilization and characterization of a high affinity ivermectin binding site from Caenorhabditis elegans.
1991,
Pubmed
Dougherty,
Cys-loop neuroreceptors: structure to the rescue?
2008,
Pubmed
Duerr,
Identified neurons in C. elegans coexpress vesicular transporters for acetylcholine and monoamines.
2001,
Pubmed
Duguet,
Recent Duplication and Functional Divergence in Parasitic Nematode Levamisole-Sensitive Acetylcholine Receptors.
2016,
Pubmed
,
Xenbase
Garcia,
Treatment for trichuriasis with oxantel.
1976,
Pubmed
Geary,
Caenorhabditis elegans: how good a model for veterinary parasites?
2001,
Pubmed
Grandemange,
Field evaluation of the efficacy and the safety of a combination of oxantel/pyrantel/praziquantel in the treatment of naturally acquired gastrointestinal nematode and/or cestode infestations in dogs in Europe.
2007,
Pubmed
Hansen,
The narrow-spectrum anthelmintic oxantel is a potent agonist of a novel acetylcholine receptor subtype in whipworms.
2021,
Pubmed
Hoagland,
Necator americanus and Ancylostoma duodenale: life history parameters and epidemiological implications of two sympatric hookworms of humans.
1978,
Pubmed
Hochedez,
Hookworm-related cutaneous larva migrans.
2007,
Pubmed
Hotez,
Human Parasitology and Parasitic Diseases: Heading Towards 2050.
2018,
Pubmed
Hotez,
Hookworm and poverty.
2008,
Pubmed
Howe,
WormBase ParaSite - a comprehensive resource for helminth genomics.
2017,
Pubmed
Howes,
Trans-1,4,5,6-tetrahydro-2-(3-hydroxystyryl)-1-methyl pyrimidine (CP-14,445), a new antiwhipworm agent.
1972,
Pubmed
Humphries,
Epidemiology of hookworm infection in Kintampo North Municipality, Ghana: patterns of malaria coinfection, anemia, and albendazole treatment failure.
2011,
Pubmed
Jimenez Castro,
Multiple drug resistance in the canine hookworm Ancylostoma caninum: an emerging threat?
2019,
Pubmed
Kearse,
Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data.
2012,
Pubmed
Keiser,
Activity of oxantel pamoate monotherapy and combination chemotherapy against Trichuris muris and hookworms: revival of an old drug.
2013,
Pubmed
Keiser,
Efficacy of current drugs against soil-transmitted helminth infections: systematic review and meta-analysis.
2008,
Pubmed
Kopp,
High-level pyrantel resistance in the hookworm Ancylostoma caninum.
2007,
Pubmed
Krepel,
Treatment of mixed Oesophagostomum and hookworm infection: effect of albendazole, pyrantel pamoate, levamisole and thiabendazole.
1993,
Pubmed
Lewis,
The genetics of levamisole resistance in the nematode Caenorhabditis elegans.
1980,
Pubmed
Li,
Ligand-binding domain of an α7-nicotinic receptor chimera and its complex with agonist.
2011,
Pubmed
Lummis,
Cis-trans isomerization at a proline opens the pore of a neurotransmitter-gated ion channel.
2005,
Pubmed
Martin,
Oxantel is an N-type (methyridine and nicotine) agonist not an L-type (levamisole and pyrantel) agonist: classification of cholinergic anthelmintics in Ascaris.
2004,
Pubmed
Martin,
Drug resistance and neurotransmitter receptors of nematodes: recent studies on the mode of action of levamisole.
2005,
Pubmed
Martin,
Mode of action of levamisole and pyrantel, anthelmintic resistance, E153 and Q57.
2007,
Pubmed
McKay,
eat-2 and eat-18 are required for nicotinic neurotransmission in the Caenorhabditis elegans pharynx.
2004,
Pubmed
Meldal,
An improved molecular phylogeny of the Nematoda with special emphasis on marine taxa.
2007,
Pubmed
Miledi,
Chloride current induced by injection of calcium into Xenopus oocytes.
1984,
Pubmed
,
Xenbase
Moser,
Efficacy and safety of oxantel pamoate in school-aged children infected with Trichuris trichiura on Pemba Island, Tanzania: a parallel, randomised, controlled, dose-ranging study.
2016,
Pubmed
Moser,
Efficacy and safety of tribendimidine, tribendimidine plus ivermectin, tribendimidine plus oxantel pamoate, and albendazole plus oxantel pamoate against hookworm and concomitant soil-transmitted helminth infections in Tanzania and Côte d'Ivoire: a randomised, controlled, single-blinded, non-inferiority trial.
2017,
Pubmed
Neveu,
Genetic diversity of levamisole receptor subunits in parasitic nematode species and abbreviated transcripts associated with resistance.
2010,
Pubmed
Nielsen,
Prediction of signal peptides and signal anchors by a hidden Markov model.
1998,
Pubmed
Okulewicz,
The impact of global climate change on the spread of parasitic nematodes.
2017,
Pubmed
Olsen,
Molecular recognition of the neurotransmitter acetylcholine by an acetylcholine binding protein reveals determinants of binding to nicotinic acetylcholine receptors.
2014,
Pubmed
Pan,
Structure of the pentameric ligand-gated ion channel ELIC cocrystallized with its competitive antagonist acetylcholine.
2012,
Pubmed
Pettersen,
UCSF Chimera--a visualization system for exploratory research and analysis.
2004,
Pubmed
Rayes,
Molecular basis of the differential sensitivity of nematode and mammalian muscle to the anthelmintic agent levamisole.
2004,
Pubmed
Raymond,
Anthelmintic actions on homomer-forming nicotinic acetylcholine receptor subunits: chicken alpha7 and ACR-16 from the nematode Caenorhabditis elegans.
2000,
Pubmed
,
Xenbase
Reynoldson,
Failure of pyrantel in treatment of human hookworm infections (Ancylostoma duodenale) in the Kimberley region of north west Australia.
1997,
Pubmed
Richards,
In vitro studies on the relative sensitivity to ivermectin of Necator americanus and Ancylostoma ceylanicum.
1995,
Pubmed
Rienzo,
Perturbation of Critical Prolines in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) Supports Conserved Gating Motions among Cys-loop Receptors.
2016,
Pubmed
,
Xenbase
Rim,
Anthelmintic effect of oxantel pamoate and pyrantel pamoate suspension against intestinal nematode infestations.
1975,
Pubmed
Rozen,
Primer3 on the WWW for general users and for biologist programmers.
2000,
Pubmed
Sali,
Comparative protein modelling by satisfaction of spatial restraints.
1993,
Pubmed
Schwarz,
The genome and transcriptome of the zoonotic hookworm Ancylostoma ceylanicum identify infection-specific gene families.
2015,
Pubmed
Soukhathammavong,
Low efficacy of single-dose albendazole and mebendazole against hookworm and effect on concomitant helminth infection in Lao PDR.
2012,
Pubmed
Speich,
Oxantel pamoate-albendazole for Trichuris trichiura infection.
2014,
Pubmed
Tang,
Genome of the human hookworm Necator americanus.
2014,
Pubmed
Touroutine,
acr-16 encodes an essential subunit of the levamisole-resistant nicotinic receptor at the Caenorhabditis elegans neuromuscular junction.
2005,
Pubmed
Traub,
Ancylostoma ceylanicum, a re-emerging but neglected parasitic zoonosis.
2013,
Pubmed
Tritten,
In vitro and in vivo efficacy of Monepantel (AAD 1566) against laboratory models of human intestinal nematode infections.
2011,
Pubmed
Tritten,
Comparison of novel and existing tools for studying drug sensitivity against the hookworm Ancylostoma ceylanicum in vitro.
2012,
Pubmed
Trott,
AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading.
2010,
Pubmed
Valley,
The methionine-aromatic motif plays a unique role in stabilizing protein structure.
2012,
Pubmed
Weaver,
Soil-transmitted helminthiases: implications of climate change and human behavior.
2010,
Pubmed
Williamson,
The nicotinic acetylcholine receptors of the parasitic nematode Ascaris suum: formation of two distinct drug targets by varying the relative expression levels of two subunits.
2009,
Pubmed
,
Xenbase
Zheng,
The Ascaris suum nicotinic receptor, ACR-16, as a drug target: Four novel negative allosteric modulators from virtual screening.
2016,
Pubmed
,
Xenbase
