Pharmacological characterization of a homomeric nicotinic acetylcholine receptor formed by Ancylostoma caninum ACR-16

Abstract

Parasitic nematode infections are treated using anthelmintic drugs, some of which target nicotinic acetylcholine receptors (nAChRs) located in different parasite tissues. The limited arsenal of anthelmintic agents and the prevalence of drug resistance imply that future defense against parasitic infections will depend on the discovery of novel targets and therapeutics. Previous studies have suggested that Ascaris suum ACR-16 nAChRs are a suitable target for the development of antinematodal drugs. In this study, we characterized the pharmacology of the Ancylostoma caninum ACR-16 receptor using two-electrode voltage-clamp electrophysiology. This technique allowed us to study the effects of cholinergic agonists and antagonists on the nematode nAChRs expressed in Xenopus laevis oocytes. Aca-ACR-16 was not sensitive to many of the existing cholinomimetic anthelmintics (levamisole, oxantel, pyrantel, and tribendimidine). 3-Bromocytisine was the most potent agonist (> 130% of the control acetylcholine current) on the Aca-ACR-16 nAChR but, unlike Asu-ACR-16, oxantel did not activate the receptor. The mean time constants of desensitization for agonists on Aca-ACR-16 were longer than the rates observed in Asu-ACR-16. In contrast to Asu-ACR-16, the A. caninum receptor was completely inhibited by DHβE and moderately inhibited by α-BTX. In conclusion, we have successfully reconstituted a fully functional homomeric nAChR, ACR-16, from A. caninum, a model for human hookworm infections. The pharmacology of the receptor is distinct from levamisole-sensitive nematode receptors. The ACR-16 homologue also displayed some pharmacological differences from Asu-ACR-16. Hence, A. caninum ACR-16 may be a valid target site for the development of anthelmintics against hookworm infections.

A Functional Comparison of Homopentameric Nicotinic Acetylcholine Receptors (ACR-16) Receptors From Necator americanus and Ancylostoma ceylanicum

“…The human hookworm ACR-16 receptors expressed relatively quickly, producing currents on a timescale and magnitude similar to those reported for other ACR-16s (Raymond et al, 2000;Abongwa et al, 2016;Charvet et al, 2018;Choudhary et al, 2019). Maximal responses were detected 48 hr post-cRNA injection and currents were detectable until oocyte quality degraded to the point of loss of membrane integrity, roughly 4-5 days after injection.…”

“…Another unexpected difference is that A. ceylanicum ACR-16 responses were in the large microamp range, whereas the N. americanus ACR-16 receptor generated currents in the nanoamp to small microamp range. Yet, the amplitude of A. caninum ACR-16 responses reported by Choudhary et al (2019) better match with those from N. americanus rather than A. ceylaniucm from its own genus. Furthermore, N. americanus ACR-16 failed to achieve maximal channel activation from nicotine (similar to Aca-and Cel-ACR-16, but dissimilar to Ace-, Peq-and Asu-ACR-16), and nicotine was ∼fivefold less potent than acetylcholine, in contrast to the other published ACR-16s.…”

“…Nematode AChRs include 4 subtypes, B-L-M-and N-AChRs, characterized by their subunit composition and preferential sensitivity to bephenium, levamisole, morantel and nicotine, respectively (Martin et al, 2005;Courtot et al, 2015). The major N-type AChR subunit is encoded by acr-16, which forms functional homopentameric receptors in the presence of the accessory protein RIC-3 (Bennett et al, 2012), and has been characterized from C. elegans (Ballivet et al, 1996;Raymond et al, 2000), Ascaris suum (Abongwa et al, 2016), Parascaris equorum (Charvet et al, 2018) and the dog hookworm Ancylostoma caninum (Choudhary et al, 2019). In C. elegans these receptors are expressed in body wall muscles and, along with the L-AChR, contribute to the acetylcholine response (Touroutine et al, 2005).…”

Caenorhabditis elegans muscle Cys-loop receptors as novel targets of terpenoids with potential anthelmintic activity

“…N-AChRs may also participate in terpenoid actions since the mutant worms lacking ACR-16 are more resistant than wild-type worms but this occurs only at low terpenoid concentrations. Interestingly, Ancylostoma caninum ACR-16 has been recently proposed as a valid target site for the development of anthelmintics against hookworm infections [39].…”

Ligand-gated ion channels: Putative target sites for anthelmintic therapy in muscle and intestine cells of parasitic nematodes

“…Heterologous expression of A. caninum ACR-16 in Xenopus oocytes showed that the receptor was insensitive to oxantel (Choudhary et al, 2019). In another study A. americanus and A.…”