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.

Keywords: Aca-ACR-16; Anthelmintic; Hookworms; Xenopus oocyte; nAChR.

Fig. 1

Amino acid sequence alignment of Aca-ACR-16 and Asu-ACR-16. The signal peptide (light brown), ligand binding loops (A to F; maroon) transmembrane regions TM1–4 (blue) and cys-loop (green) are indicated. The adjacent cysteines (in the Y–x–C–C motif) in loop-C are indicated in the black box. The negatively charged amino acids (E: Glutamic acid and D: Aspartic acid) flanking the TM2 domain are highlighted in orange. Residues involved in binding of α-BTX are highlighted in olive green. Note: The sequence of Aca-ACR-16 amplified from A. caninum larval total RNA is shorter than the WormBase sequence ANCCAN_01899 and lacks 19 amino acids (KVKEPNLFGPWENFHGDLF) between the cys-loop and loop-B. These amino acid residues are also lacking in the A. suum ACR-16 homologue

Fig. 2

Bar chart showing the effects of ancillary proteins on the expression of Aca-ACR-16 nAChR (n ≥ 6). The receptor was able to express functionally only when co-injected with Asu-RIC-3. Final column represents un-injected control oocytes

Fig. 3

Effects of nAChR agonists and antiparasitic drugs on the Aca-ACR-16 receptor. a Bar graph (mean ± SEM, %, n = 4) along with sample traces showing the effect of agonists and anthelmintics on the nAChR. The rank-order potency series when normalized to the control 100 μM ACh current was as follows: 3-bromocytisine (3-BC; 131.0 ± 18.0) > ACh (100.0 ± 0.0) > epibatidine (epi; 85.0 ± 4.0) > cytisine (cyt; 48.0 ± 9.5) > nicotine (nic; 37.0 ± 8.7) > DMPP (dimethyl-4-phenylpiperazinium; 27.0 ± 6.4) ⋙ levamisole (lev; 0.0 ± 0.0) = oxantel (oxan; 0.0 ± 0.0) = pyrantel (pyr; 0.0 ± 0.0) = morantel (mor; 0.0 ± 0.0) = choline (cho; 0.0 ± 0.0) = bephenium (beph; 0.0 ± 0.0) = tribendimidine (tbd; 0.0 ± 0.0). b Sample traces and concentration-response relationships of 3-bromocytisine and ACh for Ancylostoma caninum ACR-16. The pEC₅₀ and hill slope (n_H) values, expressed as mean ± SEM, were, respectively, 4.3 ± 0.0 and 2.5 ± 0.3 for ACh (n = 6); 5.0 ± 0.1 and 2.4 ± 0.7 for 3-BC (n = 6)

Fig. 4

Aca-ACR-16 desensitization rate constant fit. a Sample trace of response to agonist with green line signifying the desensitization fit. b Bar graph showing desensitization time constants of the Ancylostoma caninum ACR-16 nAChR in response to agonists (100 μM, n = 4). The rank order of time constants of desensitization (mean ± SEM, s) was as follows: epi (4.8 ± 0.2) > nic (4.3 ± 0.5) > cyt (3.9 ± 0.5) > ACh (3.3 ± 1.0) > 3-BC (1.5 ± 0.3). *P < 0.05, **P < 0.01; significantly different as indicated; Tukey’s multiple comparison test

Fig. 5

Effects of selected nAChR antagonists on the Aca-ACR-16. Sample trace and bar chart showing inhibition (mean ± SEM, %; n = 6) of acetylcholine-mediated currents by selected antagonists (10 μM). d-tubocurarine (d-TC), mecamylamine (mec) and dihydro-β-erythroidine (DHβE) produced almost complete inhibition of ACh mediated responses. Derquantel (der) and hexamethonium (hexa) produced moderate blockade of Aca-ACR-16 mediated ACh responses and α-BTX was the least potent antagonist. The rank-order potency series for nAChR antagonists is as follows: d-TC (100.0 ± 0.1) ≈ mec (98.8 ± 0.6) ≈ DHβE (98.8 ± 0.4) > der (72.0 ± 5.6) > hexa (52.2 ± 5.6) ≈ α-BTX (49.3 ± 5.2). Inset: magnified view of current trace showing predicted acetylcholine response in the absence of DHβE (dotted line) and inhibition of acetylcholine-mediated response in the presence of DHβE (highlighted in blue)