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Front Mol Neurosci
2020 Jan 01;13:601102. doi: 10.3389/fnmol.2020.601102.
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A Functional Comparison of Homopentameric Nicotinic Acetylcholine Receptors (ACR-16) Receptors From Necator americanus and Ancylostoma ceylanicum.
Kaji MD, Geary TG, Beech RN.
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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.
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