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Br J Pharmacol
2022 Mar 01;1796:1264-1279. doi: 10.1111/bph.15703.
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Pharmacological characterization of novel heteromeric GluCl subtypes from Caenorhabditis elegans and parasitic nematodes.
Lamassiaude N, Courtot E, Corset A, Charvet CL, Neveu C.
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BACKGROUND AND PURPOSE: Macrocyclic lactones are the most widely used broad-spectrum anthelmintic drugs for the treatment of parasitic nematodes affecting both human and animal health. Macrocyclic lactones are agonists of the nematode glutamate-gated chloride channels (GluCls). However, for many important nematode species, the GluCls subunit composition and pharmacological properties remain largely unknown. To gain new insights into GluCl diversity and mode of action of macrocyclic lactones, we identified and pharmacologically characterized receptors made of highly conserved GluCl subunits from the model nematode Caenorhabditis elegans, the human filarial nematode Brugia malayi and the horse parasite Parascaris univalens.
EXPERIMENTAL APPROACH: AVR-14, GLC-2, GLC3 and GLC-4 are the most conserved GluCl subunits throughout the Nematoda phylum. For each nematode species, we investigated the ability of these subunits to form either homomeric or heteromeric GluCls when expressed in Xenopus laevis oocytes and carried out detailed pharmacological characterization of the functional channels.
KEY RESULTS: A total of 14 GluCls were functionally reconstituted, and heteromers formation was inferred from pharmacological criteria. The GLC-2 subunit plays a pivotal role in the composition of heteromeric GluCls in nematodes. We also found a novel GluCl subtype, combining GLC-2/GLC-3 subunits, for which a high concentration of the anthelmintics ivermectin and moxidectin reversibly potentiates glutamate-induced response.
CONCLUSION AND IMPLICATIONS: This study brings new insights into the diversity of GluCl subtypes in nematodes and promotes novel drug targets for the development of the next generation of anthelmintic compounds.
FIGURE 1. Maximal likelihood tree generated with GluCl subunit deduced amino‐acid sequences from Brugia malayi (Bma),
Caenorhabditis elegans
(Cel) and Parascaris univalens (Pun). The bootstrap values (100 replicates) are indicated at each node. Scale bar represents the number of substitutions per site. Accession numbers for the sequences used in this analysis are provided in Section 2. The B. malayi,
C. elegans
and P. univalens GluCl subunit sequences investigated in the present study are highlighted in green, red and blue, respectively. The GABA receptor subunit UNC‐49B from
C. elegans
was used as an outgroup
FIGURE 2. Functional expression of GluCl subunits from
Caenorhabditis elegans
, Brugia malayi and Parascaris univalens in
Xenopus laevis
oocytes. (a,b) Box plots depict response elicited by 1‐mM glutamate application on Xenopus oocytes expressing homomeric (a) or heteromeric (b) receptors for
C. elegans
, B. malayi and P. univalens. HR (high response) corresponds to subunit combinations that led to robust expression of receptors responding to 1‐mM glutamate with peak current in the μA range. LR (low response) corresponds to subunit combinations that respond to 1‐mM glutamate with moderate currents in the nA range. NR (no response) corresponds to combinations that did not respond to 1‐mM glutamate application
FIGURE 3. Glutamate concentration–response relationships for the AVR‐14B/GLC‐2 receptors from
Caenorhabditis elegans
, Brugia malayi and Parascaris univalens. Glutamate concentration–response curves for AVR‐14B/GLC‐2 (red), AVR‐14B (blue) and GLC‐2 (green) from C. elegans (a), B. malayi (b) and P. univalens (c). Current amplitudes were normalized to the maximal effect obtained with a saturating glutamate concentration. Solid line stands for the fitted log‐logistic regression curve, and shaded area indicates 95% confidence interval. For both
C. elegans
and B. malayi, the glutamate EC50s were significantly different (P<0.05) between the heteromeric AVR‐14B/GLC‐2 receptors and the respective homomeric AVR‐14 and GLC‐2 channels
FIGURE 4. Effects of macrocyclic lactones on AVR‐14B/GLC‐2 receptors from
Caenorhabditis elegans
, Brugia malayi and Parascaris univalens. (a,c,e) Representative recording traces from a single oocyte injected with AVR‐14B and GLC‐2 of
C. elegans
(a), B. malayi (b) and P. univalens (c) induced by 1‐μM ivermectin and 1‐μM emamectin after a first application of 1‐mM glutamate. Application times are indicated by the black bars. (b,d,f) Boxplots show the effects of the macrocyclic lactones (1 μM) after 5‐s application on Cel‐AVR‐14B/GLC‐2 (c), Bma‐AVR‐14B/GLC‐2 (d) and Pun‐AVR‐14B/GLC‐2 (e). All responses were normalized to the maximum responses obtained with glutamate at 1 mM. *P < 0.05, significantly different from effects of ivermectin
FIGURE 5. Glutamate concentration–response relationships for GLC‐2/GLC‐3 receptors from
Caenorhabditis elegans
and Parascaris univalens. (a,b) Glutamate concentration–response curves for
C. elegans
GLC‐2 and the GLC‐2/GLC‐3 combination (a); P. univalens GLC‐3 and the GLC‐2/GLC‐3 combination (b). Current amplitudes were normalized to the maximal effect obtained with glutamate. Solid line stands for the fitted log‐logistic regression curve, and shaded area indicates 95% confidence interval. For both nematode species, the glutamate EC50 values were significantly different between the heteromeric and homomeric receptors
FIGURE 6. Effects of ivermectin, moxidectin and eprinomectin on GLC‐2/GLC‐3 receptors from
Caenorhabditis elegans
and Parascaris univalens. Boxplots depict 1‐μM ivermectin, moxidectin or eprinomectin effects after 5‐s application on GLC‐2/GLC‐3 from
C. elegans
and P. univalens. All currents were normalized to 1‐mM glutamate‐elicited currents. Ivermectin, moxidectin or eprinomectin applications induced higher currents in GLC‐2/GLC‐3 receptors from
C. elegans. *P < 0.05, significantly different from
C. elegans
FIGURE 7. Modulation of glutamate effect by ivermectin and moxidectin on GLC‐2/GLC‐3 receptor from Parascaris univalens. (a,b) Representative current traces induced by glutamate 100 μM followed by a co‐application of ivermectin 1 μM (a) or moxidectin 1 μM (b) with glutamate 100 μM and a final glutamate 100 μM application to monitor a potential reversible effect. Application times are indicated by the black bars. (c,d) Boxplots of summary data showing potentiating effect of ivermectin (c) and moxidectin (d) on Pun‐GLC‐2/GLC‐3 normalized and compared with the response to 100‐μM glutamate. Note that the effects of the macrocyclic lactones were reversible on washout. *P < 0.05, significantly different as indicated
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