Functional expression of a glutamate-gated chloride channel (GLC-3) from adult Brugia malayi.

Date
2018-01-01
Authors
Akanji, Bukunmi
Abongwa, Melanie
Martin, Richard
Robertson, Alan
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Abstract

Parasitic worms are causative agents for six highly prevalent neglected tropical diseases of humans which include ascariasis, lymphatic filariasis, schistosomiasis, trichuriasis, hookworm infection, and onchocerciasis that affect a high percentage of the world’s population. Resistance to available anthelminthic drugs especially for the benzimidazole anthelmintic agents (e.g., albendazole and mebendazole) and ivermectin is a serious concern (this is a real problem for veterinary medicine and a growing concern in human medicine). Previous studies involving ivermectin have shown that it has a limited action against adult filarial worms. Here, we describe the functional expression of a glutamate-gated chloride channel (GLC-3) from adult Brugia malayi, a filarial parasite. We expressed GLC-3 in Xenopus laevis oocytes and used two-electrode voltage-clamp electrophysiology to study the resulting ion channel. Application of various receptor agonists (1 mM): L-aspartate, glycine, γ-aminobutyric acid (GABA), α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) failed to activate the GLC3 receptor. Application of 1mM L-glutamate and ibotenate produced robust inward currents. Further experiments revealed that GLC-3 is a glutamate-gated channel with an EC50 of 64.8  4.01 µM and a Hill coefficient of 2.56  0.46 µM. We recorded the responses to 300 µM L-glutamate over a range of holding potentials (-80 to +20 mV) and constructed a current-voltage plot. The current-voltage relationship revealed a reversal potential (Erev) of -35.3  3.2 mV indicating the GLC-3 channel is selective for chloride ions. The GluCl channel antagonists picrotoxin and fipronil had negligible inhibitory effects on the L-glutamate responses. We investigated the ivermectin effects on the GLC-3 receptor which reveals ivermectin to be an agonist with an EC50 of 3.6 nM (pEC50 = -2.4 ± 0.4). Interestingly, we also observed that Ivermectin has an inhibitory effect on the GLC-3 glutamate response at very low concentrations, IC50 = 73 pM (pIC50 = -4.1 ± 11.4). This is in stark contrast to the previously reported positive allosteric (PAM) effects of ivermectin on many invertebrate GluCls.

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: Glutamate-gated chloride channel; Brugia malayi; Ivermectin, Picrotoxin; Fipronil; Two-electrode voltage-clamp electrophysiology. Abbreviations: GluCls, glutamate-gated chloride channels; ML, macrocyclic lactone; LGICs, ligand-gated ion channels; Bma, Brugia malayi; I-V, current-voltage; Erev, reversal potential; PCR, polymerase chain reaction; NCBI, National Center for Biotechnology Information; NEB, New England BioLabs; GABAA, γ-aminobutyric acid type A; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; NMDA, N-methyl-D-aspartate; IVM, ivermectin; PTX, picrotoxin; FIP, fipronil; DMSO, dimethyl sulfoxide.
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