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

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

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

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.</p