Mutations in the extracellular domains of glutamate‐gated chloride channel α3 and β subunits from ivermectin‐resistant Cooperia oncophora affect agonist sensitivity

Abstract

Two full-length glutamate-gated chloride channel (GluCl) cDNAs, encoding GluCla3 and GluClb subunits, were cloned from ivermectin-susceptible (IVS) and -resistant (IVR) Cooperia oncophora adult worms. The IVS and IVR GluCla3 subunits differ at three amino acid positions, while the IVS and IVR GluClb subunits differ at two amino acid positions. The aim of this study was to determine whether mutations in the IVR subunits affect agonist sensitivity. The subunits were expressed singly and in combination in Xenopus laevis oocytes. Electrophysiological whole-cell voltage-clamp recordings showed that mutations in the IVR GluCla3 caused a modest but significant threefold loss of sensitivity to glutamate, the natural ligand for GluCl receptors. As well, a significant decrease in sensitivity to the anthelmintics ivermectin and moxidectin was observed in the IVR GluCla3 receptor.Mutations in the IVR GluClb subunit abolished glutamate sensitivity. Co-expressing the IVS GluCla3 and GluClb subunits resulted in heteromeric channels that were more sensitive to glutamate than the respective homomeric channels, demonstrating co-assembly of the subunits. In contrast, the heteromeric IVR channels were less sensitive to glutamate than the homomeric IVR GluCla3 channels. The heteromeric IVS channels were significantly more sensitive to glutamate than the heteromeric IVR channels. Of the three amino acids distinguishing the IVS and IVR GluCla3 subunits, only one of them, L256F, accounted for the differences in response between the IVS and IVR GluCla3 homomeric channels.

Glutamate-gated Chloride Channels

“…Unfortunately, this deletion is within a part of the subunit, the N-terminal 40 amino acids, that had to be removed to produce the recombinant protein used for the structural studies (87), so how this deletion might reduce the effects of ivermectin on the channel is obscure. Mutations in the extracellular ␤10 strand, including the L256F mutation found in a drug-resistant isolate of the parasite Cooperia oncophora (85), and the M2-M3 linker region of AVR-14B affect the binding of ivermectin to the channel (83,93), consistent with the contacts observed between bound ivermectin and the M2-M3 linker in GLC-1 (87).…”

An Ivermectin-Sensitive Glutamate-Gated Chloride Channel from the Parasitic Nematode Haemonchus contortus

“…In both species, gene expression was detected in extrapharyngeal neurons in the head, sensory neurons, and ventral cord motor neurons and in H. contortus expression of the GluCl␣3A subunit was also detected in amphid neurons with the GluCl␣3B subunit found in three putative pharyngeal neurons. Mutations in avr-14 have been associated with avermectin resistance in C. elegans and C. oncophora (Dent et al, 2000;Njue et al, 2004). In C. oncophora, three amino acid differences, E114G, V235A, and L256F, were found between the GluCl␣3 subunits from avermectin-susceptible and -resistant worms.…”

“…The gene is alternatively spliced in most of these species to yield two subunits, GluCl␣3A and GluCl␣3B, that share a common N-terminal ligand-binding domain but differ in the C-terminal channel-forming domains. Previous studies on these subunits have indicated that the GluCl␣3B subunit can form channels that bind radiolabeled ivermectin (22,23-dihydroavermectin B1 a ) (Campbell et al, 1983) with very high affinity (Cheeseman et al, 2001) and are activated by L-glutamate-and ivermectin when expressed alone, but that the GluCl␣3A subunit cannot (Dent et al, 2000;Njue et al, 2004;Yates and Wolstenholme, 2004;Tandon et al, 2006).…”

“…The GluCl are members of the Cys-loop ligand-gated ion channel family, and there is a small family of genes encoding GluCl subunits in both freeliving and parasitic nematodes (Yates et al, 2003;Williamson et al, 2007). One member of this gene family that is widely conserved is avr-14, which has been found in Caenorhabditis elegans, Haemonchus contortus, Ascaris suum, Cooperia oncophora, the cyathostomins (parasites of horses), and the filarial parasites infecting humans and companion animals (Laughton et al, 1997;Jagannathan et al, 1999;Dent et al, 2000;Njue et al, 2004;Yates and Wolstenholme, 2004;Tandon et al, 2006). The gene is alternatively spliced in most of these species to yield two subunits, GluCl␣3A and GluCl␣3B, that share a common N-terminal ligand-binding domain but differ in the C-terminal channel-forming domains.…”

Molecular Determinants of Ivermectin Sensitivity at the Glycine Receptor Chloride Channel

“…Unfortunately, ivermectin resistance is emerging as a serious problem in nematodes and arthropods (4 -6). In several instances, resistance has been shown to be caused by mutations that reduce the GluClR ivermectin sensitivity (7)(8)(9). Insight into the binding mechanisms of ivermectin at the GluClR may contribute to the understanding of ivermectin resistance mechanisms and to the development of a much needed new generation of anthelmintic drugs.…”