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

Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, United Kingdom (S.M., A.T.R., D.M.Y., A.J.W.); and Pfizer Animal Health, Kalamazoo, Michigan (D.J.W.)

Nematode glutamate-gated chloride channels are targets of the macrocyclic lactones, the most important group of anthelmintics available. In Xenopus laevis oocytes, channels formed by the GluCl3B subunit from the parasite Haemonchus contortus were more sensitive to L-glutamate (EC₅₀ = 27.6 ± 2.7 µM) than those formed by the homologous subunit from Caenorhabditis elegans (EC₅₀ = 2.2 ± 0.12 mM). Ibotenate was a partial agonist (EC₅₀ = 87.7 ± 3.5 µM). The H. contortus channels responded to low concentrations of ivermectin (estimated EC₅₀ =0.1 ± 1.0 nM), opening slowly and irreversibly in a highly cooperative manner: the rate of channel opening was concentration-dependent. Responses to glutamate and ivermectin were inhibited by picrotoxinin and fipronil. Mutating an N-terminal domain amino acid, leucine 256, to phenylalanine increased the EC₅₀ for L-glutamate to 92.2 ± 3.5 µM, and reduced the Hill number from 1.89 ± 0.35 to 1.09 ± 0.16. It increased the K_d for radiolabeled ivermectin binding from 0.35 ± 0.1 to 2.26 ± 0.78 nM. Two other mutations (E114G and V235A) had no effect on L-glutamate activation or ivermectin binding: one (T300S) produced no detectable channel activity, but ivermectin binding was similar to wild-type. The substitution of any aromatic amino acid for Leu256 had similar effects in the radioligand binding assay. Molecular modeling studies suggested that the GluCl subunits have a fold similar to that of other Cys-loop ligand-gated ion channels and that amino acid 256 was unlikely to play a direct role in ligand binding but may be involved in mediating the allosteric properties of the receptor.

Received for publication November 7, 2008.

Accepted for publication March 24, 2009.

Address correspondence to: A. J. Wolstenholme, Dept of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK. E-mail: a.j.wolstenholme{at}bath.ac.uk