High-affinity ivermectin binding to recombinant subunits of the Haemonchus contortus glutamate-gated chloride channel

Glutamate-gated chloride channels (GluCls) are targets for the avermectin anthelmintics. A family of five GluCl subunit genes encoding seven subunits has been identified in Caenorhabditis elegans. We have previously shown that two orthologous genes in the parasite, Haemonchus contortus, encode three GluCl subunits (HcGluCl beta, Hcgbr-2A and Hcgbr-2B) with high aminoacid identity (>80%) to their C. elegans counterparts. We amplified and cloned a further subunit cDNA, HcGluCl alpha, from H. contortus eggs. Sequence comparisons suggested that this subunit was closely related to, but not orthologous with, the C. elegans GluCl alpha1, alpha2 or alpha3/GBR-2 subunits (similar to 55% amino-acid identity). The HcGluCl alpha cDNA from an ivermectin-resistant isolate contained no coding changes from the wild-type. All of the known H. contortus GluCl cDNA clones were subcloned into the expression vector pcDNA3.1 and transiently expressed in COS-7 cells. As predicted by functional data from the C. elegans orthologues, the Hcgbr-2A and HcGluCl beta subunits failed to bind [H-3]ivermectin. The Hcgbr-2B and HcGluCl alpha subunits bound [H-3]ivermectin with high affinity; the K-d values were 70 +/- 16 and 26 +/- 12 pM, respectively. This binding was inhibited by a variety of avermectins, though cold ivermectin was the most potent inhibitor of [H-3] ivermectin binding. Picrotoxin, fipronil, glutamate and GABA all failed to compete for ivermectin binding to either subunit. The affinity of [H-3]ivermectin binding to H. contortus L3 P2 larval membrane preparations was re-examined and found to be 70 +/- 7 pM. The properties of orthologous GluCl subunits are likely to be conserved across species, but the repertoire and relative importance of those subunits may vary. (C) 2001 Elsevier Science B.V. All rights reserved.