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

Glutamate-gated chloride channels (GluCls) are targets for the avermectin a nthelmintics. A family of five GluCl subunit genes encoding seven subunits has been identified in Caenorhabditis elegans. We have previously shown tha t two orthologous genes in the parasite, Haemonchus contortus, encode three GluCl subunits (HcGluCl beta, Hcgbr-2A and Hcgbr-2B) with high aminoacid i dentity (>80%) to their C. elegans counterparts. We amplified and cloned a further subunit cDNA, HcGluCl alpha, from H. contortus eggs. Sequence compa risons suggested that this subunit was closely related to, but not ortholog ous with, the C. elegans GluCl alpha1, alpha2 or alpha3/GBR-2 subunits (sim ilar to 55% amino-acid identity). The HcGluCl alpha cDNA from an ivermectin -resistant isolate contained no coding changes from the wild-type. All of t he known H. contortus GluCl cDNA clones were subcloned into the expression vector pcDNA3.1 and transiently expressed in COS-7 cells. As predicted by f unctional data from the C. elegans orthologues, the Hcgbr-2A and HcGluCl be ta 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 varie ty of avermectins, though cold ivermectin was the most potent inhibitor of [H-3] ivermectin binding. Picrotoxin, fipronil, glutamate and GABA all fail ed 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 Glu Cl subunits are likely to be conserved across species, but the repertoire a nd relative importance of those subunits may vary. (C) 2001 Elsevier Scienc e B.V. All rights reserved.