Avermectin binding in Caenorhabditis elegans. A two-state model for the avermectin binding site.

Specific binding sites for ivermectin (IVM; 22,23-dihydroavermectin-B1) were identified and characterized in a crude membrane fraction prepared from the nematode, Caenorhabditis elegans (C. elegans). Specific [3H]IVM binding was saturable with an apparent dissociation constant, Kd, of 0.26 nM and a receptor concentration of 3.53 pmol/mg protein. [3H]IVM binding in C. elegans was linear with tissue protein concentration, and optimal binding occurred within a pH range of 7.3 to 7.6. Kinetic analysis of the binding showed that the reaction proceeded by a two-step mechanism. Initially, a rapidly reversible complex was formed and, after additional incubation, this complex was transformed to a much more slowly reversible complex. Stereospecificity of [3H]IVM binding to C. elegans membranes was demonstrated by competition with a series of avermectin derivatives. The in vivo effects of IVM and its derivatives on C. elegans motility were concentration dependent and correlated well with their relative binding affinities. Several putative neurotransmitters including gamma-aminobutyric acid (GABA), carbamyl choline, taurine, glutamate and dopamine were tested and found to have no effect on IVM binding. Specific IVM binding sites were also identified in rat brain; however, the affinity was approximately 100-fold lower than that observed in C. elegans and stereospecificity studies demonstrated structural differences in the two binding sites. These results are the first direct demonstration of a specific IVM binding site in nematodes and thus are important in furthering our understanding of its mode of action.