SOLUBILIZATION AND CHARACTERIZATION OF A HIGH-AFFINITY IVERMECTIN BINDING-SITE FROM CAENORHABDITIS-ELEGANS

Ivermectin is a member of the avermectin family of compounds that are used to treat helminth and arthropod diseases in humans, domestic animals, and plants. A membrane-bound high affinity ivermectin binding site was extracted from Caenorhabditis elegans with the nonionic detergent 1-O-n-octyl-beta-D-glucopyranoside. The free-living nematode C. elegans is highly sensitive to the avermectins and was used as a model of parasitic nematodes. The membrane-bound and detergent-solubilized ivermectin binding sites are stable and exhibit high affinity binding, with dissociation constants of 0.11 nM and 0.20 nM, respectively. The maximum binding of [H-3]ivermectin is 0.54 pmol/mg of membrane protein and 0.66 pmol/mg of detergent-soluble protein. Kinetic analysis of ivermectin binding shows that the ivermectin binding sites form a slowly reversible complex with ivermectin. The rates of dissociation of [H-3]ivermectin with the solubilized and membrane-bound binding sites are 0.005 min-1 and 0.006 min-1, respectively. The association rate of the soluble binding site is 0.053 nM-1 min-1, slightly slower than that observed for the membrane-bound site, 0.074 nM-1 min-1. To characterize the ivermectin binding site, competition experiments were performed by inhibiting [H-3]ivermectin binding with several avermectin derivatives and the neurotransmitter gamma-aminobutyric acid (GABA). The order of potency was 22,23-dihydroavermectin B1a monosaccharide > 22,23-dihydroavermectin B1a aglycone > 3,4,8,9,10,11,22,23-octahydro B1 avermectin for both the membrane-bound and NOG-soluble binding sites. GABA did not compete with ivermectin binding, although it has been suggested that ivermectin acts at the GABA-gated chloride channel in some ivertebrate systems. Optimum ivermectin binding and assay conditions have been determined. The detergent-soluble ivermectin binding site appears to be negatively charged and has a pl of 4.0 and an apparent M(r) in Triton X-100 micelles of 340,000. Detergent solubilization of a high affinity ivermectin binding site will enable the subsequent purification and characterization of a putative site of ivermectin action.