High affinity binding of [³h]imidacloprid in the insect acetylcholine receptor

1-(6-Chloronicotinyl)-2-nitroimino-imidazolidine (imidacloprid or IMI) is a potent insecticide of a new chemical class considered from earlier studies to act at the insect nicotinic acetylcholine receptor. In a direct approach to the mode of action of IMI, it was radiolabeled and [³H]IMI was examined in binding studies to elucidate its pharmacological profile. [³H]IMI undergoes high affinity specific binding in house fly head P₂ membranes with 95% specific binding, a dissociation constant of 1.2 nM, and a maximal binding site capacity of 853 fmol/mg protein. The standard binding assay consisted of 1 nM [³H]IMI and 200 μg membrane protein in 50 mM NaCl, 10 mM sodium phosphate (pH 7.4) containing 0.1% Triton X-100 with incubation for 60 min at 22°C prior to filtration. The radioligand undergoes rapid biphasic association and dissociation consistent with a two-stage sequential reaction in each case. [³H]IMI binding is very sensitive to carbachol (IC₅₀ 1.9 μM) and other choline esters (IC₅₀s 0.2 to 0.5 μM for acetylcholine, propionylcholine, and butyrylcholine in the presence of paraoxon as a cholinesterase inhibitor). The pharmacological profile for [³H]IMI binding indicates inhibition by both nicotinic and muscarinic agents with IC₅₀s of 0.6 μM for (-)-nicotine, 2.2 μM for α-bungarotoxin, 30 μM for D-tubocurarine, 90 μM for atropine, 275 μM for quinuclidinyl benzilate, and 288 μM for dexetimide. Lineweaver-Burk plots establish competitive inhibition kinetics for [³H]IMI with acetylcholine, α-bungarotoxin, and quinuclidinyl benzilate. Detection of [³H]IMI binding in membrane preparations from several insects but not from the vertebrates examined is consistent with the selective toxicity of the nitromethylene and nitroimine insecticides.