Desnitroimidacloprid and nicotine binding site in rat recombinant alpha4beta2 neuronal nicotinic acetylcholine receptor

Desnitroimidacloprid (desnitro-IMI) is proposed to be a bioactivation product of imidacloprid and to bind at the same site as [(3)H]nicotine in the nicotinic acetylcholine receptor (nAChR) or mouse brain membranes. The alpha4beta2 nAChR subtype accounts for >90% of the binding sites for nicotine in rat brain. This study further characterizes the binding site for [(3)H]desnitro-IMI and [(3)H]nicotine in rat recombinant alpha4beta2 nAChR using receptor expressed in Sf9 insect cells so that the assays involved no other receptor subtypes or interference from metabolic activation and detoxification systems. The two radioligands gave the same B(max) of 7.5 pmol/mg protein and apparent K(d) values of 3.3 nM for nicotine and 8.9 nM for desnitro-IMI by Scatchard analysis at 22 degrees C. However, at 4 degrees, the observed apparent association rate is slower and dissociation rate is faster for [(3)H]desnitro-IMI than for [(3)H]nicotine and due to the rapid rate of dissociation of [(3)H]desnitro-IMI the K(d) calculated from the determined association and dissociation rates more closely approximates 1.0 for both ligands. Eight cholinergic agents and nine nicotinoids are equipotent in displacing [(3)H]desnitro-IMI and [(3)H]nicotine, with IC(50) values (nM) of 0.5 for epibatidine, 1 for cytisine, 4-6 for nicotine and desnitro-IMI, 15 for acetylcholine, and 155 for imidacloprid, with an overall correlation for inhibitor potencies of r(2) = 0.99 (n = 17). This correlation of binding site properties extends to [(3)H]nicotine in the recombinant alpha4beta2 receptor and rat brain membranes (r(2) = 0.99, n = 12). Thus, desnitro-IMI and nicotine bind with high affinity to the same site in rat recombinant alpha4beta2 neuronal nAChR. This recombinant receptor can be generated in sufficient quantities for high-throughput target site screening and structural analysis of the binding site.