Photoaffinity labeling of insect nicotinic acetylcholine receptors with a novel [H-3]azidoneonicotinoid

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel in the insect CNS and a target for major insecticides. Here we use photoaff inity labeling to approach the functional architecture of insect nAChRs. Tw o candidate 5-azido-6-chloropyridin-3-yl photoaffinity probes are evaluated for their receptor potencies: azidoneonicotinoid (AzNN) with an acyclic ni troguanidine moiety; azidodehydrothiacloprid. Compared to their non-azido p arents, both probes are of decreased potencies at Drosophila (fruit fly) an d Musca (housefly) receptors but AzNN retains full potency at the Myzus (ap hid) receptor. [H-3]AzNN was therefore radio-synthesized at high specific a ctivity (84 Ci/mmol) as a novel photoaffinity probe. [H-3]AzNN binds to a s ingle high-affinity site in Myzus that is competitively inhibited by imidac loprid and nicotine and further characterized as to its pharmacological pro file with various nicotinic ligands. [H-3]AzNN photoaffinity labeling of My zus and Homalodisca (leafhopper) detects a single radiolabeled peak in each case displaceable with imidacloprid and nicotine and with molecular masses corresponding to similar to 45 and similar to 56 kDa, respectively. The ph otoaffinity-labeled receptor in both Drosophila and Musca has imidacloprid- and nicotine-sensitive profiles and migrates at similar to 66 kDa. These p hotoaffinity-labeled polypeptides are considered to be the insecticide-bind ing subunits of native insect nAChRs.