Photoaffinity labeling of insect nicotinic acetylcholine receptors with a novel [3H]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 photoaffinity labeling to approach the functional architecture of insect nAChRs. Two candidate 5-azido-6-chloropyridin-3-yl photoaffinity probes are evaluated for their receptor potencies: azidoneonicotinoid (AzNN) with an acyclic nitroguanidine moiety; azidodehydrothiacloprid. Compared to their non-azido parents, both probes are of decreased potencies at Drosophila (fruit fly) and Musca (housefly) receptors but AzNN retains full potency at the Myzus (aphid) receptor. [3H]AzNN was therefore radiosynthesized at high specific activity (84 Ci/mmol) as a novel photoaffinity probe. [3H]AzNN binds to a single high-affinity site in Myzus that is competitively inhibited by imidacloprid and nicotine and further characterized as to its pharmacological profile with various nicotinic ligands. [3H]AzNN photoaffinity labeling of Myzus and Homalodisca (leafhopper) detects a single radiolabeled peak in each case displaceable with imidacloprid and nicotine and with molecular masses corresponding to ∼45 and ∼56 kDa, respectively. The photoaffinity-labeled receptor in both Drosophila and Musca has imidacloprid- and nicotine-sensitive profiles and migrates at ∼66 kDa. These photoaffinity-labeled polypeptides are considered to be the insecticide-binding subunits of native insect nAChRs.