[H-3]labeled affinity and photoaffinity nicotine analogues for probing brain nicotinic cholinergic receptors

A series of [H-3]labeled nicotine affinity ligands have been prepared as part of an effort to probe the nicotine binding sire on brain nicotinic cholinergic receptors (nAChR). The compounds included [H-3-1'](R,S)-5-isothiocyanonicotine, which possessed an affinity in the nmolar range, and three photoaffinity agents: [H-3-1'](R,S)-5-azidonicotine, [H-3-1'](R,S)-5-iodonicotine, and [H-3-1'](R,S)-5-aminonicotine. The nicotine analogues were evaluated for reversible and irreversible inhibition of brain nicotinic and muscarinic cholinergic receptor binding and for their interaction with various receptor subtypes. Plots of the irreversible inhibiton of [H-3]methylcarbamylcholine ([H-3]MCC) binding <(vs)under bar> concentration of unlabled (R,S)-5-isothiocyanonicotine with and without dithiothreitol revealed IC50 values of 2 x 10(-9) M and 1 x 10(-7) M, respectively, with complete inhibition of both Ligands at 1 x 10(-5) M. Photolysis of membrane preparations in the presence of 10 mu M unlabeled 5-iodonicotine, 5-azidonicotine, and 5-aminonicotine also resulted in inhibition of [H-3]MCC binding. Both [H-3-1'](R,S)-5-iodononicotine and [H-3-1'](R,S)-5-aminonicotine readily entered the brain and to a comparable degree. Aurofluorographic studies on rat brain membranes labeled with [H-3-1'](R,S)-5-isothiocyanonicotine and separated by SDS/PAGE revealed specific radiolabelling of an 84 kd protein which compared favorably with the molecular weight reported for the alpha(4) subunit of neuronal nAChR. [H-3]SCN-N and the photoaffinity nicotine analogues should prove useful in mapping the site of attachement for nicotine on the alpha subunit.