Stable expression and functional characterization of a human nicotinic acetylcholine receptor with α6β2 properties: discovery of selective antagonists

BACKGROUND AND PURPOSE Despite growing evidence that inhibition of alpha 6 beta 2-containing (alpha 6 beta 2*) nicotinic acetylcholine receptors (nAChRs) may be beneficial for the therapy of tobacco addiction, the lack of good sources of alpha 6 beta 2*-nAChRs has delayed the discovery of alpha 6 beta 2-selective antagonists. Our aim was to generate a cell line stably expressing functional nAChRs with alpha 6 beta 2 properties, to enable pharmacological characterization and the identification of novel alpha 6 beta 2-selective antagonists. EXPERIMENTAL APPROACH Different combinations of the alpha 6, beta 2, beta 3, chimeric alpha 6/3 and mutant beta 3V273S subunits were transfected in human embryonic kidney cells and tested for activity in a fluorescent imaging plate reader assay. The pharmacology of rat immune-immobilized alpha 6 beta 2*-nAChRs was determined with 125I-epibatidine binding. KEY RESULTS Functional channels were detected after co-transfection of alpha 6/3, beta 2 and beta 3V273S subunits, while all other subunit combinations failed to produce agonist-induced responses. Stably expressed alpha 6/3 beta 2 beta 3V273S-nAChR pharmacology was unique, and clearly distinct from alpha 4 beta 2-, alpha 3 beta 4-, alpha 7- and alpha 1 beta 1 delta epsilon-nAChRs. Antagonist potencies in inhibiting alpha 6/3 beta 2 beta 3V273S-nAChRs was similar to their binding affinity for rat native alpha 6 beta 2*-nAChRs. Agonist affinities for alpha 6 beta 2*-nAChRs was higher than their potency in activating alpha 6/3 beta 2 beta 3V273S-nAChRs, but their relative activities were equivalent. Focussed set screening at alpha 6/3 beta 2 beta 3V273S-nAChRs, followed by cross-screening with the other nAChRs, led to the identification of novel alpha 6 beta 2-selective antagonists. CONCLUSIONS AND IMPLICATIONS We generated a mammalian cell line stably expressing nAChRs, with pharmacological properties similar to native alpha 6 beta 2*-nAChRs, and used it to identify novel non-peptide, low molecular weight, alpha 6 beta 2-selective antagonists. We also propose a pharmacophore model of alpha 6 beta 2 antagonists, which offers a starting point for the development of new smoking cessation agents.