Stable expression and functional characterization of a human nicotinic acetylcholine receptor with alpha 6 beta 2 properties: discovery of selective antagonists

BACKGROUND AND PURPOSE Despite growing evidence that inhibition of a6b2-containing (a6b2*) nicotinic acetylcholine receptors (nAChRs) may be beneficial for the therapy of tobacco addiction, the lack of good sources of a6b2*-nAChRs has delayed the discovery of a6b2-selective antagonists. Our aim was to generate a cell line stably expressing functional nAChRs with a6b2 properties, to enable pharmacological characterization and the identification of novel a6b2-selective antagonists. EXPERIMENTAL APPROACH Different combinations of the a6, b2, b3, chimeric a6/3 and mutant b3V273S 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 a6b2*-nAChRs was determined with 125I-epibatidine binding. KEY RESULTS Functional channels were detected after co-transfection of a6/3, b2 and b3V273S subunits, while all other subunit combinations failed to produce agonist-induced responses. Stably expressed a6/3b2b3V273S-nAChR pharmacology was unique, and clearly distinct from a4b2-, a3b4-, a7- and a1b1de-nAChRs. Antagonist potencies in inhibiting a6/3b2b3V273S-nAChRs was similar to their binding affinity for rat native a6b2*-nAChRs. Agonist affinities for a6b2*-nAChRs was higher than their potency in activating a6/3b2b3V273S-nAChRs, but their relative activities were equivalent. Focussed set screening at a6/3b2b3V273S-nAChRs, followed by cross-screening with the other nAChRs, led to the identification of novel a6b2-selective antagonists. CONCLUSIONS AND IMPLICATIONS We generated a mammalian cell line stably expressing nAChRs, with pharmacological properties similar to native a6b2*-nAChRs, and used it to identify novel non-peptide, low molecular weight, a6b2-selective antagonists. We also propose a pharmacophore model of a6b2 antagonists, which offers a starting point for the development of new smoking cessation agents.