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 α6β2-containing (α6β2*) nicotinic acetylcholine receptors (nAChRs) may be beneficial for the therapy of tobacco addiction, the lack of good sources of α6β2*-nAChRs has delayed the discovery of α6β2-selective antagonists. Our aim was to generate a cell line stably expressing functional nAChRs with α6β2 properties, to enable pharmacological characterization and the identification of novel α6β2-selective antagonists. EXPERIMENTAL APPROACH Different combinations of the α6, β2, β3, chimeric α6/3 and mutant β3V273S 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 α6β2*-nAChRs was determined with 125I-epibatidine binding. KEY RESULTS Functional channels were detected after co-transfection of α6/3, β2 and β3V273S subunits, while all other subunit combinations failed to produce agonist-induced responses. Stably expressed α6/3β2β3V273S-nAChR pharmacology was unique, and clearly distinct from α4β2-, α3β4-, α7- and α1β1δe-nAChRs. Antagonist potencies in inhibiting α6/3β2β3V273S-nAChRs was similar to their binding affinity for rat native α6β2*-nAChRs. Agonist affinities for α6β2*-nAChRs was higher than their potency in activating α6/3β2β3V273S-nAChRs, but their relative activities were equivalent. Focussed set screening at α6/3β2β3V273S-nAChRs, followed by cross-screening with the other nAChRs, led to the identification of novel α6β2-selective antagonists. CONCLUSIONS AND IMPLICATIONS We generated a mammalian cell line stably expressing nAChRs, with pharmacological properties similar to native α6β2*-nAChRs, and used it to identify novel non-peptide, low molecular weight, α6β2-selective antagonists. We also propose a pharmacophore model of α6β2 antagonists, which offers a starting point for the development of new smoking cessation agents.