Capelli_2011_Br.J.Pharmacol_163_313

BACKGROUND AND PURPOSE: Despite growing evidence that inhibition of alpha6beta2-containing (alpha6beta2*) nicotinic acetylcholine receptors (nAChRs) may be beneficial for the therapy of tobacco addiction, the lack of good sources of alpha6beta2*-nAChRs has delayed the discovery of alpha6beta2-selective antagonists. Our aim was to generate a cell line stably expressing functional nAChRs with alpha6beta2 properties, to enable pharmacological characterization and the identification of novel alpha6beta2-selective antagonists. EXPERIMENTAL APPROACH: Different combinations of the alpha6, beta2, beta3, chimeric alpha6/3 and mutant beta3(V273S) 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 alpha6beta2*-nAChRs was determined with (1)(2)(5)I-epibatidine binding. KEY
RESULTS: Functional channels were detected after co-transfection of alpha6/3, beta2 and beta3(V273S) subunits, while all other subunit combinations failed to produce agonist-induced responses. Stably expressed alpha6/3beta2beta3(V273S)-nAChR pharmacology was unique, and clearly distinct from alpha4beta2-, alpha3beta4-, alpha7- and alpha1beta1deltaepsilon-nAChRs. Antagonist potencies in inhibiting alpha6/3beta2beta3(V273S) -nAChRs was similar to their binding affinity for rat native alpha6beta2*-nAChRs. Agonist affinities for alpha6beta2*-nAChRs was higher than their potency in activating alpha6/3beta2beta3(V273S)-nAChRs, but their relative activities were equivalent. Focussed set screening at alpha6/3beta2beta3(V273S)-nAChRs, followed by cross-screening with the other nAChRs, led to the identification of novel alpha6beta2-selective antagonists. CONCLUSIONS AND IMPLICATIONS: We generated a mammalian cell line stably expressing nAChRs, with pharmacological properties similar to native alpha6beta2*-nAChRs, and used it to identify novel non-peptide, low molecular weight, alpha6beta2-selective antagonists. We also propose a pharmacophore model of alpha6beta2 antagonists, which offers a starting point for the development of new smoking cessation agents.