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

ABSTRACT: BACKGROUND AND PURPOSE: Despite growing evidence that inhibition of ?6?2-containing (?6?2*) nicotinic acetylcholine receptors (nAChRs) may be beneficial for the therapy of tobacco addiction, the lack of good sources of ?6?2*-nAChRs has delayed the discovery of ?6?2-selective antagonists. Our aim was to generate a cell line stably expressing functional nAChRs with ?6?2 properties, to enable pharmacological characterization and the identification of novel ?6?2-selective antagonists. EXPERIMENTAL APPROACH: Different combinations of the ?6, ?2, ?3, chimeric ?6/3 and mutant ?3(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 ?6?2*-nAChRs was determined with ¹²?I-epibatidine binding. KEY RESULTS: Functional channels were detected after co-transfection of ?6/3, ?2 and ?3(V273S) subunits, while all other subunit combinations failed to produce agonist-induced responses. Stably expressed ?6/3?2?3(V273S)-nAChR pharmacology was unique, and clearly distinct from ?4?2-, ?3?4-, ?7- and ?1?1??-nAChRs. Antagonist potencies in inhibiting ?6/3?2?3(V273S) -nAChRs was similar to their binding affinity for rat native ?6?2*-nAChRs. Agonist affinities for ?6?2*-nAChRs was higher than their potency in activating ?6/3?2?3(V273S)-nAChRs, but their relative activities were equivalent. Focussed set screening at ?6/3?2?3(V273S)-nAChRs, followed by cross-screening with the other nAChRs, led to the identification of novel ?6?2-selective antagonists. CONCLUSIONS AND IMPLICATIONS: We generated a mammalian cell line stably expressing nAChRs, with pharmacological properties similar to native ?6?2*-nAChRs, and used it to identify novel non-peptide, low molecular weight, ?6?2-selective antagonists. We also propose a pharmacophore model of ?6?2 antagonists, which offers a starting point for the development of new smoking cessation agents.

SUBMITTER: Capelli AM 

PROVIDER: S-EPMC3087134 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature