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Stable expression and functional characterization of a human nicotinic acetylcholine receptor with α6β2 properties: discovery of selective antagonists
Author(s) -
Capelli Anna Maria,
Castelletti Laura,
Chen Yu Hua,
Van der Keyl Harjeet,
Pucci Luca,
Oliosi Beatrice,
Salvagno Cristian,
Bertani Barbara,
Gotti Cecilia,
Powell Andrew,
Mugnaini Manolo
Publication year - 2011
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.2011.01213.x
Subject(s) - epibatidine , nicotinic agonist , acetylcholine receptor , pharmacology , agonist , homomeric , methyllycaconitine , chemistry , hek 293 cells , acetylcholine , receptor , nicotinic acetylcholine receptor , biology , protein subunit , biochemistry , gene
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 β3 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 α6β2*‐nAChRs was determined with 125 I‐epibatidine binding. KEY RESULTS Functional channels were detected after co‐transfection of α6/3, β2 and β3 V273S subunits, while all other subunit combinations failed to produce agonist‐induced responses. Stably expressed α6/3β2β3 V273S ‐nAChR pharmacology was unique, and clearly distinct from α4β2‐, α3β4‐, α7‐ and α1β1δε‐nAChRs. Antagonist potencies in inhibiting α6/3β2β3 V273S ‐nAChRs was similar to their binding affinity for rat native α6β2*‐nAChRs. Agonist affinities for α6β2*‐nAChRs was higher than their potency in activating α6/3β2β3 V273S ‐nAChRs, but their relative activities were equivalent. Focussed set screening at α6/3β2β3 V273S ‐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.