title: Stable expression and functional characterization of a human nicotinic acetylcholine receptor with α6β2 properties: discovery of selective antagonists.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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 ¹²⁵I-epibatidine binding.
  
  
  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
  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.
authors.affiliation: GlaxoSmithKline Medicines Research Centre, Verona, Italy.
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Capelli
authors.forename: Laura
authors.initials: L
authors.lastname: Castelletti
authors.forename: Yu Hua
authors.initials: YH
authors.lastname: Chen
authors.forename: Harjeet
authors.initials: H
authors.lastname: Van der Keyl
authors.forename: Luca
authors.initials: L
authors.lastname: Pucci
authors.forename: Beatrice
authors.initials: B
authors.lastname: Oliosi
authors.forename: Cristian
authors.initials: C
authors.lastname: Salvagno
authors.forename: Barbara
authors.initials: B
authors.lastname: Bertani
authors.forename: Cecilia
authors.initials: C
authors.lastname: Gotti
authors.forename: Andrew
authors.initials: A
authors.lastname: Powell
authors.forename: Manolo
authors.initials: M
authors.lastname: Mugnaini
mesh_terms: D000818:Animals; D004353:Drug Evaluation, Preclinical; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D008297:Male; D008958:Models, Molecular; D009154:Mutation; D018733:Nicotinic Antagonists / Q000737:chemistry / Q000494:pharmacology*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D018733:Nicotinic Antagonists; D021122:Protein Subunits; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; C559463:alpha6beta2 nicotinic acetylcholine receptor