Stable expression and functional characterization of a @entity1 nicotinic XXXX receptor with a6b2 properties: discovery of selective antagonists.

@entity1 @entity286 @entity26331 @entity35 @entity72339 @entity934
BACKGROUND AND PURPOSE: Despite growing evidence that inhibition of a6b2-containing (a6b2*) nicotinic @entity934 receptors (nAChRs) may be beneficial for the therapy of @entity286 addiction, the lack of good sources of a6b2*-nAChRs has delayed the discovery of a6b2-selective antagonists. Our aim was to generate a cell line stably expressing functional nAChRs with a6b2 properties, to enable pharmacological characterization and the identification of novel a6b2-selective antagonists. EXPERIMENTAL APPROACH: Different combinations of the a6, b2, b3, chimeric a6/3 and mutant b3( @entity72339 ) subunits were transfected in @entity1 embryonic kidney cells and tested for activity in a fluorescent imaging plate reader assay. The pharmacology of @entity35 immune-immobilized a6b2*-nAChRs was determined with        I- @entity26331 binding. KEY RESULTS: Functional channels were detected after co-transfection of a6/3, b2 and b3( @entity72339 ) subunits, while all other subunit combinations failed to produce agonist-induced responses. Stably expressed a6/3b2b3( @entity72339 )-nAChR pharmacology was unique, and clearly distinct from a4b2-, a3b4-, a7- and a1b1    -nAChRs. Antagonist potencies in inhibiting a6/3b2b3( @entity72339 ) -nAChRs was similar to their binding affinity for @entity35 native a6b2*-nAChRs. Agonist affinities for a6b2*-nAChRs was higher than their potency in activating a6/3b2b3( @entity72339 )-nAChRs, but their relative activities were equivalent. Focussed set screening at a6/3b2b3( @entity72339 )-nAChRs, followed by cross-screening with the other nAChRs, led to the identification of novel a6b2-selective antagonists. CONCLUSIONS AND IMPLICATIONS: We generated a @entity1 cell line stably expressing nAChRs, with pharmacological properties similar to native a6b2*-nAChRs, and used it to identify novel non-peptide, low molecular weight, a6b2-selective antagonists. We also propose a pharmacophore model of a6b2 antagonists, which offers a starting point for the development of new smoking cessation agents.
