Regulation of Human α4β2 Neuronal Nicotinic Acetylcholine Receptors by Cholinergic Channel Ligands and Second Messenger Pathways

Abstract

The α4β2 nicotinic acetylcholine receptors (nAChRs), a major subtype in the brain, have been shown to be modulated by chronic treatment with nicotine. In this study, the regulation of recombinant human α4β2 nAChR subtype by (−)-nicotine and other cholinergic channel modulators was studied using human embryonic kidney 293 cells stably expressing this subunit combination. The treatment of transfected cells with (−)-nicotine and other activator ligands, including (−)-cytisine, 1,1-dimethyl-4-phenylpiperazinium, (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole, and (±)-epibatidine, resulted in concentration-dependent increases in the levels of α4β2 nAChRs. The increase in [³H]cytisine binding sites was initiated by low concentrations of (−)-nicotine (t_0.5 = 4.0 ± 0.5 hr), and totally reversible (t_0.5 = 11.7 ± 0.1 hr); and occurred with no change in ligand binding affinity. Antagonists, including dihydro-β-erythroidine, d-tubocurarine, and methyllycaconitine, also elicited significant increases in receptor levels. A good correlation was observed between theK_i values for binding inhibition and the EC₅₀ values for receptor up-regulation. Treatment of cells with mecamy-lamine, a noncompetitive antagonist, did not change receptor levels or alter (−)-nicotine-evoked up-regulation. (−)-Nicotine-evoked up-regulation was blocked by cycloheximide, suggesting a role for protein synthesis. Treatment of cells with (−)-nicotine or dihydro-β-erythroidine differentially modulated the efficacy of acetylcholine to activate cation efflux. Both 6-β-[β′(piperidino)propionyl]forskolin and phorbol-12-myristate-13-acetate increased [³H]cytisine binding sites and nAChR function and enhanced the effects of chronic (−)-nicotine treatment in a synergistic manner. These results collectively demonstrate that human α4β2 nAChRs can be differentially up-regulated by chronic treatment with nAChR ligands and activation of protein kinase A- and protein kinase C-dependent mechanisms.