Stable expression, pharmacologic properties and regulation of the human neuronal nicotinic acetylcholine α₄β₂ receptor

(-)-Nicotine, the prototypical agonist for neuronal nicotinic acetylcholine receptors (nAChR) has been shown to bind with high affinity to the rodent and avian α₄β₂ nAChR subtype. This subtype may represent a primary molecular target for some of the beneficial central nervous system effects i.e., cognitive enhancement, anxiolysis, analgesia, neuroprotection, of (-)nicotine and related ligands. However, a detailed study of the human α₄β₂ subunit combination has not yet been reported. In this study, we stably coexpressed the human neuronal α₄ and β₂ nAChR subunits in human embryonic kidney (HEK) 293 cells and studied its pharmacological and regulatory properties. [³H]Cytisine bound to stably transfected cells with high affinity (K(D) value, 0.2 ± 0.04 nM) and with a B(max) value of 1359 ± 91 fmol/mg protein. A good correlation (r = 0.98) was observed between binding affinities in transfected cells and in native neuronal preparations for a series of nAChR ligands, ⁸⁶Rb⁺ efflux studies showed that stably transfected cells express functional ion channels that are sensitive to blockade by dihydro-β-erythroidine. (±)-Epibatidine, (-)-nicotine, 1,1- dimethyl-4-phenylpiperazinium, (S)-3-methyl-5-(1-methyl-2- pyrrolidinyl)isoxazole (ABT-418), acetylcholine and (-)-cytisine stimulated ⁸⁶Rb⁺ efflux with EC₅₀ values of 0.02, 3.9, 2.5, 10, 44 and 38 μM, respectively. Treatment of transfected cells with (-)-nicotine for 7 days led to a significant increase in the density of [³H](-)-cytisine binding sites (EC₅₀ = 0.56 μM) and a significant enhancement in the sensitivity of ACh. Specific binding or (-)-nicotine-evoked cation efflux was not detected in untransfected cells. Analysis of total cellular RNA from transfected, but not untransfected cells, showed the expected fragment sizes corresponding to the human α₄ and β₂ subunit mRNA. These results demonstrate that stable expression of the human α₄β₂ nAChR subunit combination can give rise to functional ion channels that bind [³H](-)-cytisine with high affinity, exhibit homologous regulation and evoke agonist-induced cation flux with pharmacological properties consistent with native neuronal α₄β₂ nAChR.