Stable expression, pharmacologic properties and regulation of the human neuronal nicotinic acetylcholine alpha(4)beta(2) receptor

(-)-Nicotine, the prototypical agonist for neuronal nicotinic acetylcholine receptors (nAChR) has been shown to bind with high affinity to the rodent and avian alpha(4) beta(2) 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 alpha(4) beta(2) subunit combination has not yet been reported. In this study, we stably coexpressed the human neuronal alpha(4) and beta(2) nAChR subunits in human embryonic kidney (HEK) 293 cells and studied its pharmacological and regulatory properties. [H-3]Cytistine 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. Rb-86(+) efflux studies showed that stably transfected cells express functional ion channels that are sensitive to blockade by dihydro-beta-erythroidine. (+/-)-Epibatidine, (-)-nicotine, 1,1-dimethyl-4-phenylpiperazinium, (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT-418), acetylcholine and (-)-cytisine stimulated Rb-86(+) efflux with EC(50) values of 0.02, 3.9, 2.5, 10, 44 and 38 mu M, respectively. Treatment of transfected cells with (-)-nicotine for 7 days led to a significant increase in the density of [H-3](-)-cytisine binding sites (EC(50) = 0.56 mu M) 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 alpha(4) and beta(2) subunit mRNA. These results demonstrate that stable expression of the human alpha(4) beta(2) nAChR subunit combination can give rise to func tional ion channels that bind [H-3](-)-cytisine with high affinity, exhibit homologous regulation and evoke agonist-induced cation flux with pharmacological properties consistent with native neuronal alpha(4) beta(2) nAChR.