STABLE EXPRESSION, PHARMACOLOGICAL PROPERTIES AND REGULATION OF THE HUMAN NEURONAL NICOTINIC ACETYLCHOLINE ALPHA(4)BETA(2) RECEPTOR

(-)-Nicotine, the prototypical agonist for neuronal nicotinic acetylch oline receptors (nAChR) has been shown to bind with high affinity to t he rodent and avian alpha(4) beta(2) nAChR subtype. This subtype may r epresent a primary molecular target for some of the beneficial central nervous system effects i.e., cognitive enhancement, anxiolysis, analg esia, neuroprotection, of (-)nicotine and related ligands. However, a detailed study of the human alpha(4) beta(2) subunit combination has n ot yet been reported. In this study, we stably coexpressed the human n euronal alpha(4) and beta(2) nAChR subunits in human embryonic kidney (HEK) 293 cells and studied its pharmacological and regulatory propert ies. [H-3]Cytistine bound to stably transfected cells with high affini ty (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 bi nding affinities in transfected cells and in native neuronal preparati ons for a series of nAChR ligands. Rb-86(+) efflux studies showed that stably transfected cells express functional ion channels that are sen sitive to blockade by dihydro-beta-erythroidine. (+/-)-Epibatidine, (- )-nicotine, 1,1-dimethyl-4-phenylpiperazinium, (S)-3-methyl-5-(1-methy l-2-pyrrolidinyl)isoxazole (ABT-418), acetylcholine and (-)-cytisine s timulated 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 (-)-ni cotine 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 enh ancement in the sensitivity of ACh. Specific binding or (-)-nicotine-e voked cation efflux was not detected in untransfected cells. Analysis of total cellular RNA from transfected, but not untransfected cells, s howed the expected fragment sizes corresponding to the human alpha(4) and beta(2) subunit mRNA. These results demonstrate that stable expres sion 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 cat ion flux with pharmacological properties consistent with native neuron al alpha(4) beta(2) nAChR.