title: Stable expression, pharmacologic properties and regulation of the human neuronal nicotinic acetylcholine alpha 4 beta 2 receptor.
abstract: (-)-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. [3H]Cytisine bound to stably transfected cells with high affinity (KD value, 0.2 +/- 0.04 nM) and with a Bmax 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-beta-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 microM, 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 microM) 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 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 alpha 4 beta 2 nAChR.
authors.affiliation: Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, Illinois, USA.
authors.forename: M
authors.initials: M
authors.lastname: Gopalakrishnan
authors.forename: L M
authors.initials: LM
authors.lastname: Monteggia
authors.forename: D J
authors.initials: DJ
authors.lastname: Anderson
authors.forename: E J
authors.initials: EJ
authors.lastname: Molinari
authors.forename: M
authors.initials: M
authors.lastname: Piattoni-Kaplan
authors.forename: D
authors.initials: D
authors.lastname: Donnelly-Roberts
authors.forename: S P
authors.initials: SP
authors.lastname: Arneric
authors.forename: J P
authors.initials: JP
authors.lastname: Sullivan
mesh_terms: D000470:Alkaloids / Q000378:metabolism; D000818:Animals; D001392:Azocines; D001483:Base Sequence; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000502:physiology; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D009474:Neurons / Q000648:ultrastructure*; D009538:Nicotine / Q000494:pharmacology; D018722:Nicotinic Agonists / Q000494:pharmacology; D011807:Quinolizines; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011978:Receptors, Nicotinic / Q000187:drug effects* / Q000235:genetics / Q000502:physiology*; D013237:Stereoisomerism; D014162:Transfection; D014316:Tritium; D015854:Up-Regulation / Q000187:drug effects; D000093843:Quinolizidine Alkaloids
chemical_list: D000470:Alkaloids; D001392:Azocines; D018076:DNA, Complementary; D009538:Nicotine; D018722:Nicotinic Agonists; D011807:Quinolizines; D012333:RNA, Messenger; D011978:Receptors, Nicotinic; D014316:Tritium; C004712:cytisine; D000093843:Quinolizidine Alkaloids