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

Murali Gopalakrishnan, Lisa M Monteggia, David J. Anderson, Eduardo J. Molinari, Marietta Piattoni-Kaplan, Diana Donnelly-Roberts, Stephen P. Arneric, James P. Sullivan

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161 Scopus citations


(-)-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.

Original languageEnglish (US)
Pages (from-to)289-297
Number of pages9
JournalJournal of Pharmacology and Experimental Therapeutics
Issue number1
StatePublished - Jan 1 1996

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology


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