The Comparative Pharmacology and Up-Regulation of Rat Neuronal Nicotinic Receptor Subtype Binding Sites Stably Expressed in Transfected Mammalian Cells

• Published in: The Journal of pharmacology and experimental therapeutics Vol. 310; no. 1; pp. 98 - 107
• Main Authors: Xiao, Yingxian, Kellar, Kenneth J
• Format: Journal Article
• Language: English
• Published: United States American Society for Pharmacology and Experimental Therapeutics 01.07.2004
• Subjects: Animals; Binding Sites; Bridged Bicyclo Compounds, Heterocyclic - pharmacology; Carbachol - pharmacology; Cell Line; Cholinergic Agonists - pharmacology; Gene Expression - drug effects; Humans; Neurons - drug effects; Neurons - metabolism; Nicotine - pharmacology; Nicotinic Agonists - pharmacology; Pyridines - pharmacology; Rats; Receptors, Nicotinic - drug effects; Receptors, Nicotinic - metabolism; Transfection; Tritium; Up-Regulation - drug effects
• DOI: 10.1124/jpet.104.066787
• ISSN: 0022-3565

We stably transfected human embryonic kidney cells (HEK 293 cells) with genes encoding rat neuronal nicotinic receptor α2, α3, or α4 subunits in combination with the β2 or β4 subunit to generate six cell lines that express defined subunit combinations that represent potential subtypes of rat neuronal nicotinic acetylcholine receptors (nAChRs). These cell lines were designated KXα2β2, KXα2β4, KXα3β2, KXα3β4, KXα4β2, and KXα4β4. The K d values of [ 3 H](±)epibatidine ([ 3 H]EB) binding to membranes from these six cell lines ranged from ∼0.02 to 0.3 nM. The pharmacological profiles of the agonist binding sites of these putative nAChR subtypes were examined in competition studies in which unlabeled nicotinic ligands, including 10 agonists and two antagonists, competed against [ 3 H]EB. Most nicotinic ligands examined had higher affinity for the receptor subtypes containing the β2 subunit compared with those containing the β4 subunit. An excellent correlation ( r > 0.99) of the binding affinities of the 10 agonists was observed between receptors from KXα4β2 cells and from rat forebrain tissue, in which [ 3 H]EB binding represents predominantly α4β2 nAChRs. More important, the affinities ( K i values) for the two tissues were nearly identical. The densities of the binding sites of all six cell lines were increased after a 5-day exposure to (-)-nicotine or the quaternary amine agonist carbachol. These data indicate that these cell lines expressing nAChR subunit combinations should be useful models for investigating pharmacological properties and regulation of the binding sites of potential nAChR subtypes, as well as for studying the properties of nicotinic compounds.