Abstract
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 Kd values of [3H](±)epibatidine ([3H]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 [3H]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 [3H]EB binding represents predominantly α4β2 nAChRs. More important, the affinities (Ki 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.
Footnotes
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This study was supported by the National Institutes of Health Grants DA06486 and DA12976.
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Preliminary reports of this work have been presented previously: Xiao Y, Baydyuk M, Wang H, Davis HE, and Kellar KJ (2003) Pharmacology of the agonist binding site of rat neuronal nicotinic receptor subtypes expressed in HEK293 cells, in Neuronal Nicotinic Ligands and Receptors: Targets for Medication (Corrigall WM and Rapaka R eds), Bioorganic and Medicinal Chemistry Letters (in press); and Xiao Y and Kellar KJ (2002) Pharmacology of ligand binding and up-regulation of rat neuronal nicotinic receptor subtypes stably expressed in HEK 293 cells. Program 238.3, Abstract Viewer, Society of Neuroscience, 2002.
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DOI: 10.1124/jpet.104.066787.
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ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; CNS, central nervous system; [3H]EB, [3H](±)epibatidine; I-A-85380, 5-iodo-A-85380; DHβE, dihydro-β-erythroidine; DMPP, 1,1-dimethyl-4-phenylpiperazinium iodide; MLA, methyllycaconitine.
- Received February 9, 2004.
- Accepted March 11, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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