Characterization of Human α4β2-Nicotinic Acetylcholine Receptors Stably and Heterologously Expressed in Native Nicotinic Receptor-Null SH-EP1 Human Epithelial Cells

J. Brek Eaton, Jian Hong Peng, Katherine M. Schroeder, Andrew A. George, John D. Fryer, Chandra Krishnan, Lori Buhlman, Yen Ping Kuo, Ortrud Steinlein, Ronald J. Lukas

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Naturally expressed nicotinic acetylcholine receptors composed of α4 and β2 subunits (α4β2-nAChR) are the predominant form of high affinity nicotine binding site in the brain implicated in nicotine reward, mediation of nicotinic cholinergic transmission, modulation of signaling through other chemical messages, and a number of neuropsychiatric disorders. To develop a model system for studies of human α4β2-nAChR allowing protein chemical, functional, pharmacological, and regulation of expression studies, human α4 and β2 subunits were stably introduced into the native nAChR-null human epithelial cell line SH-EP1. Heterologously expressed α4β2-nAChR engage in high-affinity, specific binding of 3H-labeled epibatidine (H-EBDN; macroscopic KD = 10 pM; kon = 0.74/min/nM, koff = 0.013/min). Immunofluorescence studies show α4 and β2 subunit protein expression in virtually every transfected cell, and microautoradiographic studies show expression of 125I-labeled jodo-deschloroepibatidine binding sites in most cells. H-EBDN binding competition studies reveal high affinity for nicotinic agonists and lower affinity for nicotinic antagonists. Heterologously expressed α4β2-nAChR functional studies using 86Rb+ efflux assays indicate full efficacy of epibatidine, nicotine, and acetylcholine; partial efficacy for 1,1-dimethyl-4-phenyl-piperazinium, cytisine, and suberyldicholine; competitive antagonism by dihydro-β -erythroidine, decamethonium, and methyllycaconitine; noncompetitive antagonism by mecamylamine and eserine; and mixed antagonism by pancuronium, hexamethonium, and d-tubocurarine. These results demonstrate utility of transfected SH-EP1 cells as models for studies of human α4β2-nAChR, and they also reveal complex relationships between apparent affinities of drugs for radioligand binding and functional sites on human α4β2-nAChR.

Original languageEnglish (US)
Pages (from-to)1283-1294
Number of pages12
JournalMolecular Pharmacology
Issue number6
StatePublished - Dec 2003
Externally publishedYes


ASJC Scopus subject areas

  • Pharmacology

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