Characterization Of Human α4β2-Nicotinic Acetylcholine Receptors Stably And Heterologously Expressed In Native Nicotinic Receptor-Null Sh-Ep1 Human Epithelial Cells
Department
neurobiology
Document Type
Article
Abstract
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.
Publication Date
12-1-2003
Publication Title
Molecular Pharmacology
ISSN
0026895X
Volume
64
Issue
6
First Page
1283
Last Page
1294
Digital Object Identifier (DOI)
10.1124/mol.64.6.1283
Recommended Citation
Eaton, J. Brek; Peng, Jian Hong; Schroeder, Katherine M.; George, Andrew A.; Fryer, John D.; Krishnan, Chandra; Buhlman, Lori; Kuo, Yen Ping; Steinlein, Ortrud; and Lukas, Ronald J., "Characterization Of Human α4β2-Nicotinic Acetylcholine Receptors Stably And Heterologously Expressed In Native Nicotinic Receptor-Null Sh-Ep1 Human Epithelial Cells" (2003). Translational Neuroscience. 71.
https://scholar.barrowneuro.org/neurobiology/71