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4
2-Nicotinic Acetylcholine Receptors Stably and Heterologously Expressed in Native Nicotinic Receptor-Null SH-EP1 Human Epithelial Cells
Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona (J.B.E., J.-H.P., K.M.S., A.A.G., J.D.F., C.K., L.B., Y.-P.K., R.J.L.); and Institute for Human Genetics, Rheinische Friedrich-Wilhelms-Universitat, Bonn, Germany (O.S.)
Naturally expressed nicotinic acetylcholine receptors composed of 
4 and 
2 subunits (42-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 42-nAChR allowing protein chemical, functional, pharmacological, and regulation of expression studies, human 4 and 2 subunits were stably introduced into the native nAChR-null human epithelial cell line SHEP1. Heterologously expressed 42-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 4 and 2 subunit protein expression in virtually every transfected cell, and microautoradiographic studies show expression of 125I-labeled iodo-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 42-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--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 42-nAChR, and they also reveal complex relationships between apparent affinities of drugs for radioligand binding and functional sites on human 42-nAChR.
Received May 21, 2003; accepted July 30, 2003.
Address correspondence to: Dr. Ronald J. Lukas, Division of Neurobiology, Barrow Neurological Institute, 350 West Thomas Road, Phoenix, Arizona 85013. E-mail: rlukas{at}chw.edu
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