Characterization of human alpha 4 beta 2-nicotinic acetylcholine receptors stably and heterologously expressed in native nicotinic receptor-null SH-EP1 human epithelial cells.
Naturally expressed nicotinic acetylcholine receptors composed of alpha4 and beta2 subunits (alpha4beta2-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 alpha4beta2-nAChR allowing protein chemical, functional, pharmacological, and regulation of expression studies, human alpha4 and beta2 subunits were stably introduced into the native nAChR-null human epithelial cell line SHEP1. Heterologously expressed alpha4beta2-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 alpha4 and beta2 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 alpha4beta2-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-beta-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 alpha4beta2-nAChR, and they also reveal complex relationships between apparent affinities of drugs for radioligand binding and functional sites on human alpha4beta2-nAChR.
Eaton
J Brek
JB
Division of Neurobiology, Barrow Neurological Institute, 350 West Thomas Road, Phoenix, Arizona 85013, USA. rlukas@chw.edu
Peng
Jian-Hong
JH
Schroeder
Katherine M
KM
George
Andrew A
AA
Fryer
John D
JD
Krishnan
Chandra
C
Buhlman
Lori
L
Kuo
Yen-Ping
YP
Steinlein
Ortrud
O
Lukas
Ronald J
RJ
D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D011485:Protein Binding / Q000502:physiology; D011725:Pyridines / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000096:biosynthesis* / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics; D014162:Transfection / Q000379:methods
D019086:Bridged Bicyclo Compounds, Heterocyclic; D011725:Pyridines; D011978:Receptors, Nicotinic; C403531:nicotinic receptor alpha4beta2; C082748:epibatidine