期刊
MOLECULAR PHARMACOLOGY
卷 64, 期 6, 页码 1283-1294出版社
AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS
DOI: 10.1124/mol.64.6.1283
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资金
- NINDS NIH HHS [NS40417] Funding Source: Medline
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 SH-EP1. Heterologously expressed alpha4beta2-nAChR engage in high-affinity, specific binding of H-3-labeled epibatidine (H-EBDN; macroscopic K-D=10 pM; k(on)=0.74/min/nM, k(off)=0.013/min). Immunofluorescence studies show alpha4 and beta2 subunit protein expression in virtually every transfected cell, and microautoradiographic studies show expression of I-125-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 Rb-86(+) 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.
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