Eaton J Brek, Peng Jian-Hong, Schroeder Katherine M, George Andrew A, Fryer John D, Krishnan Chandra, Buhlman Lori, Kuo Yen-Ping, Steinlein Ortrud, Lukas Ronald J
Division of Neurobiology, Barrow Neurological Institute, 350 West Thomas Road, Phoenix, Arizona 85013, USA.
Mol Pharmacol. 2003 Dec;64(6):1283-94. doi: 10.1124/mol.64.6.1283.
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.
由α4和β2亚基组成的天然表达烟碱型乙酰胆碱受体（α4β2 - nAChR）是大脑中高亲和力尼古丁结合位点的主要形式，与尼古丁奖赏、烟碱型胆碱能传递的介导、通过其他化学信息调节信号传导以及多种神经精神疾病有关。为了开发一个用于研究人类α4β2 - nAChR的模型系统，以便进行蛋白质化学、功能、药理学和表达调控研究，将人类α4和β2亚基稳定导入天然无nAChR的人类上皮细胞系SHEP1。异源表达的α4β2 - nAChR参与3H标记的埃博霉素（H - EBDN）的高亲和力、特异性结合（宏观解离常数KD = 10 pM；结合速率常数kon = 0.74/分钟/纳摩尔，解离速率常数koff = 0.013/分钟）。免疫荧光研究显示几乎每个转染细胞中都有α4和β2亚基蛋白表达，显微放射自显影研究显示大多数细胞中有125I标记的碘去氯埃博霉素结合位点表达。H - EBDN结合竞争研究揭示了对烟碱型激动剂的高亲和力和对烟碱型拮抗剂的低亲和力。使用86Rb +外流测定法对异源表达的α4β2 - nAChR进行的功能研究表明，埃博霉素、尼古丁和乙酰胆碱具有完全效力；对1,1 - 二甲基 - 4 - 苯基哌嗪鎓、金雀花碱和辛二酰二胆碱具有部分效力；二氢β - 刺桐啶、十烃季铵和甲基lycaconitine具有竞争性拮抗作用；美加明和毒扁豆碱具有非竞争性拮抗作用；泮库溴铵、六甲铵和d - 筒箭毒碱具有混合拮抗作用。这些结果证明了转染的SH - EP1细胞作为研究人类α4β2 - nAChR模型的实用性，并且它们还揭示了药物对放射性配体结合的表观亲和力与人类α4β2 - nAChR上功能位点之间的复杂关系。
