Zhao Lingke, Kuo Yen-Ping, George Andrew A, Peng Jian-Hong, Purandare Madhuri Singh, Schroeder Katherine M, Lukas Ronald J, Wu Jie
Division of Neurology, Barrow Neurological Institute, 350 West Thomas Rd., Phoenix, AZ 85013-4496, USA.
J Pharmacol Exp Ther. 2003 Jun;305(3):1132-41. doi: 10.1124/jpet.103.048777. Epub 2003 Mar 6.
alpha 7-Nicotinic acetylcholine receptors (alpha 7-nAChRs) are broadly distributed in the central nervous system, where they play important roles in chemical and electrical signaling, and perhaps in neurite outgrowth, synaptic plasticity, and neuronal death/survival. To help elucidate their normal and pathophysiological roles, we have heterologously expressed human alpha 7-nAChR in transfected SH-EP1 human epithelial cells. Reverse transcription-polymerase chain reaction and mRNA fluorescence in situ hybridization analyses demonstrate expression of human alpha 7 subunits as messenger RNA. Patch-clamp recordings exploiting a novel strategy to prevent functional rundown of whole-cell peak current responses to repeated acute challenges with nicotinic agonists show successful expression of functional alpha 7-nAChR that mediate inward currents characterized by rapid phases of activation and inactivation. Concentration-response curves show that nicotine, acetylcholine, and choline are efficacious agonists at human alpha 7-nAChRs. Current-voltage relationships show inward rectification for agonist-induced currents. Human alpha 7-nAChRs exhibit some sensitivity to alpha 7-nAChR antagonists alpha-bungarotoxin (Bgt) or methyllycaconitine (MLA) when applied coincidentally with agonist, but much higher affinity block occurs when cells and alpha 7-nAChRs are pre-exposed to antagonists for 2 min before challenge with agonist. Both Bgt and MLA are competitive inhibitors of alpha 7-nAChR function. Whole-cell current peak amplitudes and half-times for inactivation of alpha 7-nAChR functional responses to nicotine are dramatically reduced in the absence of extracellular Ca2+, suggestive of high Ca2+ permeability of the alpha 7-nAChR channel. Thus, heterologously expressed human alpha 7-nAChR in mammalian cells have properties of native alpha 7-nAChR or of alpha 7-nAChR heterologously expressed in other systems and serve as excellent models for studies of molecular bases of alpha 7-nAChR function.
α7-烟碱型乙酰胆碱受体（α7-nAChRs）广泛分布于中枢神经系统，在化学信号和电信号传导中发挥重要作用，可能还参与神经突生长、突触可塑性以及神经元死亡/存活过程。为了帮助阐明其正常和病理生理作用，我们在转染的SH-EP1人上皮细胞中异源表达了人α7-nAChR。逆转录-聚合酶链反应和mRNA荧光原位杂交分析表明人α7亚基作为信使RNA表达。膜片钳记录采用一种新策略来防止对烟碱激动剂的重复急性刺激导致全细胞峰值电流反应功能性衰减，结果显示功能性α7-nAChR成功表达，介导以快速激活和失活相为特征的内向电流。浓度-反应曲线表明尼古丁、乙酰胆碱和胆碱是人α7-nAChRs的有效激动剂。电流-电压关系显示激动剂诱导电流的内向整流。人α7-nAChRs在与激动剂同时应用时对α7-nAChR拮抗剂α-银环蛇毒素（Bgt）或甲基lycaconitine（MLA）表现出一定敏感性，但当细胞和α7-nAChRs在激动剂刺激前预先暴露于拮抗剂2分钟时，会出现更高亲和力的阻断。Bgt和MLA都是α7-nAChR功能的竞争性抑制剂。在没有细胞外Ca2+的情况下，α7-nAChR对尼古丁功能反应的全细胞电流峰值幅度和失活半衰期显著降低，提示α7-nAChR通道具有高Ca2+通透性。因此，在哺乳动物细胞中异源表达的人α7-nAChR具有天然α7-nAChR或在其他系统中异源表达的α7-nAChR的特性，可作为研究α7-nAChR功能分子基础的优秀模型。
