Membrane potential fluorescence: A rapid and highly sensitive assay for nicotinic receptor channel function
Membrane potential fluorescence: A rapid and highly sensitive assay for nicotinic receptor channel function
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DOI：
10.1073/pnas.0630641100
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发表时间：
2003-04-15
影响因子：
11.1
通讯作者：
Daly, JW
中科院分区：
文献类型：
--
作者：
Fitch, RW;Xiao, YX;Daly, JW
七个细胞系表达天然和转染烟碱受体亚型进行了功能评价，通过使用荧光检测的基础上膜电位和钙动力学与“不洗”染料系统。在表达大鼠α 3 β 4受体的细胞系中，两种试验均提供了（+/-）-地棘蛙素、2S-（-）-尼古丁、7 R，9 S-（-）野靛碱和1，1-二甲基-4-苯基哌嗪鎓的相同效价等级顺序。烟碱拮抗剂美加明和二氢-β-赤藓定在两种测定中均抑制反应。在同一实验中评估激动剂和拮抗剂活性。激动剂似乎更有效的膜电位测定比在钙测定，而拮抗剂则匡威。膜电位试验在表达人α 4 β 2受体的K-177细胞、表达人神经节受体的IMR-32和SH-SY 5 Y细胞以及表达人神经肌肉受体的TE-671细胞中提供了稳健的响应。这些线给出了弱至中等的钙反应。此外，在表达大鼠α 4 β 2和α 4 β 4受体的细胞系中获得了膜电位反应，这些细胞系没有钙反应。因此，膜电位作为烟碱活性的灵敏测量，并且所产生的去极化可能与细胞信号传导中的钙一样重要。
Seven cell lines expressing native and transfected nicotinic receptor subtypes were evaluated functionally by using fluorescent assays based on membrane potential and calcium dynamics with "no-wash" dye systems. Both assays provided the same rank orders of potency for (+/-)-epibatidine, 2S-(-)-nicotine, 7R,9S-(-)cytisine, and 1,1-dimethyl-4-phenylpiperazinium in a cell line expressing rat alpha3beta4 receptors. Nicotinic antagonists mecamylamine and dihydro-beta-erythroidine inhibited responses in both assays. Both agonist and antagonist activity were assessed within the same experiment. Agonists seemed more potent in the membrane potential assay than in the calcium assay, whereas the converse was true for antagonists. The membrane potential assay afforded robust responses in K-177 cells expressing human alpha4beta2 receptors, in IMR-32 and SH-SY5Y cells expressing human ganglionic receptors, and in TE-671 cells expressing human neuromuscular receptors. These lines gave weak to modest calcium responses. Moreover, membrane potential responses were obtained in cell lines expressing rat alpha4beta2 and alpha4beta4 receptors, which were devoid of calcium responses. Thus, membrane potential serves as a sensitive measure of nicotinic activity, and the resulting depolarization may be as important as calcium in cell signaling.