Characterizing ligand-gated ion channel receptors with genetically encoded Ca2++ sensors.
Characterizing ligand-gated ion channel receptors with genetically encoded Ca2++ sensors.
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DOI：
10.1371/journal.pone.0016519
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发表时间：
2011-01-28
期刊：
影响因子：
3.7
通讯作者：
Taylor P
中科院分区：
文献类型：
--
作者：
Yamauchi JG;Nemecz Á;Nguyen QT;Muller A;Schroeder LF;Talley TT;Lindstrom J;Kleinfeld D;Taylor P
我们提出了一种基于细胞的系统和实验方法来表征配体门控离子通道(LGIC)的激动剂和拮抗剂的选择性，方法是开发稳定表达钙离子通透性LGIC的传感器细胞和基于遗传编码的Förster(或荧光)共振能量转移(FRET)钙传感器。特别是，我们描述了人α7和人α4β2烟碱型乙酰胆碱受体、小鼠5-HT3A 5-羟色胺受体和人α7/小鼠5-HT3A受体嵌合体的分离株。这些传感器生成了激动剂的完整浓度-反应曲线和拮抗剂的SChild曲线图，结果验证了所测试受体的已知药理作用。浓度-响应关系可以由FRET信号的初始速率或最大幅度产生。虽然这种药物荧光检测技术的分析是中等通量水平的，但它使用了一条稳定的克隆线，并且具有在多种亚型的配体门控离子通道上筛选实验室产生的作为激动剂或拮抗剂的同系物的通用性。克隆传感器系也与体内使用兼容，以间接测量内源性神经递质的受体激活。
We present a cell based system and experimental approach to characterize agonist and antagonist selectivity for ligand-gated ion channels (LGIC) by developing sensor cells stably expressing a Ca2+ permeable LGIC and a genetically encoded Förster (or fluorescence) resonance energy transfer (FRET)-based calcium sensor. In particular, we describe separate lines with human α7 and human α4β2 nicotinic acetylcholine receptors, mouse 5-HT3A serotonin receptors and a chimera of human α7/mouse 5-HT3A receptors. Complete concentration-response curves for agonists and Schild plots of antagonists were generated from these sensors and the results validate known pharmacology of the receptors tested. Concentration-response relations can be generated from either the initial rate or maximal amplitudes of FRET-signal. Although assaying at a medium throughput level, this pharmacological fluorescence detection technique employs a clonal line for stability and has versatility for screening laboratory generated congeners as agonists or antagonists on multiple subtypes of ligand-gated ion channels. The clonal sensor lines are also compatible with in vivo usage to measure indirectly receptor activation by endogenous neurotransmitters.
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影响因子：
64.8
作者：
EISELE, JL;BERTRAND, S;BERTRAND, D
通讯作者：
BERTRAND, D
DOI：
10.1073/pnas.0630641100
发表时间：
2003-04-15
影响因子：
11.1
作者：
Fitch, RW;Xiao, YX;Daly, JW
通讯作者：
Daly, JW
影响因子：
3.6
作者：
Kuryatov, Alexandre;Onksen, Jennifer;Lindstrom, Jon
通讯作者：
Lindstrom, Jon
影响因子：
17.7
作者：
Freedman, Robert;Olincy, Ann;Kem, William R.
通讯作者：
Kem, William R.
DOI：
10.1073/pnas.97.13.7260
发表时间：
2000-06-20
影响因子：
11.1
作者：
Baubet, V;Le Mouellic, H;Brûlet, P
通讯作者：
Brûlet, P