Graduate Training Program in Neuroscience, Vanderbilt University School of Medicine, Nashville, Tennessee 23235, USA.
J Biol Chem. 2012 Mar 16;287(12):8852-63. doi: 10.1074/jbc.M111.267757. Epub 2012 Jan 30.
Monoamine transporters terminate synaptic neurotransmission and are molecular targets for antidepressants and psychostimulants. Fluorescent reporters can monitor real-time transport and are amenable for high-throughput screening. However, until now, their use has mostly been successful to study the catecholamine transporters but not the serotonin (5HT) transporter. Here, we use fluorescence microscopy, electrophysiology, pharmacology, and molecular modeling to compare fluorescent analogs of 1-methyl-4-phenylpyridinium (MPP(+)) as reporters for the human serotonin transporter (hSERT) in single cells. The fluorescent substrate 4-(4-(dimethylamino)phenyl)-1-methylpyridinium (APP(+)) exhibits superior fluorescence uptake in hSERT-expressing HEK293 cells than other MPP(+) analogs tested. APP(+) uptake is Na(+)- and Cl(-)-dependent, displaced by 5HT, and inhibited by fluoxetine, suggesting APP(+) specifically monitors hSERT activity. ASP(+), which was previously used to study catecholamine transporters, is 10 times less potent than APP(+) at inhibiting 5HT uptake and has minimal hSERT-mediated uptake. Furthermore, in hSERT-expressing oocytes voltage-clamped to -60 mV, APP(+) induced fluoxetine-sensitive hSERT-mediated inward currents, indicating APP(+) is a substrate, whereas ASP(+) induced hSERT-mediated outward currents and counteracted 5HT-induced hSERT currents, indicating ASP(+) possesses activity as an inhibitor. Extra-precise ligand receptor docking of APP(+) and ASP(+) in an hSERT homology model showed both ASP(+) and APP(+) docked favorably within the active region; accordingly, comparable concentrations are required to elicit their opposite electrophysiological responses. We conclude APP(+) is better suited than ASP(+) to study hSERT transport fluorometrically.
单胺转运体终止突触神经传递，是抗抑郁药和精神兴奋剂的分子靶点。荧光报告物可实时监测转运，适用于高通量筛选。然而，到目前为止，它们的使用主要成功地用于研究儿茶酚胺转运体，但不适用于血清素（5HT）转运体。在这里，我们使用荧光显微镜、电生理学、药理学和分子建模来比较人血清素转运体（hSERT）在单细胞中的荧光类似物 1-甲基-4-苯基吡啶鎓（MPP(+)) 作为报告物。荧光底物 4-(4-(二甲基氨基)苯基）-1-甲基吡啶鎓（APP(+)) 在表达 hSERT 的 HEK293 细胞中的荧光摄取优于其他测试的 MPP(+)类似物。APP(+)摄取依赖于 Na(+)和 Cl(-)，被 5HT 置换，并被氟西汀抑制，表明 APP(+)特异性监测 hSERT 活性。ASP(+) 之前用于研究儿茶酚胺转运体，在抑制 5HT 摄取方面比 APP(+) 弱 10 倍，并且对 hSERT 介导的摄取作用最小。此外，在电压钳位至-60 mV 的表达 hSERT 的卵母细胞中，APP(+) 诱导氟西汀敏感的 hSERT 介导的内向电流，表明 APP(+) 是底物，而 ASP(+) 诱导 hSERT 介导的外向电流并抵消 5HT 诱导的 hSERT 电流，表明 ASP(+) 具有作为抑制剂的活性。APP(+) 和 ASP(+) 在 hSERT 同源模型中的超精确配体受体对接表明，ASP(+) 和 APP(+) 都有利地位于活性区域内；因此，需要相当浓度的 ASP(+) 和 APP(+) 来引起它们相反的电生理反应。我们得出结论，APP(+) 比 ASP(+) 更适合荧光测量研究 hSERT 转运。
