New fluorescent substrate enables quantitative and high-throughput examination of vesicular monoamine transporter 2 (VMAT2).
New fluorescent substrate enables quantitative and high-throughput examination of vesicular monoamine transporter 2 (VMAT2).
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DOI：
10.1021/cb400259n
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发表时间：
2013-09-20
影响因子：
4
通讯作者：
Sames D
中科院分区：
文献类型：
--
作者：
Hu G;Henke A;Karpowicz RJ Jr;Sonders MS;Farrimond F;Edwards R;Sulzer D;Sames D
囊泡单胺转运体2(VMAT2)是脑内单胺类神经递质传递系统的重要组成部分，它将单胺类神经递质从神经细胞质转运到突触小泡中，参与神经递质释放的调节。考虑到VMAT2作为药物靶点以及成像探针设计的目标的持续兴趣，我们开发了一种非常适合在细胞培养中研究VMAT2的荧光底物。在此，我们报道了一种新的荧光探针FFN206的合成和表征，它是一种优良的VMAT2底物，能够在荧光显微镜下检测完整细胞中VMAT2的活性，并在亚细胞内定位于表达VMAT2的酸性隔室，而不需要明显标记其他细胞器。VMAT2活性也可以通过微量平板阅读器进行测量。FFN206在VMAT2的表观Km为1.16±0.10µM，与多巴胺的表观Km相似。我们进一步报告了一种基于细胞的荧光检测方法的开发和验证，该方法适用于使用VMAT2转基因的HEK细胞(Z‘因子约为0.7-0.8)进行高通量筛选(HTS)，从而能够快速识别VMAT2抑制剂并在广泛的亲和力范围内测量它们的抑制常数。FFN206为细胞培养中VMAT2功能的光学检测提供了一种新的工具。
Vesicular Monoamine Transporter 2 (VMAT2) is an essential component of the monoaminergic neurotransmission system in the brain as it transports monoamine neurotransmitters from the neuronal cytosol into the synaptic vesicles and thus contributes to modulation of neurotransmitter release. Considering the continuing interest in VMAT2 as a drug target, as well as a target for the design of imaging probes, we have developed a fluorescent substrate well suited for the study of VMAT2 in cell culture. Herein, we report the synthesis and characterization of a new fluorescent probe, FFN206, as an excellent VMAT2 substrate capable of detecting VMAT2 activity in intact cells using fluorescence microscopy, with subcellular localization to VMAT2-expressing acidic compartments without apparent labeling of other organelles. VMAT2 activity can also be measured via microplate reader. The apparent Km of FFN206 at VMAT2 was found to be 1.16 ± 0.10 µM, similar to that of dopamine. We further report the development and validation of a cell-based fluorescence assay amenable to high-throughput screening (HTS) using VMAT2-transfected HEK cells (Z'-factor of approximately 0.7–0.8), enabling rapid identification of VMAT2 inhibitors and measurement of their inhibition constants over a broad range of affinities. FFN206 thus represents a new tool for optical examination of VMAT2 function in cell culture.