A fluorescent-based assay for live cell, spatially resolved assessment of vesicular monoamine transporter 2-mediated neurotransmitter transport.
A fluorescent-based assay for live cell, spatially resolved assessment of vesicular monoamine transporter 2-mediated neurotransmitter transport.
复制标题
DOI：
10.1016/j.jneumeth.2012.06.002
复制
发表时间：
2012-08-15
影响因子：
3
通讯作者：
Miller GW
中科院分区：
文献类型：
--
作者：
Bernstein AI;Stout KA;Miller GW
囊泡单胺转运体2(VMAT2；SLC18A2)将单胺包装成突触小泡。单胺稳态是高度调节的，功能障碍可能在帕金森氏病、亨廷顿病、药物成瘾和神经精神障碍中发挥作用。VMAT2的主要功能是将单胺类神经递质隔离到囊泡中，以便随后释放；它还将毒物从胞浆作用部位隔离开来。鉴定可以改变VMAT2作用的化合物可能有助于作为治疗药物来预防或逆转单胺相关的毒性。目前测量VMAT2功能的方法不能评估完整细胞的摄取。在这里，我们采用了神经递质摄取试验(分子装置)来开发一种在活的整个细胞中测量VMAT2功能的方法。这种检测方法包含一种荧光化合物，它由质膜单胺转运体转运到细胞内，并已作为一种快速、高通量、基于平板阅读器的检测这些质膜转运体功能的方法上市。我们展示了该方法的改进版本，该方法可用于可视化和测量VMAT2向囊泡的转运。建立了稳定表达多巴胺转运蛋白和mCherry-VMAT2融合蛋白的HEK293细胞株。共聚焦显微镜证实，荧光化合物被运输到mCherry阳性的隔室。此外，VMAT2特异性抑制剂四苯肼(TBZ)阻断了对mCherry阳性隔室的摄取。可以分析共焦图像以生成VMAT2活动的测量。综上所述，我们展示了一种空间分辨分析VMAT2介导的活细胞摄取的方法。
The vesicular monoamine transporter 2 (VMAT2; Slc18a2) packages monoamines into synaptic vesicles. Monoamine homeostasis is highly regulated and dysfunction may play a role in Parkinson’s disease, Huntington’s disease, drug addiction, and neuropsychiatric disorders. The primary function of VMAT2 is to sequester monoamine neurotransmitters into vesicles for subsequent release; it also sequesters toxicants away from cytosolic sites of action. Identification of compounds that modify the action of VMAT2 may be useful as therapeutic agents for preventing or reversing monoamine-related toxicity. Current methods for measuring VMAT2 function are unable to assess uptake in intact cells. Here, we adapted the Neurotransmitter Uptake Assay (Molecular Devices) to develop a measure of VMAT2 function in live whole cells. This assay contains a fluorescent compound, which is transported into cells by the plasma membrane monoamine transporters and has been marketed as a rapid, high-throughput, plate reader based assay for function of these plasma membrane transporters. We demonstrate a modified version of this assay that can be used to visualize and measure transport into vesicles by VMAT2. HEK293 cell lines stably expressing the dopamine transporter and a mCherry-VMAT2 fusion protein were generated. Confocal microscopy confirmed that the fluorescent compound is transported into mCherry-positive compartments. Furthermore, the VMAT2-specific inhibitor tetrabenazine (TBZ) blocks uptake into the mCherry-positive compartment. Confocal images can be analyzed to generate a measure of VMAT2 activity. In summary, we demonstrate a method for spatially resolved analysis of VMAT2-mediated uptake in live intact cells.
登录
查看更多内容
影响因子：
4.7
作者：
Meyer, A. C.;Horton, D. B.;Neugebauer, N. M.;Wooters, T. E.;Nickell, J. R.;Dwoskin, L. P.;Bardo, M. T.
通讯作者：
Bardo, M. T.
影响因子：
5.2
作者：
Eiden LE;Weihe E
通讯作者：
Weihe E
影响因子：
3.8
作者：
Bemis, JC;Seegal, RF
通讯作者：
Seegal, RF
影响因子：
3.8
作者：
Richardson, JR;Quan, Y;Miller, GW
通讯作者：
Miller, GW
影响因子：
56.9
作者：
Bellocchio, EE;Reimer, RJ;Edwards, RH
通讯作者：
Edwards, RH