Center for Neurodegenerative Disease, Emory University, Atlanta, GA 30322, United States; Department of Environmental Health, Rollins School of Public Health, Emory University, Atlanta, GA 30322, United States.
J Neurosci Methods. 2012 Aug 15;209(2):357-66. doi: 10.1016/j.jneumeth.2012.06.002. Epub 2012 Jun 12.
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.
囊泡单胺转运体 2（VMAT2；Slc18a2）将单胺包装到突触小泡中。单胺稳态受到高度调节，功能障碍可能在帕金森病、亨廷顿病、药物成瘾和神经精神疾病中发挥作用。VMAT2 的主要功能是将单胺神经递质隔离到小泡中以备随后释放；它还将有毒物质隔离到细胞质作用部位之外。鉴定修饰 VMAT2 作用的化合物可能有助于作为预防或逆转与单胺相关毒性的治疗剂。目前测量 VMAT2 功能的方法无法评估完整细胞中的摄取。在这里，我们改编了神经递质摄取测定法（Molecular Devices）来开发一种测量活整个细胞中 VMAT2 功能的方法。该测定法包含一种荧光化合物，该化合物通过质膜单胺转运体被转运到细胞中，并已作为一种快速、高通量、基于平板阅读器的质膜转运体功能测定法上市。我们展示了该测定法的一种改良版本，该版本可用于可视化和测量 VMAT2 介导的转运到小泡中。生成了稳定表达多巴胺转运体和 mCherry-VMAT2 融合蛋白的 HEK293 细胞系。共焦显微镜证实，荧光化合物被转运到 mCherry 阳性隔室中。此外，VMAT2 特异性抑制剂四苯嗪（TBZ）阻断了 mCherry 阳性隔室中的摄取。可以分析共焦图像以生成 VMAT2 活性的度量。总之，我们展示了一种用于分析活完整细胞中 VMAT2 介导摄取的空间分辨分析方法。
