Visualizing GABA A Receptor Trafficking Dynamics with Fluorogenic Protein Labeling.
Visualizing GABA A Receptor Trafficking Dynamics with Fluorogenic Protein Labeling.
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DOI：
10.1002/cpns.97
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发表时间：
2020-06
影响因子：
--
通讯作者：
Jacob TC
中科院分区：
文献类型：
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作者：
Lombardi JP;Kinzlmaier DA;Jacob TC
越来越多的证据表明，神经递质受体，包括离子型GABA A受体(GABAARs)，表现出高度动态的转运和细胞表面的流动性。受管制的来往于地表的贩运是GABAAR神经传递的关键决定因素。胞吐递送的受体在质膜上横向扩散，通过受体与突触下支架的相互作用，在突触处发生拴系和运动减少。在从突触扩散出去后，受体在突触外位置被依赖于笼蛋白的内吞作用内化，并可以循环回到细胞膜或在溶酶体中降解。为了研究神经元中这些关键转运事件的动力学，我们开发了基于含有双标记γ2亚单位(γ2PHFAP)的受体与荧光染料相结合的新的光学方法。具体地说，GABAARγ2亚基被一个对pH敏感的绿色荧光蛋白和一个荧光素激活肽标记。FAP允许用荧光染料标记受体，这些染料在结合到FAP之前是光学无声的。将FAP/荧光成像与细胞器标记相结合，可以在从体外癫痫模型到药物暴露范例的基本条件和情景下，新颖而准确地测量受体在细胞内的周转和积聚。在这里，我们提供了一种方案来跟踪和量化从神经元表面到内小体和溶酶体的受体。该方案很容易适用于细胞系和原代细胞，允许快速定量测量受体表面水平和内吞后转运决策。
It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAARs), exhibit highly dynamic trafficking and cell surface mobility. Regulated trafficking to and from the surface is a critical determinant of GABAAR neurotransmission. Receptors delivered by exocytosis diffuse laterally in the plasma membrane, with tethering and reduced movement at synapses occurring through receptor interactions with the sub-synaptic scaffold. After diffusion away from synapses, receptors are internalized by clathrin-dependent endocytosis at extrasynaptic sites and can be either recycled back to the cell membrane or degraded in lysosomes. To study the dynamics of these key trafficking events in neurons, we have developed novel optical methods based around receptors containing a dual-tagged γ2 subunit (γ2pHFAP) in combination with fluorogen dyes. Specifically, the GABAAR γ2 subunit is tagged with a pH-sensitive green fluorescent protein and a fluorogen-activating peptide (FAP). The FAP allows receptor labeling with fluorogen dyes that are optically silent until bound to the FAP. Combining FAP/fluorescent imaging with organelle labeling allows novel and accurate measurement of receptor turnover and accumulation into intracellular compartments under basal conditions and scenarios ranging from in vitro seizure models to drug exposure paradigms. Here we provide a protocol to track and quantify receptors in transit from the neuronal surface to endosomes and lysosomes. This protocol is readily applicable to cell lines and primary cells, allowing rapid quantitative measurements of receptor surface levels and post-endocytic trafficking decisions.