Bats Cecile, Coombs Ian D, Farrant Mark, Cull-Candy Stuart G
Department of Neuroscience, Physiology and Pharmacology, University College London, Gower Street, London WC1E 6BT, UK.
Department of Neuroscience, Physiology and Pharmacology, University College London, Gower Street, London WC1E 6BT, UK.
Neuroscience. 2025 Jul 10;578:81-90. doi: 10.1016/j.neuroscience.2024.08.036. Epub 2024 Aug 30.
In mammalian central neurons AMPARs are clustered at glutamatergic synapses where they mediate fast excitatory transmission. In addition to four pore-forming subunits (GluA1-4), AMPARs contain auxiliary transmembrane AMPAR regulatory proteins (γ2, γ3, γ4, γ5, γ7 or γ8) whose incorporation can vary between neuron types, brain regions, and stages of development. As well as modulating the functional properties of AMPARs, these auxiliary subunits play a central role in AMPAR trafficking. Directly visualizing TARPs could therefore provide a valuable insight into mechanisms underlying these processes. Although antibodies are routinely used for the detection of surface proteins, our experience suggests anti-TARP antibodies are too bulky to access their target, possibly due to close interactions between the extracellular domains of TARP and AMPAR subunits. We therefore assessed the utility of a small monovalent probe - fluorescent α-bungarotoxin (α-Btx) - for TARP labelling in living neurons. We inserted the bungarotoxin binding site (BBS) within the extracellular domain of TARPs to enable their detection in cells exposed to fluorescent α-Btx. Focusing on the prototypical TARP γ2, we demonstrate that the small size of fluorescent α-Btx allows it to bind to the BBS-tagged TARP when associated with AMPARs. Importantly, labelled γ2 enhances AMPAR function in the same way as unmodified γ2. In living neurons, fluorescent α-Btx-labelled γ2 associates with AMPAR clusters at synapses. As a proof-of-principle, we employed our method to compare the surface trafficking of γ2 and γ7 in cerebellar stellate neurons. Our approach provides a simple way to visualize TARPs within AMPARs in living cells.
在哺乳动物中枢神经元中，α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体（AMPARs）聚集在谷氨酸能突触处，介导快速兴奋性传递。除了四个形成孔道的亚基（GluA1-4）外，AMPARs还包含辅助跨膜AMPAR调节蛋白（γ2、γ3、γ4、γ5、γ7或γ8），其整合情况在不同神经元类型、脑区和发育阶段会有所不同。这些辅助亚基除了调节AMPARs的功能特性外，在AMPAR转运中也起着核心作用。因此，直接可视化跨膜AMPAR调节蛋白（TARPs）可以为这些过程背后的机制提供有价值的见解。尽管抗体通常用于检测表面蛋白，但我们的经验表明，抗TARP抗体体积太大，无法接近其靶点，这可能是由于TARP的细胞外结构域与AMPAR亚基之间的紧密相互作用所致。因此，我们评估了一种小的单价探针——荧光α-银环蛇毒素（α-Btx）——在活神经元中标记TARPs的效用。我们将银环蛇毒素结合位点（BBS）插入TARPs的细胞外结构域内，以便在暴露于荧光α-Btx的细胞中对其进行检测。以典型的TARP γ2为重点，我们证明荧光α-Btx的小尺寸使其在与AMPARs结合时能够与带有BBS标签的TARP结合。重要的是，标记的γ2与未修饰的γ2一样增强了AMPAR功能。在活神经元中，荧光α-Btx标记的γ2与突触处的AMPAR簇相关联。作为原理验证，我们采用我们的方法比较了γ2和γ7在小脑星状神经元中的表面转运。我们的方法提供了一种在活细胞中可视化AMPARs内TARPs的简单方法。
