Super-resolution imaging of potassium channels with genetically encoded EGFP.
Super-resolution imaging of potassium channels with genetically encoded EGFP.
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使用基因编码的 EGFP 对钾通道进行超分辨率成像。
DOI：
10.1101/2023.10.13.561998
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Hansen,ScottB
中科院分区：
文献类型：
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作者：
Call,IsabelleM;Bois,JulianL;Hansen,ScottB
质膜是脂质和蛋白质的良好组织结构，分割成大小在200 nm以下的脂质隔室。这种特定的空间模式对于蛋白质的功能至关重要，需要超分辨率成像来阐明。在这里，我们建立了基因编码的增强型绿色荧光蛋白（EGFP），当结合直接光学重建显微镜（dSTORM），跟踪剪切和胆固醇诱导的纳米级图案的钾通道过表达的HEK 293 T细胞。利用dSTORM中的EGFP（EGFP-STORM），我们的研究结果表明，胆固醇将TWIK相关钾通道（TREK-1）的C-末端导向富含神经酰胺的脂质神经节苷脂（GM 1）簇。在没有C-末端的情况下，通道与磷脂酰肌醇4，5-二磷酸（PIP 2）簇缔合。类似地，来自星形胶质细胞的胆固醇将EGFP标记的内向整流钾（Kir）通道重新定位为GM 1簇。在没有胆固醇的情况下，通道与PIP 2脂质对齐。我们推断胆固醇与Kir的相互作用隔离了通道，将其与其激活的脂质PIP 2分离。从根本上说，基因编码的EGFP标签应该使任何蛋白质都适合dSTORM分析。
The plasma membrane is a well-organized structure of lipids and proteins, segmented into lipid compartments under 200 nm in size. This specific spatial patterning is crucial for the function of proteins and necessitates super-resolution imaging for its elucidation. Here, we establish that the genetically encoded enhanced green fluorescent protein (EGFP), when combined with direct optical reconstruction microscopy (dSTORM), tracks shear- and cholesterol-induced nanoscopic patterning of potassium channels overexpressed in HEK293T cells. Leveraging EGFP in dSTORM (EGFP-STORM), our findings indicate that cholesterol directs the C-terminus of TWIK-related potassium channel (TREK-1) to ceramide-enriched lipid ganglioside (GM1) clusters. In the absence of the C-terminus, the channel associates with phosphatidylinositol 4,5-bisphosphate (PIP2) cluster. Similarly, cholesterol derived from astrocytes repositions EGFP-tagged inward-rectifying potassium (Kir) channels into GM1 clusters. Without cholesterol, the channel aligns with PIP2 lipids. We deduce that cholesterol’s interaction with Kir sequesters the channel, separating it from its activating lipid PIP2. Fundamentally, a genetically encoded EGFP tag should make any protein amenable to dSTORM analysis.