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首页> 美国卫生研究院文献>The Journal of General Physiology >Light inactivation of water transport and protein–protein interactions of aquaporin–Killer Red chimeras
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Light inactivation of water transport and protein–protein interactions of aquaporin–Killer Red chimeras

机译：水运输的光灭活和水通道蛋白-杀手红嵌合体的蛋白质-蛋白质相互作用

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摘要

Aquaporins (AQPs) have a broad range of cellular and organ functions; however, nontoxic inhibitors of AQP water transport are not available. Here, we applied chromophore-assisted light inactivation (CALI) to inhibit the water permeability of AQP1, and of two AQP4 isoforms (M1 and M23), one of which (M23) forms aggregates at the cell plasma membrane. Chimeras containing Killer Red (KR) and AQPs were generated with linkers of different lengths. Osmotic water permeability of cells expressing KR/AQP chimeras was measured from osmotic swelling–induced dilution of cytoplasmic chloride, which was detected using a genetically encoded chloride-sensing fluorescent protein. KR-AQP1 red fluorescence was bleached rapidly (∼10% per second) by wide-field epifluorescence microscopy. After KR bleaching, KR-AQP1 water permeability was reduced by up to 80% for the chimera with the shortest linker. Remarkably, CALI-induced reduction in AQP4-KR water permeability was approximately twice as efficient for the aggregate-forming M23 isoform; this suggests intermolecular CALI, which was confirmed by native gel electrophoresis on cells coexpressing M23-AQP4-KR and myc-tagged M23-AQP4. CALI also disrupted the interaction of AQP4 with a neuromyelitis optica autoantibody directed against an extracellular epitope on AQP4. CALI thus permits rapid, spatially targeted and irreversible reduction in AQP water permeability and interactions in live cells. Our data also support the utility of CALI to study protein–protein interactions as well as other membrane transporters and receptors.
机译：水通道蛋白（AQP）具有广泛的细胞和器官功能。但是，尚无AQP水传输的无毒抑制剂。在这里，我们应用了生色团辅助光灭活（CALI）来抑制AQP1和两种AQP4亚型（M1和M23）的水渗透性，其中一种（M23）在细胞质膜上形成聚集体。含有杀手红（KR）和AQP的嵌合体是用不同长度的接头生成的。表达KR / AQP嵌合体的细胞的渗透水渗透率是通过渗透溶胀诱导的细胞质氯化物稀释来测量的，这是使用遗传编码的氯化物敏感荧光蛋白检测到的。 KR-AQP1红色荧光通过宽视场荧光显微镜快速漂白（约每秒10％）。 KR漂白后，对于连接子最短的嵌合体，KR-AQP1的水渗透率降低了80％。值得注意的是，CALI诱导的AQP4-KR透水性降低对形成聚集体的M23同工型的效率大约是两倍。这暗示了分子间CALI，这通过共表达M23-AQP4-KR和myc标签的M23-AQP4的细胞的天然凝胶电泳证实。 CALI还破坏了AQP4与针对AQP4上细胞外表位的视神经脊髓炎自身抗体的相互作用。因此，CALI可以迅速，在空间上有针对性地，不可逆地降低AQP的水渗透性和活细胞中的相互作用。我们的数据还支持CALI在研究蛋白质间相互作用以及其他膜转运蛋白和受体方面的实用性。

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