Fluorescence microscopy of piezo1 in droplet hydrogel bilayers

Mechanosensitive ion channels are membrane gated pores which are activated by mechanical stimuli. The focus of this study is on Piezo1, a newly discovered, large, mammalian, mechanosensitive ion channel, which has been linked to diseases such as dehydrated hereditary stomatocytosis (<i>Xerocytosis</i>) and lymphatic dysplasia. Here we utilize an established <i>in-vitro</i> artificial bilayer system to interrogate single Piezo1 channel activity. The droplet-hydrogel bilayer (DHB) system uniquely allows the simultaneous recording of electrical activity and fluorescence imaging of labelled protein. We successfully reconstituted fluorescently labelled Piezo1 ion channels in DHBs and verified activity using electrophysiology in the same system. We demonstrate successful insertion and activation of hPiezo1-GFP in bilayers of varying composition. Furthermore, we compare the Piezo1 bilayer reconstitution with measurements of insertion and activation of KcsA channels to reproduce the channel conductances reported in the literature. Together, our results showcase the use of DHBs for future experiments allowing simultaneous measurements of ion channel gating while visualising the channel proteins using fluorescence.

机械敏感离子通道（Mechanosensitive ion channels）是一类可被机械刺激激活的膜门控孔道。本研究聚焦于新近发现的大型哺乳动物源机械敏感离子通道Piezo1，该通道与脱水遗传性口形红细胞增多症（Xerocytosis）及淋巴发育异常等疾病密切相关。本研究利用已成熟建立的体外（in vitro）人工双层膜系统，探究单个Piezo1通道的活性。液滴-水凝胶双层膜（droplet-hydrogel bilayer, DHB）系统可实现标记蛋白的电活动记录与荧光成像同步进行，具备独特的实验优势。我们成功将荧光标记的Piezo1离子通道重组至DHB中，并通过同一系统内的电生理学实验验证了其通道活性。我们证实了人源Piezo1-绿色荧光蛋白融合体（hPiezo1-GFP）可在不同组成的双层膜中完成有效插入与激活。此外，我们将Piezo1的双层膜重组实验与KcsA通道的插入及活性检测结果进行对比，成功复现了文献中报道的通道电导值。综上，本研究结果展示了DHB在未来实验中的应用价值，即可同步实现离子通道门控机制的检测与通道蛋白的荧光可视化。