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 (Xerocytosis) and lymphatic dysplasia. Here we utilize an established in-vitro 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.

机械敏感离子通道是由机械刺激激活的膜门控孔。这项研究的重点是Piezo1，这是一种新发现的，大型的哺乳动物机械敏感离子通道，已与脱水遗传性气孔细胞增多症（Xerocytosis）和淋巴发育不良等疾病相关。在这里，我们利用已建立的体外方法人工双层系统询问单个Piezo1通道的活动。液滴-水凝胶双层（DHB）系统独特地允许同时记录电活动和标记蛋白的荧光成像。我们成功地重建了DHBs中荧光标记的Piezo1离子通道，并在同一系统中使用电生理学验证了活性。我们证明了hPiezo1-GFP在不同组成的双层中的成功插入和激活。此外，我们比较了Piezo1双层重构与KcsA通道插入和激活的测量，以再现文献中报道的通道电导。总之，我们的结果证明了DHB在未来的实验中的使用，从而可以同时测量离子通道门控，同时使用荧光可视化通道蛋白。