Fluorescence Microscopy of Piezo1 in Droplet Hydrogel Bilayers

Abstract

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.

Piezo1 mechanosensitive channels: what are they and why are they important

“…Though similar to the bacterial MscL and MscS channels, Piezo1 also functions in reconstituted planar bilayers (Jaggers et al 2019; Syeda et al 2016), and critical role of lipids for the Piezo1 channel activity has clearly been established (Cox et al 2016b). Recent experimental work conducted in our laboratory demonstrated how purified Piezo1 could be activated by applying pipette suction in patch clamp experiments on reconstituted proteoliposomes (Fig.…”