Mechanosensitive PIEZO1 and PIEZO2 ion channels consist of a molecular assembly of three identical subunits. Yet, PIEZO1 and PIEZO2 subunits are structurally similar and are co-expressed by a discrete number of cells, suggesting that the PIEZO subunits could also form heterotrimers. Here, using bimolecular fluorescence complementation (BiFC), we first show that co-expressing PIEZO1 and PIEZO2 subunits, C-terminally tagged with complementary fragments of mNeonGreen2 (mNG2), rescues green fluorescence at the cell membrane of transfected HEK293 cells. We next split the calcium indicator GCaMP to show that the PIEZO1/PIEZO2 heteromeric assemblies are capable of eliciting calcium influx in response to a mechanical stimulus, suggesting that these heteromeric assemblies also function as mechanosensitive channels. We next used FLIM-FRET to demonstrate the physical proximity between the C termini of PIEZO1 and PIEZO2 subunits and to probe the stoichiometry of the PIEZO1/PIEZO2 heteromers. In conclusion, our multi-pronged approach provides compelling structural and functional evidence that the over-expression of PIEZO1 and PIEZO2 subunits into HEK293 cells promote the formation of heteromeric, membrane-bound, and mechanosensitive channels. Our findings challenge the current view that PIEZO channels exclusively exist as homotrimers and will prompt further studies to explore the existence and potential physiological roles of PIEZO heteromers in vivo.
Chandrasekharan et al. (Sun,) studied this question.