Long-range retrograde mechanical coupling in the PIEZO1 channel

Abstract

Mechanosensitive PIEZO channels possess a unique bowl-like structure spanning approximately 25 nm in diameter, making them some of the largest membrane proteins known to date. We had previously identified gating-associated conformational changes in two discrete regions of the N-terminal mechanosensory domain of PIEZO1 by temporally correlating fluorescence signals from genetically-inserted probes (cyclic permuted GFPs) with fluid shear stress-induced channel activation, measured using calcium imaging. Here, we show that signals from both probes are largely unchanged by mutations known to accelerate or slow down PIEZO1 inactivation, suggesting our probes capture gating transitions from resting to open/inactivated states. Likewise, application of the mechanosensitive channel inhibitor GsMtx4 does not inhibit these signals, despite the ability of the toxin to inhibit shear stress-induced PIEZO1-dependent calcium responses. Remarkably, shear stress-induced signals emitted by the most distal probe, near the N-terminus, are strongly inhibited in a redox-dependent manner by introducing a previously reported inhibitory disulfide bridge in the C-terminal central pore region, located more than 10 nm away from the probe. Whereas anterograde mechanical coupling from sensory domains to pore domains forms a prevailing paradigm for stimulus-mediated ion channel activation, our results suggest that this coupling also exists in the retrograde direction in PIEZO1, from the pore to the mechanosensory domain.