Figure 1. Physical constrictions in the CTD play only moderate roles in Piezo1 inactivation.

(A) Topology diagram and cryo-EM structure of Piezo1 (PDB: 6BPZ). IH, inner pore helix; OH, outer pore helix. (B) Side view and top view of the Piezo1 pore region from three cryo-EM structures showing the location of the MF and PE constrictions. (C) Top view close-up of the MF and PE constrictions in Piezo1 (PDB: 6BPZ). (D) Representative whole-cell MA current traces and quantification of MA current inactivation rate (τ_inact) in HEK293T^ΔP1 cells expressing Piezo1 with mutations at the M2493 F2494 (MF) site (n = 7–9 cells). E_hold = −80 mV. ***p<0.001; NS, not significant, p>0.05, one-way ANOVA with Holm-Sidak’s correction. (E and F) Representative whole-cell MA current traces and quantification of MA current inactivation for WT Piezo1 and P2536G/E2537G mutant. **p<0.001, unpaired t-test. (G) Quantification of peak MA current amplitude (I_peak) at different indentation depths for WT Piezo1 and P2536G/E2537G mutant. ***p<0.001, two-way ANOVA. Data are mean ± SEM.

Figure 1—figure supplement 1. Mutations at the Piezo1 PE site accelerate deactivation of MA current.

(A and B) Representative whole-cell MA current traces recorded in HEK293T^ΔP1 cells expressing Piezo1 with mutations at the putative inactivation gate (L2475S/V2476S) and PE constriction (P2536G/E2537G). E_hold = −80 mV. Current traces were normalized to peak current amplitude to highlight the differences in the kinetics of persistent current decay. Blue arrows indicate points of measurements of current amplitude at the end of mechanical stimulus (I_post) and 100 ms after the removal of mechanical stimulus (I_remaining). (C) Quantification of I_remaining/I_post for the indicated mPiezo1 mutants (***p<0.001, unpaired t-test). Data are mean ± SEM.