(A) Left panel, amino acid sequence alignment of the Piezo1 inner helix (IH) from different species. A cluster of five conserved hydrophobic residues in the middle are highlighted. Red and blue dots indicate hydrophobic residues facing and pointing away from the pore, respectively. Right panel, cryo-EM structure of the Piezo1 inner helix (PDB: 6BPZ) showing the hydrophobic residues in the left panel. (B) Representative whole-cell MA current traces and quantification of MA current inactivation rate (τinact) in HEK293TΔP1 cells expressing Piezo1 with mutations in the hydrophobic cluster in the inner helix (n = 8–22 cells). Ehold = −80 mV. ***p<0.001; NS, not significant, p>0.05, one-way ANOVA with Dunnet’s correction. (C–E) Quantification of peak MA current amplitude (Ipeak) at different indentation depths (C), apparent indentation threshold of MA current activation (D) and MA current rise time (E) for WT and mutant Piezo1. NS, not significant, p>0.05, one-way ANOVA with Dunnet’s correction. (F) Peak MA current-voltage relationship in response to mechanical indentation at 9 μm for WT Piezo1 or indicated mutants. Insets show representative traces of whole-cell MA currents evoked at Ehold ranging from −100 mV to +100 mV, in 20 mV increments. (G) Quantification of the reversal potential (Erev) from current-voltage plots in (F). NS, not significant, p>0.05, one-way ANOVA with Dunnet’s correction. (H) Quantification of MA current inactivation rate for WT or mutant Piezo1 at different voltages. Data are mean ± SEM.