The slow-inactivation phenotype of the human PIEZO1 mutant R2456H is exacerbated by the removal of plasma membrane cholesterol via MBCD. The R2456H mutation was introduced in the human PIEZO1-1591-GFP clone, henceforth referred to as hPIEZO1 R2456H. (A) Example trace of a cell-attached recording from transiently overexpressed human hPIEZO1 R2456H mutant. (B) Example trace of a cell-attached recording from transiently overexpressed human hP1 R2456H treated with 5 mM MBCD for 30 min in serum-free medium. (C) Histogram quantifying the impact of 5-mM MBCD treatment on the normalized steady-state current. hPIEZO1 R2456H (n = 12); hPIEZO1 R2456H + 5 mM MBCD (n = 20). (D) This effect was accompanied by a longer latency to activation of the peak current at all pressures after treatment with MBCD. Control (n = 6), MBCD (n = 8). (E) Pressure–response curve of hPIEZO1 R2456H and MBCD-treated hPIEZO1 R2456H showing Boltzmann curve fits. hPIEZO1 R2456H (n = 5), hPIEZO1 R2456H + MBCD (n = 8). (F) Quantification of P₅₀ values obtained from Boltzmann fits analyzed in E. Statistical analysis was performed using one-way ANOVA (****, P < 0.0001; n = 1 represents the average of minimum three recordings from the same cell). Error bars in all graphs represent SEM.