Skip to main content
. 2020 Jun 24;152(8):e201912515. doi: 10.1085/jgp.201912515

Figure 1.

Figure 1.

Modulation of PIEZO1-GFP activity in cell-attached patches of HEK293T cells in response to manipulation of cholesterol domains in the plasma membrane. (A–D) Example recordings from cell attached patches in response to the application of negative-pressure pulses of HEK293T cells stably expressing PIEZO1-GFP at +65 mV pipette voltage. Currents shown are from control cells incubated in serum-free medium for 30 min (A), cells treated with 100 µg/ml water-soluble cholesterol/MBCD complex (B), cells incubated in serum-free medium supplemented with 5 mM MBCD (C), or supplemented with 80 µM dynasore (D). The first pressure pulse and its associated current are highlighted in red. (E) Pressure–response curves comparing the effects of each compound on PIEZO1-GFP pressure sensitivity. Peak current values (I) are normalized to the maximal current (Imax) within the same recording and fitted with a Boltzmann distribution curve. (F) Midpoint activation pressures (P50) producing 50% of the maximal current response from each Boltzmann curve analyzed in E (one-way ANOVA; **, P < 0.005; n.s., not significant; n = 7–10). (G) Latency time in milliseconds of the PIEZO1-GFP response measured between the start of negative-pressure pulse and the onset of the maximal current within the same recording (n = 3, where each is the average of at least three consecutive pressure protocols). Average latencies are displayed for pulse pressures between −20 and −70 mmHg. (H) Average maximal current from cell-attached patches generated from each cell in all conditions. (I) Quantification of the mean steady-state current produced by each cell. The average current measured over the last 100 ms of the pressure stimulus was normalized to the maximal current within the same cell (n = 7 cells per condition). The steady-state current for each cell is the average mean steady-state current from a minimum of three pressure pulses between −40 and −60 mmHg. All data represent mean ± SEM; statistical analysis was performed using one-way ANOVA (**, P < 0.005; ***, P < 0.0005).