(A) Stimulus protocol and representative currents from a cell-attached HEK293t cell patch expressing mouse Piezo1-IRES-GFP. The test pulse for these currents was −10 mmHg (thick purple line); holding potential was −80 mV. Inset shows test currents at magnified scale. (B) Peak current-pressure relationships for test pulses (0 to −50mmHg, Δ5 mmHg) following 5 s 0 mmHg, +5 mmHg and +10 mmHg prepulses.. All data points are mean ± s.e.m. N = 8 cell-attached patches (pcDNA) and 11 cell-attached patches (Piezo1). (C) Mean patch curvature as a function of time during protocol performed shown in (A). Representative images of one individual patch are shown above. Each patch was tested with no prepulse (0 mmHg), a +5 mmHg prepulse, and a +10 mmHg prepulse at each test pressure before advancing to the next test pressure. Grey markers show inverse radius during rest periods (0 mmHg, between stimuli), purple markers show inverse radius during 300 ms test pulses (0 to −50 mmHg, Δ5 mmHg), orange markers show inverse radius during +5 mmHg or +10 mmHg prepulse. All data points are mean ± s.e.m. N = 11 for cell-attached patches. (D–F) Normalized current-tension relationships obtained from protocol shown in (A) using no prepulse (0 mmHg) (D), +5 mmHg prepulse (E) and +10 mmHg prepulse (F). Currents from individual patches are normalized to the maximal response for each patch. Data were pooled and binned (bin width 1 mN/m); bars represent mean ± s.e.m. N = 11 patches. Binned data were fit with a Boltzmann function I = Imax/(1+exp(-(T-T50/k)) where I is normalized current, Imax is the plateau, T is tension, T50 is the tension of half-maximal activation, and k is the slope factor. The standard deviation of the normalized amplitude for each bin was used to weight the fit. Fit parameters for no prepulse (0 mmHg): Imax = 0.84±0.02, T50 = 2.2±0.1 mN/m, k = 0.8±0.1. For +5 mmHg prepulse: Imax = 0.85±0.01, T50 = 1.4±0.1 mN/m, k = 0.7±0.1. For +10 mmHg prepulse: Imax = 0.70±0.04, T50 = 1.8±0.2 mN/m, k = 1.1±0.2. (G) Fits from D-F overlayed (solid line) with 95% confidence intervals (dashed lines).