Fig. 5

PIEZO chimeras respond to membrane stretch. a Chimeric PIEZO channels were constructed by fusing the N-terminal region of one protein to the pore domain of the other protein including the CED region. PIEZO1, PIEZO2, and the chimeric channels were overexpressed in N2a ^{Piezo1−/−} cells (Supplementary Fig. 3). Cells were clamped at −60 mV and subjected to soma indentation. b The maximum current amplitude (normalized for the capacitance) is plotted. The time course of inactivation for each construct was fitted to a mono-exponential function. PIEZO1 inactivation was approximately threefold slower than PIEZO2 and the chimeric receptor was P1/P2 (ANOVA, dF = 25, Dunnett’s post-hoc test P < 0.023). The P1/P2 chimera showed a current amplitude and a time course of inactivation similar to PIEZO2. The P2/P1 chimera showed a current amplitude and a time course of inactivation similar to PIEZO1. c Typical response of the chimeric channels and wild-type PIEZO1 to membrane stretch in outside-out patches. Outside-out patches pulled from cells overexpressing PIEZO2 did not respond to stretch stimulation. d Maximal current level recorded in outside-out patches at −60 mV. Current levels for P2/P1 were significantly different from PIEZO1 patches (n = 10, Student’s t-test, P = 0.0003, dF = 25. e Pressure–response relationships of the chimeric channels are not different from PIEZO1 (P1/P2 37.6 ± 4.6 mmHg, 10 cells; PIEZO1 38.9 ± 3.1 mmHg, 21 cells; P2/P1 42.4 ± 4.7 mmHg, 7 cells, Anova Dunnet’s post-test P = 0.78, dF = 32). f The decay of inactivation for pressure-mediated responses at +60 and −60 mV is plotted for the chimeric and PIEZO1 channels. Also for pressure-mediated responses, the kinetic of inactivation of PIEZO1 remained approximately threefold slower than the chimeric channel P1/P2 (P1/P2 26.3 ± 6.2 ms, 10 cells; PIEZO1 60.9 ± 5.1 ms at −60 mV, 9 cells, Student’s t-test, P = 0.0025, dF = 17). The P2/P1 chimera did not show any inactivation properties. g Example traces of tail current protocols (at 70 mmHg) for chimeric channels ranging from 0 to 150 mV, followed by a repolarization step to −60 mV. Tail currents (−60 mV) from individual cells were normalized to their maximum and plotted against voltage (n = 10). Pooled data mean ± SEM are shown