Failure of deactivation in PIEZO1^WT/M-R and PIEZO1^M-R/M-R single channels. Data are for single-channel activity measured by the cell-attached patch technique applied to freshly isolated RBCs of WT (A-B), PIEZO1^WT/M-R (C-D), and PIEZO1^M-R/M-R (E-H) mice. A fast pressure-clamp system applied brief (200 ms) negative pressure pulses to the patches, as indicated below the original exemplar current traces (A,C,E,G,H). Dashed horizontal lines indicate current levels for closed channels (c) and open channels (o, or o1-4 for multiple channel openings in panels C and G). Constant voltage of +80 mV was applied to the patch pipette. (A,E) Data are considered to be for patches each containing only 1 PIEZO1 channel; (C,G) patches contain multiple PIEZO1 channels. (B,D,F) Amplitude histogram analysis for the exemplar traces, showing plots of the frequency of detection of events (N) at the current amplitudes indicated on the x-axis, where zero current indicates no current flowing in the circuit. The closed channel state appears as a small negative current peaking at −0.5 (B), −0.7 (D), and −0.4 pA (F). (Bi) The small amount of open channel activity at −2.5 pA is shown in an expansion. Raw data are shown with superimposed Gaussian fits in green (WT), blue (PIEZO1^WT/M-R), and red (PIEZO1^M-R/M-R). Representative of n = 6, 5, and 4 for WT, PIEZO1^WT/M-R, and PIEZO1^M-R/M-R, respectively. (H) An exemplar recording from a PIEZO1^M-R/M-R RBC patch with 2.5 μM GsMTx4 in the extracellular solution (n = 5).