Figure 1.
Slow kinetics of RBC PIEZO1 mice and slow recovery in PIEZO1WT/M-R and PIEZO1M-R/M-R mice. (A-C) Membrane potential (Vm) recordings obtained using the perforated-patch, whole-cell mode applied to freshly isolated RBCs from WT (A), PIEZO1WT/M-R (B), and PIEZO1M-R/M-R (C) mice. RBCs were exposed to 20 µL·s−1 fluid flow for 20 seconds, as indicated by the shaded areas. (D) Summary data for experiments of the type shown in panels A-C. Presented is the peak (maximum [max]) change in Vm of WT, PIEZO1WT/M-R, and PIEZO1M-R/M-R RBC counts after exposure to flow and Vm at 1 to 5 minutes after flow. Averaged data are displayed as means ± standard deviation, and each data point is shown. WT: n = 20 (max, 1 minute), 16 (2 minutes), 13 (3 minutes), and 9 (4 and 5 minutes); PIEZO1WT/M-R: n = 13 (max), 12 (1 minute), 11 (2 minutes), 10 (3 minutes), and 9 (4 and 5 minutes); and PIEZO1M-R/M-R: n = 14 (max, 1 minute), 13 (2 minutes), 11 (3 minutes), 9 (4 minutes), and 8 (5 minutes). (E) As in panel D, except with 2.5 μM GsMTx4 in the extracellular solution. WT: n = 5 (max, 1 minute, 2 minutes, 3 minutes), 4 (4 minutes), and 3 (5 minutes); PIEZO1WT/M-R: n = 7 (max, 1 minute), 6 (2 minutes), 4 (3 and 4 minutes), 3 (5 minutes); and PIEZO1M-R/M-R: n = 8 (max), 6 (1 minute), 5 (2 minutes), 4 (3 minutes), and 3 (4 and 5 minutes). (F-H) Ionic current recordings obtained using the perforated-patch technique in whole-cell, voltage-clamp mode applied to freshly isolated RBCs from WT (F), PIEZO1WT/M-R (G), and PIEZO1M-R/M-R mice (H). RBCs were exposed to 20 µL·s−1 fluid flow for 20 seconds, as indicated by the shaded areas. Holding voltage was −80 mV. (I) Summary data for experiments of the type shown in panels F-H. Presented is the peak (max) change in current (ΔI) of WT, PIEZO1WT/M-R RBCs after exposure to flow and then the ΔI at 1 and 2 minutes after flow. Averaged data are means ± standard deviation, and individual data points are shown. WT: n = 23 (max, 1 minute) and 15 (2 minutes); PIEZO1WT/M-R: n = 12 (max, 1 minute) and 10 (2 minutes); and PIEZO1M-R/M-R: n = 18 (max), 15 (1 minute), and 11 (2 minutes). (J) As in panel I, except with 2.5 μM GsMTx4 in the extracellular solution. WT: n = 5 (max, 1 minute, 2 minutes); PIEZO1WT/M-R: n = 4 (max, 1 minute), 3 (2 minutes); and PIEZO1M-R/M-R: n = 4 (max, 1 minute, 2 minutes). Statistical analysis by one-way ANOVA with Bonferroni’s post hoc test indicated: (D,I) significant decay of the WT (**P < .01; *** P < .001) but not mutant RBC responses; (D) significant increase in the peak response in PIEZO1M-R/M-R RBCs compared with WT (P < .05); (E) no significant effects of flow in WT or PIEZO1WT/M-R, but significant hyperpolarization at 1 minute compared with the peak in PIEZO1M-R/M-R (P < .01); and (J) no significant effects of flow in WT or PIEZO1WT/M-R but significant outward current at 1 and 2 minutes compared with peak in PIEZO1M-R/M-R (P < .001).

Slow kinetics of RBC PIEZO1 mice and slow recovery in PIEZO1WT/M-R and PIEZO1M-R/M-R mice. (A-C) Membrane potential (Vm) recordings obtained using the perforated-patch, whole-cell mode applied to freshly isolated RBCs from WT (A), PIEZO1WT/M-R (B), and PIEZO1M-R/M-R (C) mice. RBCs were exposed to 20 µL·s−1 fluid flow for 20 seconds, as indicated by the shaded areas. (D) Summary data for experiments of the type shown in panels A-C. Presented is the peak (maximum [max]) change in Vm of WT, PIEZO1WT/M-R, and PIEZO1M-R/M-R RBC counts after exposure to flow and Vm at 1 to 5 minutes after flow. Averaged data are displayed as means ± standard deviation, and each data point is shown. WT: n = 20 (max, 1 minute), 16 (2 minutes), 13 (3 minutes), and 9 (4 and 5 minutes); PIEZO1WT/M-R: n = 13 (max), 12 (1 minute), 11 (2 minutes), 10 (3 minutes), and 9 (4 and 5 minutes); and PIEZO1M-R/M-R: n = 14 (max, 1 minute), 13 (2 minutes), 11 (3 minutes), 9 (4 minutes), and 8 (5 minutes). (E) As in panel D, except with 2.5 μM GsMTx4 in the extracellular solution. WT: n = 5 (max, 1 minute, 2 minutes, 3 minutes), 4 (4 minutes), and 3 (5 minutes); PIEZO1WT/M-R: n = 7 (max, 1 minute), 6 (2 minutes), 4 (3 and 4 minutes), 3 (5 minutes); and PIEZO1M-R/M-R: n = 8 (max), 6 (1 minute), 5 (2 minutes), 4 (3 minutes), and 3 (4 and 5 minutes). (F-H) Ionic current recordings obtained using the perforated-patch technique in whole-cell, voltage-clamp mode applied to freshly isolated RBCs from WT (F), PIEZO1WT/M-R (G), and PIEZO1M-R/M-R mice (H). RBCs were exposed to 20 µL·s−1 fluid flow for 20 seconds, as indicated by the shaded areas. Holding voltage was −80 mV. (I) Summary data for experiments of the type shown in panels F-H. Presented is the peak (max) change in current (ΔI) of WT, PIEZO1WT/M-R RBCs after exposure to flow and then the ΔI at 1 and 2 minutes after flow. Averaged data are means ± standard deviation, and individual data points are shown. WT: n = 23 (max, 1 minute) and 15 (2 minutes); PIEZO1WT/M-R: n = 12 (max, 1 minute) and 10 (2 minutes); and PIEZO1M-R/M-R: n = 18 (max), 15 (1 minute), and 11 (2 minutes). (J) As in panel I, except with 2.5 μM GsMTx4 in the extracellular solution. WT: n = 5 (max, 1 minute, 2 minutes); PIEZO1WT/M-R: n = 4 (max, 1 minute), 3 (2 minutes); and PIEZO1M-R/M-R: n = 4 (max, 1 minute, 2 minutes). Statistical analysis by one-way ANOVA with Bonferroni’s post hoc test indicated: (D,I) significant decay of the WT (**P < .01; *** P < .001) but not mutant RBC responses; (D) significant increase in the peak response in PIEZO1M-R/M-R RBCs compared with WT (P < .05); (E) no significant effects of flow in WT or PIEZO1WT/M-R, but significant hyperpolarization at 1 minute compared with the peak in PIEZO1M-R/M-R (P < .01); and (J) no significant effects of flow in WT or PIEZO1WT/M-R but significant outward current at 1 and 2 minutes compared with peak in PIEZO1M-R/M-R (P < .001).

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