Figure 2: Erythrocytes of patient II.1 display severely decreased PIEZO1 function.

Erythrocytes loaded with Fluo-4 were subjected to mechanical or chemical PIEZO1 stimulation. (a) Schematic of experimental set-up for mechanical stimulation of erythrocytes via negative pipette pressure. (b) Representative response of a control (healthy volunteer) red blood cell to mechanical stimulation, indicated by shaded areas on the trace. Magnitude of negative pressure is indicated above the shaded sections in mm Hg. (c) Average±s.e.m. of responses to negative pipette pressures in four different healthy volunteer individuals are shown as normalized to the response to the calcium ionophore A23187 (4 μM). (d) Average calcium responses of erythrocytes from the four volunteer individuals (depicted in different colours) to the PIEZO1 activator Yoda1 (5 and 15 μM). Cells were stimulated with the compound via whole-chamber perfusion after allowing them to settle and loosely attach to the bottom of the chamber. (e) Red blood cells from I.1, I.2 and II.2 were stimulated mechanically with negative pipette pressures where indicated by shaded grey bars. Numbers above grey bars denote pressure applied in mm Hg. Yoda1 and the calcium ionophore A23187 were applied via whole-chamber perfusion at the end of each measurement. (f) Average±s.e.m. of individual responses normalized to the calcium ionophore A23187 (4 μM). While every parental red blood cell responded to a stimulation greater than −10 mm Hg, no responses were observed to pressures as high as −35 mm Hg in the cells of patient II.2. Statistical comparison of I.1 and I.2 (n=5 in both groups) was performed using the Student’s t-test and were found to be not significantly different at the 0.05 level, P=0.21. (g) Red blood cells were stimulated with Yoda1 (15 μM) and the calcium ionophore A23187 (4 μM) as described in d. Background-subtracted Fluo-4 fluorescence intensities from individual cells from I.1, I.2 and II.2 are shown. (h) Average magnitude of calcium responses to 15 μM Yoda1 from g is shown for n=42, 29 and 45 cells of I.1, I.2 and II.2, respectively. One-way analysis was used for statistical comparison. II.2 responses were significantly different from I.1 and I.2, with P values of 2.53E−31 and 1.59E−23, respectively (Bonferroni means comparison test). AFU: arbitrary fluorescence unit.