Morphological description and functional characterization of PIEZO1 variants. Morphological description and functional characterization of cation channel activity in (A) PIEZO1:pIle2462Met RBCs and (B) PIEZO1:pVal1223Ile RBCs. In both panels: distribution of cell projected area (a) and cell circularity (b) from control (blue) and patient (red) RBCs, measured from peripheral blood smears and quantified using ImageJ (n > 1000 cells for both cell types). Circularity is defined as 4π.[area/perimeter2]. (c) Content in cell water (left), Na⁺ (middle) and K⁺ (right) of control (blue) and patient (red) RBCs. Averages ± SD from n = 3 experimental replicates. (d-e) Monitoring of membrane potential using the carbonyl cyanide 3-chlorophenylhydrazone (CCCP) technique. (d) Control (blue) and patient (red) RBCs in a normal Ringer solution, followed by addition of A23187 (10 µM, arrows). (e) Control (blue) and patient (red) RBCs in an low ionic strength (LIS) solution, followed by addition of Ca²⁺ (1 mM arrows). At the end of experiment, zero is obtained by lysing the cells with Triton X100 in 3 M NaCl.