Figure 1.

Functional properties of PIEZO1 V598M erythrocytes. (A–B) K⁺ influx into wild-type (WT) and heterozygous mutant PIEZO1 V598M RBC was measured as baseline unidirectional ⁸⁶Rb⁺ influx at 37°C in the presence of ouabain (0.1 mM) and bumetanide (1 mM), and in the absence (white bars, WT; gray bars, V598M) or presence (red bars) of 200 nM senicapoc (SCP). Rates were calculated from ⁸⁶Rb⁺ uptakes measured at 2 and 10 minutes after initiation of influx. The senicapoc-sensitive fraction of K⁺ influx (B) represents KCNN4 activity, and was elevated in V598M RBC. Values in panels A and B are means from 2 triplicate experiments (^∗P < 0.05; ^∗∗P < 0.01 comparing V598M vs WT). (C–F) ADVIA120 hemoanalyzer profiles showing YODA-1 effects on RBC mean cell volume (MCV; C, E) and mean corpuscular hemoglobin concentration (MCHC; D, F). Freshly isolated and washed RBC (WT [C, D] or heterozygous PIEZO1 V598M [E, F]) were resuspended at 10% cytocrit in normal saline containing 1.5 mM CaCl₂, 10 mM glucose, 0.1 mM ouabain, and 1 mM bumetanide and incubated at room temperature in the presence of 15 μM YODA-1 in the absence (green squares) or presence of 200 nM senicapoc (red triangles). Control red cells of both genotypes were exposed to neither YODA-1 nor senicapoc (black diamonds). Results in panels C–F are single experiments, each representative of 2 with similar results. (G-I) Single channel characteristics of cell-attached patches on RBC of the affected proband II:1 (V598M) and of an unaffected family member (WT). Symmetric bath and pipette solutions included (in mM) 140 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, 10 mM Na HEPES, pH 7.4.¹ (G) Representative cell-attached RBC patch traces (2.5 seconds) in unstimulated conditions show low-channel activity in a patch from an unaffected family member (WT, upper trace), in contrast to the high spontaneous channel activity in a patch from the HX proband II:1 heterozygous for PIEZO1 V598M (lower trace). −Vp = −50 mV. (H) Single channel current-voltage relation of a representative cell-attached patch on an HX proband II:1 red cell heterozygous for PIEZO1 V598M. Single channel slope conductance was 19.6 pS with extrapolated reversal potential at +1 mV, r² = 0.99. (I) NPo (measured from 10 seconds sweeps) was 1.39 ± 0.30 in cell-attached patches on 3 RBC from HX proband II:1, in contrast to an NPo of 0.05 ± 0.03 in patches on RBC from an unaffected family member (P = 0.02). Single channel recordings from RBC were performed as previously¹ with seal resistances of >5 Gigohms. Blood samples used in these studies were shipped on coldpack from Sao Paulo and arrived in Boston on the 2nd day post-venisection. Cells were immediately washed and used for experiments. Washed cells were also stored in choline storage solution until additional experimental use on the 3rd and 4th day post-venisection. RBC = red blood cells.