Hereditary Xerocytosis: Differential Behavior of PIEZO1 Mutations in the N-Terminal Extracellular Domain Between Red Blood Cells and HEK Cells

Abstract

Hereditary Xerocytosis, a rare hemolytic anemia, is due to gain of function mutations in PIEZO1, a non-selective cation channel activated by mechanical stress. How these PIEZO1 mutations impair channel function and alter red blood cell (RBC) physiology, is not completely understood. Here, we report the characterization of mutations in the N-terminal part of the protein (V598M, F681S and the double mutation G782S/R808Q), a part of the channel that was subject of many investigations to decipher its role in channel gating. Our data show that the electrophysiological features of these PIEZO1 mutants expressed in HEK293T cells are different from previously characterized PIEZO1 mutations that are located in the pore or at the C-terminal extracellular domain of the protein. Although RBC with PIEZO1 mutations showed a dehydrated phenotype, the activity of V598M, F681S or R808Q in response to stretch was not significantly different from the WT channels. In contrast, the G782S mutant showed larger currents compared to the WT PIEZO1. Interestingly, basal activity of all the mutated channels was not significantly altered at the opposite of what was expected according to the decreased water and cation contents of resting RBC. In addition, the features of mutant PIEZO1 expressed in HEK293 cells do not always correlate with the observation in RBC where PIEZO1 mutations induced a cation leak associated with an increased conductance. Our work emphasizes the role of the membrane environment in PIEZO1 activity and the need to characterize RBC permeability to assess pathogenicity to PIEZO1 mutants associated with erythrocyte diseases.

Keywords: KCNN4; PIEZO1; hereditary xerocytosis; red blood cell; stomatocytosis.

FIGURE 1

(A) Ektacytometry profiles of RBC from three independent index-cases with clinical and biological phenotypes typical of hereditary xerocytosis, which were previously described. (B) water, (C) K⁺, and (D) Na⁺ content of control or mutant RBC, measured in resting conditions 24 h after blood withdrawal. Statistical analyzes of the data were done with a Kruskal and Wallis test followed by a Dunn’s post-test. The p value is given on each figure. There were no significant differences (p < 0.05) between mutants except the Na⁺ content between V598M and F681S, p = 0.03.

FIGURE 2

(A) Representative Western blot (WB) from three different cell labeling done on three different batches of transfected cells. (B) Quantification of PIEZO1 mutant expression related to WT. A ratio of Na⁺/K⁺ ATPase signal for each mutant versus WT was calculated for each WB and the PIEZO1 signal was corrected according to this internal standard. The plot illustrates the ratio Mutant/WT PIEZO1 calculated after five different WB (means ± sem). Statistical analyzes were done with Kruskal and Wallis test and differences were not significant (p > 0.05). (C) Stretch-activated currents of PIEZO1 WT and mutants, expressed in HEK293T cells, recorded in cell-attached configuration. Bars represent the mean ± sem, and the number of cells tested/the number of cell transfections is shown as n in the figures. **p < 0.01; *p < 0.05 vs. WT, two-way analysis of variance with Bonferroni’s post hoc. (D) Basal activity of PIEZO1. Basal activity calculated from the current traces without additional negative pipette pressure application, showing no significant differences between eGFP without PIEZO1, WT, and mutant constructs. Bars represent the mean ± sem, and the number of cells tested is shown in the bottom of the bars. n.s., not significantly different, one-way analysis of variance with Bonferroni’s post hoc.

FIGURE 3

(A) Representative traces of currents induced by stepwise negative pipette pressure (0 to −80 mmHg, Δ10 mmHg) recorded in HEK293T cells expressing WT and mutants. Stretch-activated currents of PIEZO1 WT and mutants-expressing HEK293T cells recorded in cell-attached configuration. (B) Inactivation time constant (τ) of currents from PIEZO1 WT and mutants. The top trace shows a representative current fitted by an exponential decay at −60 and −80 mmHg. τ is obtained from the fit. (C) Near steady-state currents from PIEZO1 WT and mutants. The steady-state current values were obtained from the fit at the end of the pressure pulse. Currents recorded in cell-attached configuration.