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.
Highlights
Gain-of-function mutations in PIEZO1 have been linked to Dehydrated Hereditary Stomatocytosis (DHSt), a rare red blood cell (RBC) disease known as hereditary xerocytosis (Zarychanski et al, 2012; Albuisson et al, 2013; Andolfo et al, 2013)
Na+ content was significantly higher (∼10–15%) in the F681S and the double mutant (Figure 1D). This Na+ increase did not fully compensate the K+ loss, and there is a decrease in monovalent cation content (Na+ + K+), which correlates with the RBC dehydration
It was shown in our previous work, that patients’ RBC cation leak is associated with a rise in cell membrane conductance (Rapetti-Mauss et al, 2017): a transient current following Goldman-Hodgkin-Katz law was observed in patients’ RBC, which is not observed in control RBC. This current is thought to be indicative of increased PIEZO1 activity in RBC with the PIEZO1 mutations V598M, F681S and G782S/R808Q
Summary
Gain-of-function mutations in PIEZO1 have been linked to Dehydrated Hereditary Stomatocytosis (DHSt), a rare red blood cell (RBC) disease known as hereditary xerocytosis (Zarychanski et al, 2012; Albuisson et al, 2013; Andolfo et al, 2013). PIEZO1 is a non-selective cation channel, activated by mechanical stimuli, that has been proposed to link mechanical forces to RBC calcium permeability (Coste et al, 2012; Cahalan et al, 2015). In RBC, this transient activation of PIEZO1 in response to membrane stretch increases intracellular calcium concentration and stimulates the Ca2+ activated K+ channel, KCNN4 (or Gardos channel), that allows K+ to leave the cell. The high anion conductance of RBC membrane allows Cl− flux, leading to a net KCl efflux. This loss of osmolytes from the cytosol is accompanied by water leaving the cell, resulting in RBC dehydration. Gainof-function mutations in PIEZO1 have been associated with RBC dehydration through excessive KCNN4 stimulation (Rapetti-Mauss et al, 2017)
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