PIEZO1-driven echinocytosis in red blood cells is independent of calcium and water fluxes

In red blood cells (RBCs), mechanosensitive PIEZO1 channel activation is traditionally thought to induce a large Ca 2+ influx, leading to activation of the Gárdos channel (KCNN4), cell dehydration, and echinocytosis. Yoda1 is the most commonly used pharmacological agent to activate PIEZO1 independently of shear stress. In this study, we investigated the effects of chemical PIEZO1 activation in the presence and absence of extracellular Ca 2+ . We demonstrate that, even in the absence of extracellular Ca 2+ , PIEZO1 activation induces significant potassium (K + ) efflux and sodium (Na + ) influx, without associated water fluxes. Moreover, PIEZO1 activation rapidly triggers morphological changes in RBCs, including echinocytosis and spherocytosis, which occur independently of (i) Ca 2+ , (ii) KCNN4 activation or (iii) ion or water fluxes. Importantly, these shape changes critically depend on cytoskeletal dynamics, as disruption of actin polymerization or inhibition of non-muscle myosin IIA markedly alters the effects of chemical PIEZO1 activation. Mechanical stimulation experiments further highlight the physiological relevance of PIEZO1 in regulating RBC deformability, demonstrating that although Ca 2+ is important for RBC deformability, its interaction with KCNN4 is not required over short time scales. Collectively, these findings underscore the central role of PIEZO1 in RBC physiology, emphasizing its involvement in the regulation of RBC volume, morphology, and deformability, with potential implications for RBC function under both physiological and pathological conditions.