Ca2+-dependent pathways controlled by Piezo1 activity in human erythroid progenitor cell line: focus on KCa3.1 channels, ANO6 lipid scramblases and pannexins-1

Piezo1 is a mechanosensitive Ca 2+ -permeable ion channel that plays a crucial role in the Ca 2+ signaling processes of human red blood cells (RBCs). Ca 2+ influx through Piezo1 controls the activity of various molecules that are crucial for RBC physiology and pathophysiology, including Ca 2+ -activated K + channels of intermediate conductance (KCa3.1, or Gardos channels), ANO6 lipid scramblases and pannexins-1 (Panx1). Erythropoiesis, the differentiation of erythropoietic stem cells in the bone marrow to myeloid progenitor cells and then to mature (RBCs, is traditionally studied using different blood cell lines as models. Among them, K562 cells, a human chronic myeloid leukemia cell line, are multipotent progenitors of hematopoietic cells that can be differentiated along the erythroid lineage, thus making these cells an invaluable model for studying human erythropoiesis. As K562 is an immortalized cell line obtained from a specific donor, the putative differences in the relevant pathways between K562 cells and human RBCs should be identified and taken into account. Here, we aimed to reveal and compare the functional interactions between Ca 2+ influx through Piezo1 and Gardos channel, ANO6 and Panx1 activities in K562 cells and RBCs. Database analysis was performed to confirm the expression of genes of interest and putative mutations in K562 cells. Mutations were confirmed using Sanger sequencing. Piezo1 activity in the plasma membrane was stimulated by a selective Piezo1 agonist, Yoda1. KCa3.1 activation was detected using light microscopy and single-channel patch-clamp analysis. PS exposure was detected by AnV fluorescent staining. Presence of Panx1 was shown using RT-PCR, Western Blot and immunofluorescence. K562 differentiation was induced by cytarabine-hemin treatment and confirmed by a benzidine test. Panx1 activation was probed using dye uptake assay and whole-cell patch-clamp recordings. We found that, similar to RBCs, both Piezo1-KCa3.1 and Piezo1-ANO6 functional links are present in K562 cells, whereas the Piezo1-Panx1 axis is not functional because Panx1 cannot be activated by Ca 2+ in this cell line. Thus, the physiologically relevant pathways associated with Ca 2+ -induced Panx1 activity in K562 cells may significantly differ from those that are functional in human RBCs.