Role of Piezo1 in Terminal Density Reversal of Red Blood Cells

Density reversal of senescent red blood cells has been known for a long time, yet the identity of the candidate ion transporter(s) causing the senescent cells to swell is still elusive. While performing fractionation of RBCs from healthy individuals in Percoll density gradient and characterization of the separated fractions, we identified a subpopulation of cells in low-density fraction (1.02% ± 0.47) showing signs of senescence such as loss of membrane surface area associated with a reduction in band 3 protein abundance, and Phosphatidylserine (PS) exposure to the outer membrane. In addition, we found that these cells are overloaded with Na⁺ and Ca²⁺. Using a combination of blockers and activators of ion pumps and channels, we revealed reduced activity of Plasma membrane Ca²⁺ ATPase and an increase in Ca²⁺ and Na⁺ leaks through ion channels in senescent-like cells. Our data revealed that Ca²⁺ overload in these cells is a result of reduced PMCA activity and facilitated Ca²⁺ uptake via a hyperactive Piezo1 channel. However, we could not exclude the contribution of other Ca²⁺-permeable ion channels in this scenario. In addition, we found, as a universal mechanism, that an increase in intracellular Ca²⁺ reduced the initially high selectivity of Piezo1 channel for Ca²⁺ and allowed higher Na⁺ uptake, Na⁺ accumulation, and swelling.