S-glutathionylation inhibits plasma membrane Ca2+ ATPase 4b activity in erythrocytes from healthy subjects and sickle cell patients

Plasma membrane Ca2+ ATPase (PMCA) in red blood cells (RBCs) is sensitive to changes in redox state, however, the mechanism of its redox sensitivity is still elusive. In RBCs, two different isoforms of PMCA, e.g. PMCA4b and PMCA1, are present. In the present study, we explored the possibility of PMCA S-glutathionylation upon depletion of intracellular GSH with diamide in healthy RBCs. We report for the first time the presence of S-glutathionylated cysteine residues in PMCA4b. In contrast, we did not find any glutathionylated cysteine residues on PMCA1 upon diamide treatment. S-glutathionylation of PMCA4b was associated with suppression of the maximal hydrolytic activity of the enzyme along with inhibition in the Ca2+ efflux. However, the pump’s activity could be restored upon treatment with DTT. The enzyme inhibition concurred with S-glutathionylation of the Cys-24, -55, -537, and -632. Upon incubation of RBC ghosts with GSSG, we observed inhibited PMCA activity, that can be partially prevented by prior incubation with calmodulin. This indicates a probable mechanism of S-glutathionylation-driven PMCA inhibition as well as the importance of Cys-537 presence. Additionally, we also found PMCA4b is S-glutathionylated on Cys-537 in sickling RBCs, indicating a possible pathophysiologic role of this particular post-translational modification. Despite suppressed PMCA4b activity, diamide treatment causes a decrease in the intracellular free Ca2+ level. This is due either to the inhibition of the Piezo1 channel activity, that may either be caused by the direct effect of diamide on Piezo1 itself, or to the modification of the cytoskeletal protein(s) upon S-glutathionylation, which can interfere with the mechanosensation of Piezo1.