Effect of Chloride Channel Inhibitors on Cytosolic Ca2+ Levels and Ca2+-Activated K+ (Gardos) Channel Activity in Human Red Blood Cells

DIDS, NPPB, tannic acid (TA) and AO1 are widely used inhibitors of Cl- channels. Some Cl- channel inhibitors (NPPB, DIDS, niflumic acid) were shown to affect phosphatidylserine (PS) scrambling and, thus, the life span of human red blood cells (hRBCs). Since a number of publications suggest Ca2+ dependence of PS scrambling, we explored whether inhibitors of Cl- channels (DIDS, NPPB) or of Ca2+-activated Cl- channels (DIDS, NPPB, TA, AO1) modified intracellular free Ca2+ concentration ([Ca2+](i)) and activity of Ca2+-activated K+ (Gardos) channel in hRBCs. According to Fluo-3 fluorescence in flow cytometry, a short treatment (15 min, +37 A degrees C) with Cl- channels inhibitors decreased [Ca2+](i) in the following order: TA > AO1 > DIDS > NPPB. According to forward scatter, the decrease of [Ca2+](i) was accompanied by a slight but significant increase in cell volume following DIDS, NPPB and AO1 treatments. TA treatment resulted in cell shrinkage. According to whole-cell patch-clamp experiments, TA activated and NPPB and AO1 inhibited Gardos channels. The Cl- channel blockers further modified the alterations of [Ca2+](i) following ATP depletion (glucose deprivation, iodoacetic acid, 6-inosine), oxidative stress (1 mM t-BHP) and treatment with Ca2+ ionophore ionomycin (1 mu M). The ability of the Cl- channel inhibitors to modulate PS scrambling did not correlate with their influence on [Ca2+](i) as TA and AO1 had a particularly strong decreasing effect on [Ca2+](i) but at the same time enhanced PS exposure. In conclusion, Cl- channel inhibitors affect Gardos channels, influence Ca2+ homeostasis and induce PS exposure of hRBCs by Ca2+-independent mechanisms.