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 °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 μM). 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.