Role of Ca²⁺-activated K⁺ channels in human erythrocyte apoptosis

Abstract

Exposure of erythrocytes to the Ca2+ ionophore ionomycin has recently been shown to induce cell shrinkage, cell membrane blebbing, and breakdown of phosphatidylserine asymmetry, all features typical of apoptosis of nucleated cells. Although breakdown of phosphatidylserine asymmetry is thought to result from activation of a Ca2+-sensitive scramblase, the mechanism and role of cell shrinkage have not been explored. The present study was performed to test whether ionomycin-induced activation of Ca2+-sensitive Gardos K+ channels and subsequent cell shrinkage participate in ionomycin-induced breakdown of phosphatidylserine asymmetry of human erythrocytes. According to on-cell patch-clamp experiments, ionomycin (1 microM) induces activation of inwardly rectifying K+-selective channels in the erythrocyte membrane. Fluorescence-activated cell sorter analysis reveals that ionomycin leads to a significant decrease of forward scatter, reflecting cell volume, an effect blunted by an increase of extracellular K+ concentration to 25 mM and exposure to the Gardos K+ channel blockers charybdotoxin (230 nM) and clotrimazole (5 microM). As reflected by annexin binding, breakdown of phosphatidylserine asymmetry is triggered by ionomycin, an effect again blunted, but not abolished, by an increase of extracellular K+ concentration and exposure to charybdotoxin (230 nM) and clotrimazole (5 microM). Similar to ionomycin, glucose depletion leads (within 55 h) to annexin binding of erythrocytes, an effect again partially reversed by an increase of extracellular K+ concentration and exposure to charybdotoxin. K-562 human erythroleukemia cells similarly respond to ionomycin with cell shrinkage and annexin binding, effects blunted by antisense, but not sense, oligonucleotides against the small-conductance Ca2+-activated K+ channel isoform hSK4 (KCNN4). The experiments disclose a novel functional role of Ca2+-sensitive K+ channels in erythrocytes, i.e., their participation in regulation of erythrocyte apoptosis.

Stimulation of human red blood cells leads to Ca2+-mediated intercellular adhesion

“…As illustrated in Fig. 3b, during that is not related to the PS exposure [16,17]. Therefore, RBCs were stained with annexin-V-FITC conjugates in order to visualize PS exposure on the surface of RBCs.…”

PGE2 in the regulation of programmed erythrocyte death

“…25 Beyond that the cellular loss of K þ participates in the triggering of erythrocyte scramblase. 19 Along those lines, apoptotic death of nucleated cells has similarly been shown to be fostered by cellular loss of K þ .…”

“…Ca 2 þ entry further activates Ca 2 þ -sensitive K þ channels, 17,18 leading to or augmenting cell shrinkage. 19 Ca 2 þ may enter erythrocytes through Ca 2 þ -permeable cation channels, 20,21 which are activated following removal of external Cl À , osmotic shock by increase of extracellular osmolarity, oxidative stress by addition of t-butylhydroperoxide (t-BHP) and energy depletion by removal of extracellular glucose. 5,6 Activation of those channels triggers breakdown of phosphatidylserine asymmetry and subsequent erythrocyte death through increased Ca 2 þ leakage into the cell.…”

Hemin-induced suicidal erythrocyte death

“…Cytosolic Ca 2+ concentration is increased by activation of Ca 2+ -permeable cation channels [20,21,32,41]. The increase in cytosolic Ca 2+ activity leads to activation of Ca 2+ -sensitive K + channels [9,14], which in turn results in exit of KCl with osmotically obliged water and thus in cell shrinkage [45]. The Ca 2+ sensitivity of phospholipid scrambling is increased by ceramide [42].…”