Erythrocytes lack nuclei and mitochondria, the organelles important for apoptosis of nucleated cells. However, following increase of cytosolic Ca 2 þ activity, erythrocytes undergo cell shrinkage, cell membrane blebbing and breakdown of phosphatidylserine asymmetry, all features typical for apoptosis in nucleated cells. The same events are observed following osmotic shock, an effect mediated in part by activation of Ca 2 þ -permeable cation channels. However, erythrocyte death following osmotic shock is blunted but not prevented in the absence of extracellular Ca 2 þ pointing to additional mechanisms. As shown in this study, osmotic shock (950 mOsm) triggers sphingomyelin breakdown and formation of ceramide. The stimulation of annexin binding following osmotic shock is mimicked by addition of ceramide or purified sphingomyelinase and significantly blunted by genetic (aSM-deficient mice) or pharmacologic (50 lM 3,4-dichloroisocoumarin) knockout of sphingomyelinase. The effect of ceramide is blunted but not abolished in the absence of Ca 2 þ . Conversely, osmotic shock-induced annexin binding is potentiated in the presence of sublethal concentrations of ceramide. In conclusion, ceramide and Ca 2 þ entry through cation channels concert to trigger erythrocyte death during osmotic shock.

Intraerythrocyte growth of the malaria parasite Plasmodium falciparum induces a Ca²⁺-permeable unselective cation conductance in the host cell membrane which is inhibited by ethylisopropylamiloride (EIPA) and is paralleled by an exchange of K⁺ by Na⁺ in the host cytosol. The present study has been performed to elucidate the functional significance of the electrolyte exchange. Whole-cell patch-clamp experiments confirmed the Ca²⁺ permeability and EIPA sensitivity of the Plasmodium falciparum induced cation channel. In further experiments, ring stage-synchronized parasites were grown in vitro for 48 h in different test media. Percentage of Plasmodium-infected and phosphatidylserine-exposing erythrocytes was measured with FACS analysis by staining with the DNA-dye Syto16 and annexin V, respectively. The increase of infected cells was not significantly affected by an 8 h replacement of NaCl in the culture medium with Na-gluconate but was significantly blunted by replacement of NaCl with KCl, NMDG-Cl or raffinose. Half maximal growth was observed at about 25 mM Na⁺. The increase of infected cells was further inhibited by EIPA (IC₅₀< 10 µM) and at low extracellular free Ca²⁺. Infected cells displayed significantly stronger annexin binding, an effect mimicked by exposure of noninfected erythrocytes to oxidative stress (1 mM t-butylhydroperoxide for 15 min) or to Ca²⁺ ionophore ionomycin (1 µM for 60 min). The observations indicate that parasite growth requires the entry of both, Na⁺ and Ca²⁺ cations into the host erythrocyte probably through the EIPA sensitive cation channel. Ca²⁺ entry further induces break-down of the phospholipid asymmetry in the host membrane.

Intraerythrocyte survival of the malaria pathogen Plasmodium falciparumdepends on the induction of the new-permeability-pathways (NPPs) in the host cell membrane. NPPs are characterized as anion- and organic osmolyte-permeable channels which also exhibit a low but significant permeability for inorganic cations. To disclose the electrophyiologial properties of this infection-induced cation permeability whole-cell currents were recorded inPlasmodium falciparum-infected human erythrocytes (pRBC) using bath and pipette solutions with low Cl^- concentrations. The data disclose a nonselective cation conductance (G_cat) which activated upon removal of extracellular Cl^-. Upon activation, G_cat was 0.3 ± 0.05 nS (n=16) in control RBC and 2.0 ± 0.3 nS (n = 32) in pRBC indicating an induction of G_cat during the infection. G_cat of pRBC exibited a relative permselectivity for monovalent cations of Cs⁺ñK⁺>Na⁺>Li⁺ (P_Na/P_K ñ 0.5) with a significant permeability for Ca²⁺. G_cat of pRBC was inhibited by NPPs blockers (furosemide and NPPB) and cation channel blockers (amiloride, EIPA, GdCl₃) with the highest sensitivity to EIPA (IC₅₀ñ0.5µM). Most importantly, the blocker sensitivities differed between the infection-induced anion conductances and G_cat suggesting that G_cat and the anion conductances represent different channel proteins which in concert build up the NPPs.

The course of malaria does not only depend on the virulence of the parasite Plasmodium but also on properties of host erythrocytes. Here, we show that infection of erythrocytes from human sickle cell trait (HbA/S) carriers with ring stages of P. falciparum led to significantly enhanced PGE₂ formation, Ca²⁺ permeability, annexin-A7 degradation, phosphatidylserine (PS) exposure at the cell surface, and clearance by macrophages. P. berghei-infected erythrocytes from annexin-A7-deficient (annexin-A7^-/-) mice were more rapidly cleared than infected wildtype cells. Accordingly, P. berghei-infected annexin-A7^-/- mice developed less parasitemia than wildtype mice. The cyclooxygenase inhibitor aspirin decreased erythrocyte PS exposure in infected annexin-A7^-/- mice and abolished the differences of parasitemia and survival between the genotypes. Conversely, the PGE₂-agonist sulprostone decreased parasitemia and increased survival of wild type mice. In conclusion, PS exposure on erythrocytes results in accelerated clearance of Plasmodium ring stage-infected HbA/S or annexin-A7^-/- erythrocytes and thus confers partial protection against malaria in vivo.