Verena B. Brand

Dependence of Plasmodium falciparum in vitro growth on the cation permeability of the human host erythrocyte.

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.

Electrophysiological Properties of the Plasmodium falciparum-Induced Cation Conductance of Human Erythrocytes

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.

Accelerated Clearance of Plasmodium-infected Erythrocytes in Sickle Cell Trait and Annexin-A7 Deficiency

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.

Channel-induced apoptosis of infected host cells—the case of malaria

Infection of erythrocytes by the malaria pathogen Plasmodium falciparum leads to activation of several distinct anion channels and a non-selective, Ca2+-permeable cation channel. All channel types are presumably activated by the oxidative stress generated by the pathogen. Similar or identical channels are activated by oxidation of non-infected erythrocytes. Activation of the non-selective cation channel allows entry of Ca2+ and Na+, both of which are required for intracellular growth of the pathogen. The entry of Ca2+ stimulates an intraerythrocytic scramblase that facilitates bi-directional phospholipid migration across the bilayer, resulting in breakdown of the phosphatidylserine asymmetry of the cell membrane. The exposure of phosphatidylserine at the outer surface of the cell membrane is presumably followed by binding to phosphatidylserine receptors on macrophages and subsequent phagocytosis of the affected erythrocyte. The lysosomal degradation may eventually eliminate the pathogen. The channel may thus play a dual role in pathogen survival. Absence of the channels is not compatible with pathogen growth, enhanced channel activity accelerates erythrocyte “apoptosis” that may represent a host defence mechanism serving to eliminate infected erythrocytes.

Purinoceptors are involved in the induction of an osmolyte permeability in malaria-infected and oxidized human erythrocytes

In human erythrocytes, infection by the malaria parasite Plasmodium falciparum or oxidative stress induces a new organic osmolyte and anion permeability. To examine a role for autocrine purinoceptor signaling during this induction process, erythrocytic purinoceptor expression, and ATP release were determined. Furthermore, using pharmacological and genetic approaches the dependence on purinoceptor signaling of osmolyte permeability and Plasmodium development, both in vitro and in vivo, were assessed. Extracellular ATP did not induce an osmolyte permeability in non‐infected or non‐oxidized erythrocytes. ATP and other purinoceptor agonists increased the induction of osmolyte permeability during infection or oxidation as measured by isosmotic hemolysis and patch‐clamp recording. Purinoceptor antagonists and apyrase decreased the induced permeability. The observed pharmacology suggested the involvement of P2Y purinoceptors. Accordingly, human erythrocytes expressed P2Y1 protein. Moreover, P2Y1 deficient mouse erythrocytes exhibited a delayed appearance of the osmolyte permeability during P. berghei infection‐ or oxidation compared with wild‐type erythrocytes. Furthermore, the nonspecific purinoceptor antagonist suramin decreased in vitro growth and DNA/RNA amplification of P. falciparum in human erythrocytes and decreased in vivo growth of P. berghei. P. berghei developed slower in P2Y1‐deficient mice in vivo compared with wild‐type animals. In conclusion, induction of the osmolyte permeability in Plasmodium‐infected erythrocytes involves autocrine purinoceptor signaling.

Organic Osmolyte Permeabilities of the Malaria-induced Anion Conductances in Human Erythrocytes

Infection of human erythrocytes with the malaria parasite Plasmodium falciparum induces new permeability pathways (NPPs) in the host cell membrane. Isotopic flux measurements demonstrated that the NPP are permeable to a wide variety of molecules, thus allowing uptake of nutrients and release of waste products. Recent patch-clamp recordings demonstrated the infection-induced up-regulation of an inwardly and an outwardly rectifying Cl− conductance. The present experiments have been performed to explore the sensitivity to cell volume and the organic osmolyte permeability of the two conductances. It is shown that the outward rectifier has a high relative lactate permeability (Plactate/PCl = 0.4). Sucrose inhibited the outward-rectifier and abolished the infection-induced hemolysis in isosmotic sorbitol solution but had no or little effect on the inward-rectifier. Furosemide and NPPB blocked the outward-rectifying lactate current and the sorbitol hemolysis with IC50s in the range of 0.1 and 1 μM, respectively. In contrast, the IC50s of NPPB and furosemide for the inward-rectifying current were >10 μM. Osmotic cell-shrinkage inhibited the inwardly but not the outwardly rectifying conductance. In conclusion, the parasite-induced outwardly-rectifying anion conductance allows permeation of lactate and neutral carbohydrates, whereas the inward rectifier seems largely impermeable to organic solutes. All together, these data should help to resolve ongoing controversy regarding the number of unique channels that exist in P. falciparum–infected erythrocytes.

A high specificity and affinity interaction with serum albumin stimulates an anion conductance in malaria-infected erythrocytes.

The intraerythrocytic development of P. falciparum induces New Permeability Pathways (NPP) in the membrane of the parasitized erythrocyte which provide the parasite with nutrients, adjust the erythrocyte electrolyte composition to the needs of the parasite, and dispose of metabolic waste products and osmolytes. Patch-clamp recordings identified inwardly and outwardly rectifying (OR) anion conductances in the host erythrocyte membrane as electrophysiological correlate of the NPP. The OR conductance is regulated by serum. Here we show that serum albumin (SA) stimulated OR-generated Cl- and lactate outward currents with an EC50 of approximately 100 nM while other proteins such as ovalbumin or casein did not. The stimulatory efficacy did not differ between fatty acid free bovine SA and recombinant human SA and disruption of the SA tertiary structure abolished the effect suggesting that intact SA protein and not other bound factors interact with the erythrocyte membrane. Taken together, the data indicate a high affinity and specificity interaction of native SA with the parasitized erythrocytes which might underlie the observed dependence of P. falciparum growth in vitro on SA.

Permselectivity and pH-dependence of Plasmodium falciparum-induced anion currents in human erythrocytes

Intraerythrocytic survival of the malaria pathogen Plasmodium falciparum requires delivery of nutrients and disposal of waste products across the host erythrocyte membrane. Recent patch-clamp experiments have demonstrated inwardly and outwardly rectifying anion conductances in infected but not in control erythrocytes. A ClC-2-generated fraction of the inwardly rectifying current is activated by cell swelling and presumably subserves host cell volume regulation. In contrast, the outwardly rectifying current is insensitive to cell volume but allows the passage of lactate and is involved in the transport of nutrients. The present study was performed to characterize the permselectivity and pH sensitivity of the anion conductances using whole-cell recording. The outwardly rectifying and the inwardly rectifying currents exhibited permselectivities of Cl−≥Br−≈I−>SCN− and SCN−>I−>Br−>Cl−, respectively, as evident from the reversal potentials recorded under biionic conditions. While the inwardly rectifying current was not affected significantly by alterations of pH between 6.0 and 8.4, the outward rectifier was inhibited strongly by alkalinization to pH≥7.8. Fluxes of 14C-lactate and parasite growth were decreased markedly by the increase of bath pH, an effect that may at least in part be due to inhibition of the outward rectifier and subsequently impaired transport across the erythrocyte membrane.