Stéphane Egée

A stretch-activated anion channel is up-regulated by the malaria parasite Plasmodium falciparum

A recent study on malaria‐infected human red blood cells (RBCs) has shown induced ion channel activity in the host cell membrane, but the questions of whether they are host‐ or parasite‐derived and their molecular nature have not been resolved. Here we report a comparison of a malaria‐induced anion channel with an endogenous anion channel in Plasmodium falciparum‐infected human RBCs. Ion channel activity was measured using the whole‐cell, cell‐attached and excised inside‐out configurations of the patch‐clamp method. Parasitised RBCs were cultured in vitro, using co‐cultured uninfected RBCs as controls. Unstimulated uninfected RBCs possessed negligible numbers of active anion channels. However, anion channels could be activated in the presence of protein kinase A (PKA) and ATP in the pipette solution or by membrane deformation. These channels displayed linear conductance (∼15 pS), were blocked by known anion channel inhibitors and showed the permeability sequence I− > Br− > Cl−. In addition, in less than 5 % of excised patches, an outwardly rectifying anion channel (∼80 pS, outward conductance) was spontaneously active. The host membrane of malaria‐infected RBCs possessed spontaneously active anion channel activity, with identical conductances, pharmacology and selectivity to the linear conductance channel measured in stimulated uninfected RBCs. Furthermore, the channels measured in malaria‐infected RBCs were shown to have a low open‐state probability (Po) at positive potentials, which explains the inward rectification of membrane conductance observed when using the whole‐cell configuration. The data are consistent with the presence of two endogenous anion channels in human RBCs, of which one (the linear conductance channel) is up‐regulated by the malaria parasite P. falciparum.

Modulation of whole-cell currents in Plasmodium falciparum-infected human red blood cells by holding potential and serum

Recent electrophysiological studies have identified novel ion channel activity in the host plasma membrane of Plasmodium falciparum‐infected human red blood cells (RBCs). However, conflicting data have been published with regard to the characteristics of induced channel activity measured in the whole‐cell configuration of the patch‐clamp technique. In an effort to establish the reasons for these discrepancies, we demonstrate here two factors that have been found to modulate whole‐cell recordings in malaria‐infected RBCs. Firstly, negative holding potentials reduced inward currents (i.e. at negative potentials), although this result was highly complex. Secondly, the addition of human serum increased outward currents (i.e. at positive potentials) by approximately 4‐fold and inward currents by approximately 2‐fold. These two effects may help to resolve the conflicting data in the literature, although further investigation is required to understand the underlying mechanisms and their physiological relevance in detail.

Red cell investigations: Art and artefacts

Red blood cell research is important for both, the clinical haematology, such as transfusion medicine or anaemia investigations, and the basic research fields like exploring general membrane physiology or rheology. Investigations of red blood cells include a wide spectrum of methodologies ranging from population measurements with a billion cells evaluated simultaneously to single-cell approaches. All methods have a potential for pitfalls, and the comparison of data achieved by different technical approaches requires a consistent set of standards. Here, we give an overview of common mistakes using the most popular methodologies in red blood cell research and how to avoid them. Additionally, we propose a number of standards that we believe will allow for data comparison between the different techniques and different labs. We consider biochemical analysis, flux measurements, flow cytometry, patch-clamp measurements and dynamic fluorescence imaging as well as emerging single-cell techniques, such as the use of optical tweezers and atomic force microscopy.

Local Membrane Deformations Activate Ca2+-Dependent K+ and Anionic Currents in Intact Human Red Blood Cells

Background The mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cells diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents. Methodology/Principal Findings The cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patch-pipette. K+ and Cl− currents were strictly dependent on the presence of Ca2+. The Ca2+-dependent currents were transient, with typical decay half-times of about 5–10 min, suggesting the spontaneous inactivation of a stretch-activated Ca2+ permeability (PCa). These results indicate that local membrane deformations can transiently activate a Ca2+ permeability pathway leading to increased [Ca2+]i, secondary activation of Ca2+-sensitive K+ channels (Gardos channel, IK1, KCa3.1), and hyperpolarization-induced anion currents. Conclusions/Significance The stretch-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca2+-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca2+ content of red cells in certain hereditary anemias such as thalassemia and sickle cell anemia.

Plasmodium falciparum STEVOR proteins impact erythrocyte mechanical properties

Infection of erythrocytes with the human malaria parasite, Plasmodium falciparum, results in dramatic changes to the host cell structure and morphology. The predicted functional localization of the STEVOR proteins at the erythrocyte surface suggests that they may be involved in parasite-induced modifications of the erythrocyte membrane during parasite development. To address the biologic function of STEVOR proteins, we subjected a panel of stevor transgenic parasites and wild-type clonal lines exhibiting different expression levels for stevor genes to functional assays exploring parasite-induced modifications of the erythrocyte membrane. Using this approach, we show that stevor expression impacts deformability of the erythrocyte membrane. This process may facilitate parasite sequestration in deep tissue vasculature.

Malaria parasite Plasmodium gallinaceum up‐regulates host red blood cell channels

The properties of the malaria parasite‐induced permeability pathways in the host red blood cell have been a major area of interest particularly in the context of whether the pathways are host‐ or parasite‐derived. In the present study, the whole‐cell configuration of the patch‐clamp technique has been used to show that, compared with normal cells, chicken red blood cells infected by Plasmodium gallinaceum exhibited a 5–40‐fold larger membrane conductance, which could be further increased up to 100‐fold by raising intracellular Ca2+ levels. The increased conductance was not due to pathways with novel electrophysiological properties. Rather, the parasite increased the activity of endogenous 24 pS stretch‐activated non‐selective cationic (NSC) and 62 pS calcium‐activated NSC channels, and, in some cases, of endogenous 255 pS anionic channels.

Toward a unifying model of malaria-induced channel activity

Infection of RBC by the malaria parasite Plasmodium falciparum activates, at the trophozoite stage, a membrane current 100- to 150-fold larger than in uninfected RBC. This current is carried by small anion channels initially described in supraphysiological ion concentrations (1.115 M Cl−) and named plasmodial surface anion channels (PSAC), suggesting their plasmodial origin. Our results obtained with physiological ion concentrations (0.145 M Cl−) support the notion that the parasite-induced channels represent enhanced activity versions of anion channels already present in uninfected RBCs. Among them, an 18-pS inwardly rectifying anion channel (IRC) and a 4- to 5-pS small conductance anion channel (SCC) were present in most single-channel recordings of infected membranes. The aim of this study was to clarify disparities in the reported electrophysiological data and to investigate possible technical reasons why these discrepancies have arisen. We demonstrate that PSAC is the supraphysiological correlate of the SCC and is inhibited by Zn2+, suggesting that it is a ClC-2 channel. We show that in physiological solutions 80% of the membrane conductance in infected cells can be accounted for by IRC and 20% can be accounted for by SCC whereas in supraphysiological conditions the membrane conductance is almost exclusively carried by SCC (PSAC) because the IRC is functionally turned off.

Anti-Plasmodium activity of ceramide analogs

BackgroundSphingolipids are key molecules regulating many essential functions in eukaryotic cells and ceramide plays a central role in sphingolipid metabolism. A sphingolipid metabolism occurs in the intraerythrocytic stages of Plasmodium falciparum and is associated with essential biological processes. It constitutes an attractive and potential target for the development of new antimalarial drugs.MethodsThe anti-Plasmodium activity of a series of ceramide analogs containing different linkages (amide, methylene or thiourea linkages) between the fatty acid part of ceramide and the sphingoid core was investigated in culture and compared to the sphingolipid analog PPMP (d,1-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol). This analog is known to inhibit the parasite sphingomyelin synthase activity and block parasite development by preventing the formation of the tubovesicular network that extends from the parasitophorous vacuole to the red cell membrane and delivers essential extracellular nutrients to the parasite.ResultsAnalogs containing methylene linkage showed a considerably higher anti-Plasmodium activity (IC50 in the low nanomolar range) than PPMP and their counterparts with a natural amide linkage (IC50 in the micromolar range). The methylene analogs blocked irreversibly P. falciparum development leading to parasite eradication in contrast to PPMP whose effect is cytostatic. A high sensitivity of action towards the parasite was observed when compared to their effect on the human MRC-5 cell growth. The toxicity towards parasites did not correlate with the inhibition by methylene analogs of the parasite sphingomyelin synthase activity and the tubovesicular network formation, indicating that this enzyme is not their primary target.ConclusionsIt has been shown that ceramide analogs were potent inhibitors of P. falciparum growth in culture. Interestingly, the nature of the linkage between the fatty acid part and the sphingoid core considerably influences the antiplasmodial activity and the selectivity of analogs when compared to their cytotoxicity on mammalian cells. By comparison with their inhibitory effect on cancer cell growth, the ceramide analogs might inhibit P. falciparum growth through modulation of the endogenous ceramide level.

Volume-activated DIDS-sensitive whole-cell chloride currents in trout red blood cells.

1 The nystatin‐perforated whole‐cell recording mode of the patch‐clamp technique was used to investigate the membrane conductance of trout (Oncorhynchus mykiss) red blood cells in the steady state, 5 min after exposure to hyposmotic medium and 10 min after return to normal isosmotic medium. 2 Whole‐cell I–V relations showed outward rectification when red blood cells were bathed in isosmotic (320 mosmol l−1) saline solution and the patch pipette was filled with 117 mm KCl. The membrane conductance was 2.58 ± 0.59 nS (number of experiments, n=18) between 0 and 100 mV and 1.32 ± 0.19 nS (n=18) between 0 and −100 mV. Removal of Cl− from the extracellular side or incubation with the CL−1 channel blocker DIDS caused a reduction in whole‐cell membrane conductance by more than 50%, indicating that the membrane current was generated by Cl− ions. The remaining conductance was voltage independent and probably due to non‐selective cation conductance. 3 The membrane conductance increased approximately 2‐fold after cell swelling induced by exposure to hyposmotic saline solution (215 mosmol I1). This effect was abolished in Cl−1‐free hyposmotic medium or in the presence of DIDS. 4 The return to isosmotic solution produced a fall in membrane conductance to, or below, control values. 5 We conclude that trout red blood cells possess a significant Cl− conductance in the steady state which is reversibly activated during cell swelling and contributes to volume recovery.

Effects of elevated intracellular calcium on the osmotic fragility of human red blood cells

High throughput methodologies that measure the distribution of osmotic fragilities in red blood cell populations have enabled the investigation of dynamic changes in red cell homeostasis and membrane permeability in health and disease. The common assumption in the interpretation of dynamic changes in osmotic fragility curves is that left or right shifts reflect a decreased or increased hydration state of the cells, respectively, allowing direct inferences on membrane transport from osmotic fragility measurements. However, the assumed correlation between shifts in osmotic fragility and hydration state has never been directly explored, and may prove invalid in certain conditions. We investigated here whether this correlation holds for red cells exposed to elevated intracellular calcium. The results showed that elevated cell calcium causes a progressive increase in osmotic fragility with minimal contribution from cell hydration (<8%). Loss of membrane area by the release of 160+/-40nm diameter (mean+/-SD) vesicles is shown to be a major contributor, but may not account for the full non-hydration component. The rest must reflect a specific calcium-induced lytic vulnerability of the membrane causing rupture before the cells attain their maximal spherical volumes. The implications of these findings are discussed.