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Abstract Background Sphingolipids 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. Methods The 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. Results Analogs 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. Conclusions It 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.

Adenosine Triphosphatase (ATPase) Phospholipid Transporting 11C gene (ATP11C) encodes the major phosphatidylserine (PS) flippase in human red blood cells (RBCs). Flippases actively transport phospholipids (e.g., PS) from the outer to the inner leaflet to establish and maintain phospholipid asymmetry of the lipid bilayer of cell membranes. This asymmetry is crucial for survival since externalized PS triggers phagocytosis by splenic macrophages. Here we report on pathophysiological consequences of decreased flippase activity, prompted by a patient with hemolytic anemia and hemizygosity for a novel c.2365C > T p.(Leu789Phe) missense variant in ATP11C. ATP11C protein expression was strongly reduced by 58% in patient-derived RBC ghosts. Furthermore, functional characterization showed only 26% PS flippase activity. These results were confirmed by recombinant mutant ATP11C protein expression in HEK293T cells, which was decreased to 27% compared to wild type, whereas PS-stimulated ATPase activity was decreased by 57%. Patient RBCs showed a mild increase in PS surface exposure when compared to control RBCs, which further increased in the most dense RBCs after RBC storage stress. The increase in PS was not due to higher global membrane content of PS or other phospholipids. In contrast, membrane lipid lateral distribution showed increased abundance of cholesterol-enriched domains in RBC low curvature areas. Finally, more dense RBCs and subtle changes in RBC morphology under flow hint toward alterations in flow behavior of ATP11C-deficient RBCs. Altogether, ATP11C deficiency is the likely cause of hemolytic anemia in our patient, thereby underlining the physiological role and relevance of this flippase in human RBCs., (© 2023 The Authors. American Journal of Hematology published by Wiley Periodicals LLC.)

Haemoglobin S polymerization in the red blood cells (RBCs) of individuals with sickle cell anaemia (SCA) can cause RBC sickling and cellular alterations. Piezo1 is a mechanosensitive protein that modulates intracellular calcium (Ca 2+ ) influx, and its activation has been associated with increased RBC surface membrane phosphatidylserine (PS) exposure. Hypothesizing that Piezo1 activation, and ensuing Gárdos channel activity, alter sickle RBC properties, RBCs from patients with SCA were incubated with the Piezo1 agonist, Yoda1 (0.1-10 μM). Oxygen-gradient ektacytometry and membrane potential measurement showed that Piezo1 activation significantly decreased sickle RBC deformability, augmented sickling propensity, and triggered pronounced membrane hyperpolarization, in association with Gárdos channel activation and Ca 2+ influx. Yoda1 induced Ca 2+ -dependent adhesion of sickle RBCs to laminin, in microfluidic assays, mediated by increased BCAM binding affinity. Furthermore, RBCs from SCA patients that were homo-/heterozygous for the rs59446030 gain-of-function Piezo1 variant demonstrated enhanced sickling under deoxygenation and increased PS exposure. Thus, Piezo1 stimulation decreases sickle RBC deformability, and increases the propensities of these cells to sickle upon deoxygenation and adhere to laminin. Results support a role of Piezo1 in some of the RBC properties that contribute to SCA vaso-occlusion, indicating that Piezo1 may represent a potential therapeutic target molecule for this disease., (© 2023 British Society for Haematology and John Wiley & Sons Ltd.)

PIEZO1 is a mechanosensitive non-selective cation channel, present in many cell types including Red Blood Cells (RBCs). Together with the Gárdos channel, PIEZO1 forms in RBCs a tandem that participates in the rapid adjustment of the cell volume. The pharmacology allowing functional studies of the roles of PIEZO1 has only recently been developed, with Yoda1 as a widely used PIEZO1 agonist. In 2018, Yoda1 analogues were developed, as a step towards an improved understanding of PIEZO1 roles and functions. Among these, Dooku1 was the most promising antagonist of Yoda1-induced effects, without having any ability to activate PIEZO1 channels. Since then, Dooku1 has been used in various cell types to antagonize Yoda1 effects. In the present study using RBCs, Dooku1 shows an apparent IC 50 on Yoda1 effects of 90.7 µM, one order of magnitude above the previously reported data on other cell types. Unexpectedly, it was able, by itself , to produce entry of calcium sufficient to trigger Gárdos channel activation. Moreover, Dooku1 evoked a rise in intracellular sodium concentrations, suggesting that it targets a non-selective cation channel. Dooku1 effects were abolished upon using GsMTx4, a known mechanosensitive channel blocker, indicating that Dooku1 likely targets PIEZO1. Our observations lead to the conclusion that Dooku1 behaves as a PIEZO1 agonist in the RBC membrane, similarly to Yoda1 but with a lower potency. Taken together, these results show that the pharmacology of PIEZO1 in RBCs must be interpreted with care especially due to the unique characteristics of RBC membrane and associated cytoskeleton., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hatem, Poussereau, Gachenot, Pérès, Bouyer and Egée.)

Severe malarial anemia (SMA) is the main cause of malaria-associated infant mortality in malaria endemic countries. One major factor that contributes to SMA is the accumulation of uninfected red blood cells (uRBCs) in the spleen. We report the activation of adhesion molecules Lutheran/basal cell adhesion molecule (Lu/BCAM) and CD44 on uRBCs from Plasmodium falciparum in vitro cultures and patients with malaria that mediates adherence to the splenic extracellular matrix (ECM) components laminin-α5 and hyaluronic acid (HA), respectively. This tight ECM-adhesion molecule interaction was associated with elevated intracellular Ca2+ levels, increased shedding of microvesicles, and Lu/BCAM clustering on altered uRBCs. Moreover, we observed that a soluble parasite-derived factor promoted the adhesive phenotype of uRBCs, as the incubation of RBCs with filtered malaria-conditioned medium reproduced the same adhesive effect in malaria culture-derived uRBCs. Eventually, Lu/BCAM and CD44 activation facilitate the adherence to ECM components of the red pulp, resulting in the enhanced splenic retention of uRBCs. Our results suggest a novel adhesion molecule-dependent mechanism that augments malaria-induced anemia., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Over 95% of Polycythemia Vera (PV) patients carry the V617F mutation in the tyrosine kinase Janus kinase 2 (JAK2), resulting in uncontrolled erythroid proliferation and a high risk of thrombosis. Using mass spectrometry, we analyzed the RBC membrane proteome and showed elevated levels of multiple Ca 2+ binding proteins as well as endoplasmic-reticulum-residing proteins in PV RBC membranes compared with RBC membranes from healthy individuals. In this study, we investigated the impact of JAK2 V617F on (1) calcium homeostasis and RBC ion channel activity and (2) protein expression and sorting during terminal erythroid differentiation. Our data from automated patch-clamp show modified calcium homeostasis in PV RBCs and cell lines expressing JAK2 V617F , with a functional impact on the activity of the Gárdos channel that could contribute to cellular dehydration. We show that JAK2 V617F could play a role in organelle retention during the enucleation step of erythroid differentiation, resulting in modified whole cell proteome in reticulocytes and RBCs in PV patients. Given the central role that calcium plays in the regulation of signaling pathways, our study opens new perspectives to exploring the relationship between JAK2 V617F , calcium homeostasis, and cellular abnormalities in myeloproliferative neoplasms, including cellular interactions in the bloodstream in relation to thrombotic events.

Handbooks of physiology state that the strategy adopted by red blood cells (RBCs) to preserve cell volume is to maintain membrane permeability for cations at its minimum. However, enhanced cation permeability can be measured and observed in specific physiological and pathophysiological situations such as in vivo senescence, storage at low temperature, sickle cell anemia and many other genetic defects affecting transporters, membrane or cytoskeletal proteins. Among cation pathways, cation channels are able to dissipate rapidly the gradients that are built and maintained by the sodium and calcium pumps. These situations are very well-documented but a mechanistic understanding of complex electrophysiological events underlying ion transports is still lacking. In addition, non-selective cation (NSC) channels present in the RBC membrane have proven difficult to molecular identification and functional characterization. For instance, NSC channel activity can be elicited by Low Ionic Strength conditions (LIS): the associated change in membrane potential triggers its opening in a voltage dependent manner. But, whereas this depolarizing media produces a spectacular activation of NSC channel, Gárdos channel-evoked hyperpolarization's have been shown to induce sodium entry through a pathway thought to be conductive and termed P cat . Using the CCCP method, which allows to follow fast changes in membrane potential, we show here (i) that hyperpolarization elicited by Gárdos channel activation triggers sodium entry through a conductive pathway, (ii) that chloride conductance inhibition unveils such conductive cationic conductance, (iii) that the use of the specific chloride conductance inhibitor NS3623 (a derivative of Neurosearch compound NS1652), at concentrations above what is needed for full anion channel block, potentiates the non-selective cation conductance. These results indicate that a non-selective cation channel is likely activated by the changes in the driving force for cations rather than a voltage dependence mechanism per se ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Monedero Alonso, Pérès, Hatem, Bouyer and Egée.)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

To ensure the transport of nutrients necessary for their survival, Plasmodium falciparum parasites increase erythrocyte permeability to diverse solutes. These new permeation pathways (NPPs) have been extensively characterized in the pathogenic asexual parasite stages, however the existence of NPPs has never been investigated in gametocytes, the sexual stages responsible for transmission to mosquitoes. Here, we show that NPPs are still active in erythrocytes infected with immature gametocytes and that this activity declines along gametocyte maturation. Our results indicate that NPPs are regulated by cyclic AMP (cAMP) signaling cascade, and that the decrease in cAMP levels in mature stages results in a slowdown of NPP activity. We also show that NPPs facilitate the uptake of artemisinin derivatives and that phosphodiesterase (PDE) inhibitors can reactivate NPPs and increase drug uptake in mature gametocytes. These processes are predicted to play a key role in P. falciparum gametocyte biology and susceptibility to antimalarials.

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Cell movement or motility is essential for a large variety of processes. Fertilization and host cells invasion by parasites are among the mostly studied models so far. Body of evidences into the literature raises the question that common mechanisms may be found in the sequential events that lead to cell motility in these two particular models. This short review aims at highlighting these common features by comparing knowledge on motile forms of Plasmodium falciparum and one of the best known motile cell namely the spermatozoa. Emphasis will be done on the substantial changes affecting the biochemical, electrophysiological and functional properties of both models., (Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

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.

Plasmodium falciparum relies on anion channels activated in the erythrocyte membrane to ensure the transport of nutrients and waste products necessary for its replication and survival after invasion. The molecular identity of these anion channels, termed "new permeability pathways" is unknown, but their currents correspond to up-regulation of endogenous channels displaying complex gating and kinetics similar to those of ligand-gated channels. This report demonstrates that a peripheral-type benzodiazepine receptor, including the voltage dependent anion channel, is present in the human erythrocyte membrane. This receptor mediates the maxi-anion currents previously described in the erythrocyte membrane. Ligands that block this peripheral-type benzodiazepine receptor reduce membrane transport and conductance in P falciparum-infected erythrocytes. These ligands also inhibit in vitro intraerythrocytic growth of P falciparum. These data support the hypothesis that dormant peripheral-type benzodiazepine receptors become the "new permeability pathways" in infected erythrocytes after up-regulation by P falciparum. These channels are obvious targets for selective inhibition in anti-malarial therapies, as well as potential routes for drug delivery in pharmacologic applications.

During the past three decades, electrophysiological studies revealed that human red blood cell membrane is endowed with a large variety of ion channels. The physiological role of these channels, if any, remains unclear; they do not participate in red cell homeostasis which is rather based on the almost total absence of cationic permeability and minute anionic conductance. They seem to be inactive in the "resting cell." However, when activated experimentally, ion channels can lead to a very high single cell conductance and potentially induce disorders, with the major risks of fast dehydration and dissipation of gradients. Could there be physiological conditions under which the red cell needs to activate these high conductances, or are ion channels relics of a function lost in anucleated cells? It has been demonstrated that they play a key role in diseases such as sickle cell anemia or malaria. This short overview of ion channels identified to-date in the human red cell membrane is an attempt to propose a dynamic role for these channels in circulating cells in health and disease., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Historically, the anion transport through the human red cell membrane has been perceived to be mediated by Band 3, in the two-component concept with the large electroneutral anion exchange accompanied by the conductance proper, which dominated the total membrane conductance. The status of anion channels proper has never been clarified, and the informations obtained by different groups of electrophysiologists are rather badly matched. This study, using the cell-attached configuration of the patch-clamp technique, rationalizes and explains earlier confusing results by demonstrating that the diversity of anionic channel activities recorded in human erythrocytes corresponds to different kinetic modalities of a unique type of maxi-anion channel with multiple conductance levels and probably multiple gating properties and pharmacology, depending on conditions. It demonstrates the role of activator played by serum in the recruitment of multiple new conductance levels showing very complex kinetics and gating properties upon serum addition. These channels, which seem to be dormant under normal physiological conditions, are potentially activable and could confer a far higher anion conductance to the red cell than the ground leak mediated by Band 3.

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 cell's 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 Ca(2+). The Ca(2+)-dependent currents were transient, with typical decay half-times of about 5-10 min, suggesting the spontaneous inactivation of a stretch-activated Ca(2+) permeability (PCa). These results indicate that local membrane deformations can transiently activate a Ca(2+) permeability pathway leading to increased [Ca(2+)](i), secondary activation of Ca(2+)-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 Ca(2+)-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca(2+) content of red cells in certain hereditary anemias such as thalassemia and sickle cell anemia.

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., (2009 Elsevier Ltd. All rights reserved.)

By replicating within red blood cells, malaria parasites are largely hidden from immune recognition; however, in the cells, nutrients are limiting and hazardous metabolic end products can rapidly accumulate. Therefore, to survive within erythrocytes, parasites alter the permeability of the host plasma membrane, either by upregulating existing transporters or by creating new permeation pathways. Recent electrophysiological studies of Plasmodium-infected erythrocytes have demonstrated that membrane permeability is mediated by transmembrane transport through ion channels in the infected erythrocyte. This article discusses the evidence and controversies concerning the nature of these channels and surveys the potential role of phosphorylation in activating anion channels that could be important in developing novel strategies for future malarial chemotherapies.

In this paper, we provide an update on cation channels in nucleated chicken erythrocytes. Patch-clamp techniques were used to further characterize the two different types of cation channels present in the membrane of chicken red blood. In the whole-cell mode, with Ringer in the bath and internal K+ saline in the pipette solution, the membrane conductance was generated by cationic currents, since the reversal potential was shifted toward cations equilibrium when the impermeant cation NMDG was substituted to small cations. The membrane conductance could be increased by application of mechanical deformation or by the addition of agonists of the cAMP-dependent pathway. At the unitary level, two different types of cationic channels were revealed and could account for the cationic conductance observed in whole-cell configuration. One of them belongs to the family of stretch-activated cationic channel showing changes in activity under conditions of membrane deformation, whereas the second one belongs to the family of the cAMP activated cationic channels. These two channels could be distinguished according to their unitary conductances and drug sensitivities. The stretch-activated channel was sensitive to Gd(3+) and the cAMP-dependent channel was sensitive to flufenamic acid. Possible role of these channels in cell volume regulation process is discussed.

Malaria symptoms occur during Plasmodium falciparum development into red blood cells. During this process, the parasites make substantial modifications to the host cell in order to facilitate nutrient uptake and aid in parasite metabolism. One significant alteration that is required for parasite development is the establishment of an anion channel, as part of the establishment of New Permeation Pathways (NPPs) in the red blood cell plasma membrane, and we have shown previously that one channel can be activated in uninfected cells by exogenous protein kinase A. Here, we present evidence that in P. falciparum-infected red blood cells, a cAMP pathway modulates anion conductance of the erythrocyte membrane. In patch-clamp experiments on infected erythrocytes, addition of recombinant PfPKA-R to the pipette in vitro, or overexpression of PfPKA-R in transgenic parasites lead to down-regulation of anion conductance. Moreover, this overexpressing PfPKA-R strain has a growth defect that can be restored by increasing the levels of intracellular cAMP. Our data demonstrate that the anion channel is indeed regulated by a cAMP-dependent pathway in P. falciparum-infected red blood cells. The discovery of a parasite regulatory pathway responsible for modulating anion channel activity in the membranes of P. falciparum-infected red blood cells represents an important insight into how parasites modify host cell permeation pathways. These findings may also provide an avenue for the development of new intervention strategies targeting this important anion channel and its regulation.

Electrophysiological studies on human RBCs have been difficult due to fragility and small size of cells, and little is known of ionic conductive pathways present in the RBC membrane in health and disease. We report on anionic channels in cells of healthy donors (control) and cystic fibrosis (CF) patients. Anion channel activity (8-12 pS, linear) was induced in cell-attached configuration by forskolin (50 microM) and in excised inside-out configuration by PKA (100 nM) and ATP (1 mM) but control and CF RBCs differed by their respective kinetics and gating properties. These channels were permeable to ATP (100 mM, symmetrical Tris-ATP). These data suggest either the existence of two different anionic channel types or regulation of a single channel type either by the CFTR (cystic fibrosis transmembrane regulator) protein or by different cytosolic factors. Another anionic channel type displaying outward rectification (approximately 80 pS, outward conductance) was present in 30% of CF cell patches but was not observed in normal cell patches. The frequently recorded activity of this channel in CF patches suggests a down-regulation in normal RBCs.

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 Zn(2+), 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.

The altered permeability characteristics of erythrocytes infected with malaria parasites have been a source of interest for over 30 years. Recent electrophysiological studies have provided strong evidence that these changes reflect transmembrane transport through ion channels in the host erythrocyte plasma membrane. However, conflicting results and differing interpretations of the data have led to confusion in this field. In an effort to unravel these issues, the groups involved recently came together for a week of discussion and experimentation. In this article, the various models for altered transport are reviewed, together with the areas of consensus in the field and those that require a better understanding.

The electrophysiological study of red blood cells (RBCs), using the patch-clamp technique, has been going through a renaissance with the recent discovery of novel channel activity in the host plasma membrane of Plasmodium falciparum-infected human RBCs (S.A. Desai et al., Nature 406, 1001-1005, 2000; S.M. Huber et al., EMBO J. 21 (2002) 22-30; S. Egee et al., J. Physiol. 542 (2002) 795-801). This arose from the finding that malaria-infected RBCs have altered permeability characteristics due to the induction of new permeation pathways (NPPs) (H. Ginsburg, Novartis Foundation Symposium 226 (1999) 99-108; K. Kirk, Physiol. Rev. 81 (2001) 495-537), which are defined, using non-electrophysiological techniques, as having the general characteristics of anion channels (i.e. high anion permeability, linear concentration dependence, inability to distinguish between stereo-isomers of permeant solutes). Discovering potent and specific inhibitors of the NPPs is an important therapeutic challenge, but too many questions remain unanswered: do the NPPs correspond to a single path or multiple pathways? Are they parasite-derived proteins? Are they up-regulated or modified endogenous quiescent red blood cell proteins? This article concerns the identification of different types of anionic channels that are expressed in malaria-infected human RBCs. Implications regarding the presence of these different types of channels in infected RBCs and their functional significance are discussed.

Background: Sphingolipids 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., Methods: The 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., Results: Analogs 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., Conclusions: It 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.

As the intraerythrocytic stage of the human malarial parasite, Plasmodium falciparum, matures, the plasma membrane of the host red blood cell (RBC) becomes increasingly permeable to a variety of physiologically relevant solutes via the induction of new permeation pathways (NPPs) (H. Ginsburg, Novartis Foundation Symposium 226, 99-108,1999; K. Kirk, Physiol. Rev. 81, 495-537, 2001). Although permeable to cationic and electroneutral solutes, transport studies have shown that the NPPs exhibit the general properties of anion channels and recent electrophysiological studies, using the patch-clamp technique, have demonstrated that anion channels are activated in the plasma membrane of the RBC following infection (S.A. Desai et al., Nature 406, 1001-1005, 2000; S.M. Huber et al., EMBO J. 21, 22-30,2002; S. Egee et al., J. Physiol. 542, 795-801, 2002). In this paper, we review the features of the anionic channels that we have observed in both uninfected and malaria-infected human RBCs, the data that suggest that the NPPs are endogenous to the RBC membrane, and present new evidence, which suggests that the mechanism of induction of the NPPs, used by the parasite, involves phosphorylation steps.

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.

The patch-clamp technique was used to demonstrate the presence of ATP-sensitive K(+) channels and Ca(2+)-activated K(+) channels in lamprey ( Petromyzon marinus) red blood cell membrane. Whole-cell experiments indicated that the membrane current under isosmotic (285 mosmol l(-1)) conditions is carried by K(+). In the inside-out configuration an ATP-sensitive K(+) channel (70-80 pS inward, 35-40 pS outward) was present in 35% of patches. Application of ATP to the intracellular side reduced unitary current with half-maximal inhibition in the range 10-100 microM. A block was obtained with 100 microM lidocaine and inhibition was obtained with 0.5 mM barium acetate. A Ca(2+)-activated K(+) channel (25-30 pS inward, 10-15 pS outward) was present in 57% of patches. Inhibition was produced by 10 mM TEA and 500 nM apamin and sensitivity to Ba(2+) was lower than for ATP-sensitive channels. No spontaneous channel activity was recorded in the cell-attached configuration under isotonic conditions. With hypotonic saline 68% of patches showed spontaneous single-channel activity, and, of 75 active patches, 66 cell-attached patches showed channel activity corresponding to Ca(2+)-activated K(+) channels.

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 (P(o)) 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.

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 Ca(2+) 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.

(1) An outwardly rectifying chloride channel (ORCC) of large conductance has been detected under isotonic conditions (320 mosM 1(-1)) in the plasma membrane of trout red blood cells (RBCs) using the excised inside-out configuration. The channel, with a permeability ratio P(Cl)/Pcation of 12, was inhibited by the Cl- channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) (50 microM), and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) (100 microM) in the bathing solution. (2) In hypotonic conditions (215 mosM 1(-1)), 44% of cell-attached patches showed spontaneous single channel activity identified as nonselective cationic (NSC) channels. A second group, corresponding to 7% of cell-attached patches, showed spontaneous activity corresponding to a channel type presenting outward rectification and anionic selectivity. Finally, 49% of patches displayed a complex spontaneous signal corresponding to the superimposition of inward and outward currents probably due to activation of different channel types. (3) Giga-seals obtained without suction in intact cells under isotonic conditions possessed NSC channels that were quiescent but which could be activated either by mechanical deformation of cell membrane or by hypotonic cell swelling. (4) Hypotonically swollen RBCs exhibited regulatory volume decrease (RVD) over 3 h, which was linked to a fivefold to sixfold increase in unidirectional fluxes of K+, a net loss of intracellular K+ and net gain of extracellular Na+. RVD and the hypotonically activated, unidirectional K+ influx continued after replacement of Cl- by methylsulfonate (MeSF) albeit more slowly. (5) The NSC channel inhibitor, barium, and the Cl- channel inhibitor, NPPB, both inhibited the RVD response by approximately 50% in Cl- containing saline. When Cl- was replaced by MeSF, the inhibition was > 90% suggesting that NSC channels and ORCC play key roles in the chloride-independent component of RVD.

1. The cell-attached and excised inside-out configurations of the patch-clamp technique were used to demonstrate the presence of two different types of ion channels in the membrane of trout red blood cells under isotonic and normoxic conditions, in the absence of hormonal stimulation. The large majority (93%) of successful membrane seals allowed observation of at least one channel type. 2. In the cell-attached mode with Ringer solution in the bath and Ringer solution, 145 mM KCl or 145 NaCl in the pipette, a channel of intermediate conductance (15-25 pS at clamped voltage, Vp = 0 mV) was present in 85% of cells. The single channel activity reversed between 5 and 7 mV positive to the spontaneous membrane potential. A small conductance channel of 5-6 pS and +5 mV reversal potential was also present in 62% of cells. 3. After excision into the inside-out configuration (with 145 mM KCl or NaCl, pCa 8 in the bath, 145 mM KCl or NaCl, pCa 3 in the pipette) the intermediate conductance channel was present in 439 out of 452 successful seals. This channel was spontaneously active in 90% of patches and in the other 10% of patches the channel was activated by suction. The current-voltage relationship showed slight inward rectification. The channel conductance was in the range 15-20 pS between -60 and 0 mV and increased to 25-30 pS between 0 and 60 mV, with a reversal potential close to zero. Substitution of K+ for Na+ in the pipette or in the bath did not significantly change the single channel conductance. Dilution of the bathing solution KCl concentration shifted the reversal potential towards the Nernst equilibrium for cations. Substitution of N-methyl-D-glucamine (NMDG) for K+ or Na+ in the bath almost abolished the outward current whilst the divalent cation Ca2+ permeated the channel with a higher permeability than K+ and Na+. Inhibition of channel openings was obtained with flufenamic acid, quinine, gadolinium or barium. Taken together these data demonstrate that the intermediate conductance channel belongs to a class of non-selective cation (NSC) channels. 4. In excised patches, under the same control conditions, the conductance of the small conductance non-rectifying channel was 8.6 +/- 0.8 pS (n = 12) between -60 and +60 mV and the reversal potential was close to 0 mV. This channel could be blocked by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) but not by flufenamic acid, DIDS, barium or gadolinium. Selectivity and substitution experiments made it possible to identify this channel as a non-rectifying small conductance chloride (SCC) channel.

Several membrane ion transporters playing a role in gas transport and exchanges, cell volume regulation and intracellular acid-base regulation have been identified in fish red blood cells (RBCs). This short review focuses on Na+/K+ATPase and its role in establishing the ionic gradients across the membrane, on the Cl-/HCO3- exchanger and its key role in respiration and possibly in inducing a chloride conductance, on the Na+/H+ exchanger and the recent advances on its molecular mechanisms of activation and regulation, on the different types of K-Cl cotransports, the different hypotheses and suggested models and their role in cell volume regulation. There is no evidence in the literature for ionic channels in fish RBCs. We present original data obtained with the patch-clamp technique that shows for the first time the existence of a DIDS-sensitive chloride anionic conductance measured in whole cell configuration and the presence of a stretch-activated nonselective cationic channel recorded in cell-attached and excised inside-out configuration. The part played by these ionic conductances is discussed in relation with their possible involvement in volume regulation.

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- 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 l-1). This effect was abolished in Cl(-)-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.