Erythrocyte peripheral type benzodiazepine receptor/voltage-dependent anion channels are upregulated by Plasmodium falciparum.

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.

Ion channels in human red blood cell membrane: actors or relics?

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.

Anion conductance of the human red cell is carried by a maxi-anion channel.

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.

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 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.

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.

Anion channels in Plasmodium-falciparum-infected erythrocytes and protein kinase A.

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.

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 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.

Chloride channels in normal and cystic fibrosis human erythrocyte membrane.

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.

Electrophysiological studies of malaria parasite-infected erythrocytes: current status.

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.

Three types of spontaneously active anionic channels in malaria-infected human red blood cells.

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.

Anti-Plasmodium activity of ceramide analogs.

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.

Anionic channels in malaria-infected human red blood cells.

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.

Plasmodium falciparum-induced channels.

To survive within a red blood cell, the malaria parasite alters dramatically the permeability of the host's plasma membrane (allowing the uptake of essential nutrients and the removal of potentially hazardous metabolites). The pathway(s) responsible for the increased permeability have been proposed as putative chemotherapeutic targets and/or selective routes for antimalarial agents that target the internal parasite. This review covers our current understanding of this parasite-induced phenomenon in Plasmodium falciparum-infected human red blood cells. In particular, recent electrophysiological studies, using the patch-clamp technique, are reviewed.

Plasmodium falciparum and the permeation pathway of the host red blood cell.

Invasion of red blood cells by malaria parasites leads to a huge increase in solute traffic across the membrane of a normally tight cell. Recent electrophysiological investigations strongly support earlier evidence from transport and pharmacological studies that the permeability pathway, which the parasite induces in the host cell membrane, is an anion-selective channel. This article analyzes the evidence and controversies concerning the nature of this channel, surveys the main open questions and suggests directions for future research in this area.

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.

ATP-sensitive K+ and Ca2+-activated K+ channels in lamprey ( Petromyzon marinus) red blood cell membrane.

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 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 (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.