The plasmodial surface anion channel is functionally conserved in divergent malaria parasites

The plasmodial surface anion channel (PSAC), a novel ion channel induced on human erythrocytes infected with Plasmodium falciparum, mediates increased permeability to nutrients and presumably supports intracellular parasite growth. Isotope flux studies indicate that other malaria parasites also increase the permeability of their host erythrocytes, but the precise mechanisms are unknown. Channels similar to PSAC or alternative mechanisms, such as the upregulation of endogenous host transporters, might fulfill parasite nutrient demands. Here we evaluated these possibilities with rhesus monkey erythrocytes infected with Plasmodium knowlesi, a parasite phylogenetically distant from P. falciparum. Tracer flux and osmotic fragility studies revealed dramatically increased permeabilities paralleling changes seen after P. falciparum infection. Patch-clamp of P. knowlesi-infected rhesus erythrocytes revealed an anion channel with striking similarities to PSAC: its conductance, voltage-dependent gating, pharmacology, selectivity, and copy number per infected cell were nearly identical. Our findings implicate a family of unusual anion channels highly conserved on erythrocytes infected with various malaria parasites. Together with PSAC's exposed location on the host cell surface and its central role in transport changes after infection, this conservation supports development of antimalarial drugs against the PSAC family.     

Ion and nutrient uptake by malaria parasite-infected erythrocytes

Erythrocytes infected with malaria parasites have increased permeability to diverse organic and inorganic solutes. While these permeability changes have been known for decades, the molecular basis of transport was unknown and intensively debated. CLAG3, a parasite protein previously thought to function in cytoadherence, has recently been implicated in formation of the plasmodial surface anion channel (PSAC), an unusual small conductance ion channel that mediates uptake of most solutes. Consistent with transport studies, the clag genes are conserved in all plasmodia but are absent from other genera. The encoded protein is integral to the host membrane, as also predicted by electrophysiology. An important question is whether functional channels are formed by CLAG3 alone or through interactions with other proteins. In either case, gene identification should advance our understanding of parasite biology and may lead to new therapeutics.     

Malaria parasite clag3 genes determine channel-mediated nutrient uptake by infected red blood cells

Development of malaria parasites within vertebrate erythrocytes requires nutrient uptake at the host cell membrane. The plasmodial surface anion channel (PSAC) mediates this transport and is an antimalarial target, but its molecular basis is unknown. We report a parasite gene family responsible for PSAC activity. We used high-throughput screening for nutrient uptake inhibitors to identify a compound highly specific for channels from the Dd2 line of the human pathogen P. falciparum. Inheritance of this compound's affinity in a Dd2 × HB3 genetic cross maps to a single parasite locus on chromosome 3. DNA transfection and in vitro selections indicate that PSAC-inhibitor interactions are encoded by two clag3 genes previously assumed to function in cytoadherence. These genes are conserved in plasmodia, exhibit expression switching, and encode an integral protein on the host membrane, as predicted by functional studies. This protein increases host cell permeability to diverse solutes.     

How many functional transport pathways does Plasmodium falciparum induce in the membrane of its host erythrocyte?

Intraerythrocytic malaria parasites induce considerable change in the permeability of the membrane of their host cell. Using classical techniques of radiolabel uptake and iso-osmotic lysis, the permeability characteristics of the host-cell membrane have been determined. In a recent analysis of these results, we concluded that there are at least two types of channel that conform to the data: a low copy number (four channels per cell) type that mediates the transport of cations, anions and most other osmolytes that were tested, and a high copy number (300-400 channels per cell) type that is an anion channel that could also mediate the translocation of purine nucleosides. Patch-clamping experiments using cells infected with Plasmodium falciparum reveal 200-1000 anion channels of more than one type that are of host-cell endogenous provenance. Recent reports show that parasites can grow normally in erythrocytes that lack these endogenous agencies and in which the anion channels are not expressed, although their osmolyte permeability is present. We suggest that only the latter type of channel is important for normal development of the parasite.     

Specific inhibition of the plasmodial surface anion channel by dantrolene

The plasmodial surface anion channel (PSAC), induced on human erythrocytes by the malaria parasite Plasmodium falciparum, is an important target for antimalarial drug development because it may contribute to parasite nutrient acquisition. However, known antagonists of this channel are quite nonspecific, inhibiting many other channels and carriers. This lack of specificity not only complicates drug development but also raises doubts about the exact role of PSAC in the well-known parasite-induced permeability changes. We recently identified a family of new PSAC antagonists structurally related to dantrolene, an antagonist of muscle Ca++ release channels. Here, we explored the mechanism of dantrolene's actions on parasite-induced permeability changes. We found that dantrolene inhibits the increased permeabilities of sorbitol, two amino acids, an organic cation, and hypoxanthine, suggesting a common pathway shared by these diverse solutes. It also produced parallel reductions in PSAC single-channel and whole-cell Cl- currents. In contrast to its effect on parasite-induced permeabilities, dantrolene had no measurable effect on five other classes of anion channels, allaying concerns of poor specificity inherent to other known antagonists. Our studies indicate that dantrolene binds PSAC at an extracellular site distinct from the pore, where it inhibits the conformational changes required for channel gating. Its affinity for this site depends on ionic strength, implicating electrostatic interactions in dantrolene binding. In addition to the potential therapeutic applications of its derivatives, dantrolene's specificity and its defined mechanism of action on PSAC make it a useful tool for transport studies of infected erythrocytes.     

Two Distinct Mechanisms of Transport Through the Plasmodial Surface Anion Channel

The plasmodial surface anion channel (PSAC) is a voltage-dependent ion channel on erythrocytes infected with malaria parasites. To fulfill its presumed function in parasite nutrient acquisition, PSAC is permeant to a broad range of charged and uncharged solutes; it nevertheless excludes Na⁺ as required to maintain erythrocyte osmotic stability in plasma. Another surprising property of PSAC is its small single-channel conductance (<3 pS in isotonic Cl^?) in spite of broad permeability to bulky solutes. While exploring the mechanisms underlying these properties, we recently identified interactions between permeating solutes and PSAC inhibitors that suggest the channel has more than one route for passage of solutes. Here, we explored this possibility with 22 structurally diverse solutes and found that each could be classified into one of two categories based on effects on inhibitor affinity, the temperature dependence of these effects and a clear pattern of behavior in permeant solute mixtures. The clear separation of these solutes into two discrete categories suggests two distinct mechanisms of transport through this channel. In contrast to most other broad-permeability channels, selectivity in PSAC appears to be complex and cannot be adequately explained by simple models that invoke sieving through rigid, noninteracting pores.     

Characterization of permeation pathways in the plasma membrane of human erythrocytes infected with early stages of Plasmodium falciparum: association with parasite development

Human intraerythrocytic malarial parasites (Plasmodium falciparum) induce permeability changes in the membrane of their host cells. The differential permeability of infected erythrocytes at various stages of parasite growth, in combination with density gradient centrifugation, was used to fractionate parasitized cells according to their developmental stage. By this method it was possible to obtain cell fractions consisting essentially of erythrocytes infected with the youngest parasite stage (i.e., rings). These preparations were used for the measurement of transport of various solutes. It is shown that permeabilization of host erythrocyte membrane appears as early as 6 h after parasite invasion of the erythrocyte and increases gradually with parasite maturation. Since the selectivity for several different solutes and the enthalpy of activation of transport remain unaltered with maturation-related increase of permeability, it is concluded that the number of transport agencies in the host cell membrane increases with parasite maturation. Evidence is presented to indicate the need for parasite protein synthesis as an essential factor for the generation of the new permeability pathways.     

Plasmodium falciparum activates endogenous Cl(-) channels of human erythrocytes by membrane oxidation.

Intraerythrocytic survival of the malaria parasite Plasmodium falciparum requires that host cells supply nutrients and dispose of waste products. This solute transport is accomplished by infection-induced new permeability pathways (NPP) in the erythrocyte membrane. Here, whole-cell patch-clamp and hemolysis experiments were performed to define properties of the NPP. Parasitized but not control erythrocytes constitutively expressed two types of anion conductances, differing in voltage dependence and sensitivity to inhibitors. In addition, infected but not control cells hemolyzed in isosmotic sorbitol solution. Both conductances and hemolysis of infected cells were inhibited by reducing agents. Conversely, oxidation induced identical conductances and hemolysis in non-infected erythrocytes. In conclusion, P.falciparum activates endogenous erythrocyte channels by applying oxidative stress to the host cell membrane.     

Plasmodium falciparum regulatory subunit of cAMP-dependent PKA and anion channel conductance

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.     

Plasmodium induces swelling-activated ClC-2 anion channels in the host erythrocyte

Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum depends on delivery of nutrients. Moreover, infection challenges cell volume constancy of the host erythrocyte requiring enhanced activity of cell volume regulatory mechanisms. Patch clamp recording demonstrated inwardly and outwardly rectifying anion channels in infected but not in control erythrocytes. The molecular identity of those channels remained elusive. We show here for one channel type that voltage dependence, cell volume sensitivity, and activation by oxidation are identical to ClC-2. Moreover, Western blots and FACS analysis showed protein and functional ClC-2 expression in human erythrocytes and erythrocytes from wild type (Clcn2(+/+)) but not from Clcn2(-/-) mice. Finally, patch clamp recording revealed activation of volume-sensitive inwardly rectifying channels in Plasmodium berghei-infected Clcn2(+/+) but not Clcn2(-/-) erythrocytes. Erythrocytes from infected mice of both genotypes differed in cell volume and inhibition of ClC-2 by ZnCl(2) (1 mm) induced an increase of cell volume only in parasitized Clcn2(+/+) erythrocytes. Lack of ClC-2 did not inhibit P. berghei development in vivo nor substantially affect the mortality of infected mice. In conclusion, activation of host ClC-2 channels participates in the altered permeability of Plasmodium-infected erythrocytes but is not required for intraerythrocytic parasite survival.     

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 (P(lactate)/P(Cl) = 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 IC(50)s in the range of 0.1 and 1 microM, respectively. In contrast, the IC(50)s of NPPB and furosemide for the inward-rectifying current were >10 microM. 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.     

Increased Ca++ uptake by erythrocytes infected with malaria parasites: Evidence for exported proteins and novel inhibitors

Malaria parasites export many proteins into their host erythrocytes and increase membrane permeability to diverse solutes. Although most solutes use a broad‐selectivity channel known as the plasmodial surface anion channel, increased Ca⁺⁺ uptake is mediated by a distinct, poorly characterised mechanism that appears to be essential for the intracellular parasite. Here, we examined infected cell Ca⁺⁺ uptake with a kinetic fluorescence assay and the virulent human pathogen, Plasmodium falciparum. Cell surface labelling with N‐hydroxysulfosuccinimide esters revealed differing effects on transport into infected and uninfected cells, indicating that Ca⁺⁺ uptake at the infected cell surface is mediated by new or altered proteins at the host membrane. Conditional knockdown of PTEX, a translocon for export of parasite proteins into the host cell, significantly reduced infected cell Ca⁺⁺ permeability, suggesting involvement of parasite‐encoded proteins trafficked to the host membrane. A high‐throughput chemical screen identified the first Ca⁺⁺ transport inhibitors active against Plasmodium‐infected cells. These novel chemical scaffolds inhibit both uptake and parasite growth; improved in vitro potency at reduced free [Ca⁺⁺] is consistent with parasite killing specifically via action on one or more Ca⁺⁺ transporters. These inhibitors should provide mechanistic insights into malaria parasite Ca⁺⁺ transport and may be starting points for new antimalarial drugs.     

Voltage-dependent inactivation of the plasmodial surface anion channel via a cleavable cytoplasmic component

Erythrocytes infected with malaria parasites have increased permeability to ions and various nutrient solutes, mediated by a parasite ion channel known as the plasmodial surface anion channel (PSAC). The parasite clag3 gene family encodes PSAC activity, but there may also be additional unidentified components of this channel. Consistent with a lack of clag3 homology to genes of other ion channels, PSAC has a number of unusual functional properties. Here, we report that PSAC exhibits an unusual form of voltage-dependent inactivation. Inactivation was readily detected in the whole-cell patch-clamp configuration after steps to negative membrane potentials. The fraction of current that inactivates, its kinetics, and the rate of recovery were all voltage-dependent, though with a modest effective valence (0.7±0.1 elementary charges). These properties were not affected by solution composition or charge carrier, suggesting inactivation intrinsic to the channel protein. Intriguingly, inactivation was absent in cell-attached recordings and took several minutes to appear after obtaining the whole-cell configuration, suggesting interactions with soluble cytosolic components. Inactivation could also be largely abolished by application of intracellular, but not extracellular protease. The findings implicate inactivation via a charged cytoplasmic channel domain. This domain may be tethered to one or more soluble intracellular components under physiological conditions.     

The New Permeability Pathways Induced by the Malaria Parasite in the Membrane of the Infected Erythrocyte: Comparison of Results Using Different Experimental Techniques

The membrane of erythrocytes infected with malaria parasites is highly permeable to a large variety of solutes, including anions, carbohydrates, amino acids, nucleosides, organic and inorganic cations and small peptides. The altered permeability is presumed to be due to the activation of endogenous dormant channels, the new permeability pathways. The latter have been studied by different techniques—isosmotic lysis and tracer fluxes—and recently by patch-clamping. Here we analyze all available published data and we show that there is generally a good agreement between the two first methods. From the fluxes we calculate the number of channels per cell using reasonable assumptions as to the radius of the channel, and assuming that penetration through the channel is by diffusion through a water-filled space. The number of channels so calculated is <10 for most solutes, but ~400 for anions and the nucleosides thymidine and adenosine. This latter number is not far from that calculated from patch-clamp experiments. However, the anion flux measured directly by tracer is an order of magnitude larger than expected from conductance measurements. We conclude that the new permeability pathways consist of two types of channels; one is present in small number, and is charge- and size-selective. The other type is about 100-fold more abundant and is anion-selective, but does not admit non-electrolytes other than perhaps nucleosides.     

A two-compartment model of osmotic lysis in Plasmodium falciparum-infected erythrocytes

We recently identified a voltage-dependent anion channel on the surface of human red blood cells (RBCs) infected with the malaria parasite, Plasmodium falciparum. This channel, the plasmodial erythrocyte surface anion channel (PESAC), likely accounts for the increased permeability of infected RBCs to various small solutes, as assessed quantitatively with radioisotope flux and patch-clamp studies. Whereas this increased permeability has also been studied by following osmotic lysis of infected cells in permeant solutes, these experiments have been limited to qualitative comparisons of lysis rates. To permit more quantitative examination of lysis rates, we have developed a mathematical model for osmotic fragility of infected cells based on diffusional uptake via PESAC and the two-compartment geometry of infected RBCs. This model, combined with a simple light scattering assay designed to track osmotic lysis precisely, produced permeability coefficients that match both previous isotope flux and patch-clamp estimates. Our model and light scattering assay also revealed Michaelian kinetics for inhibition of PESAC by furosemide, suggesting a 1:1 stoichiometry for their interaction.     

Altered plasmodial surface anion channel activity and in vitro resistance to permeating antimalarial compounds

Erythrocytes infected with malaria parasites have increased permeability to various solutes. These changes may be mediated by an unusual small conductance ion channel known as the plasmodial surface anion channel (PSAC). While channel activity benefits the parasite by permitting nutrient acquisition, it can also be detrimental because water-soluble antimalarials may more readily access their parasite targets via this channel. Recently, two such toxins, blasticidin S and leupeptin, were used to select mutant parasites with altered PSAC activities, suggesting acquired resistance via reduced channel-mediated toxin uptake. Surprisingly, although these toxins have similar structures and charge, we now show that reduced permeability of one does not protect the intracellular parasite from the other. Leupeptin accumulation in the blasticidin S-resistant mutant was relatively preserved, consistent with retained in vitro susceptibility to leupeptin. Subsequent in vitro selection with both toxins generated a double mutant parasite having additional changes in PSAC, implicating an antimalarial resistance mechanism for water-soluble drugs requiring channel-mediated uptake at the erythrocyte membrane. Characterization of these mutants revealed a single conserved channel on each mutant, albeit with distinct gating properties. These findings are consistent with a shared channel that mediates uptake of ions, nutrients and toxins. This channel's gating and selectivity properties can be modified in response to in vitro selective pressure.     

Infection by Trypanosoma cruzi Enhances Anion Conductance in Rat Neonatal Ventricular Cardiomyocytes

Recent studies on malaria-infected erythrocytes have shown increased anion channel activity in the host cell membrane, increasing the exchange of solutes between the cytoplasm and exterior. In the present work, we addressed the question of whether another intracellular protozoan parasite, Trypanosoma cruzi, alters membrane transport systems in the host cardiac cell. Neonatal rat cardiomyocytes were cultured and infected with T. cruzi in vitro. Ion currents were measured by patch-clamp technique in the whole-cell configuration. Two small-magnitude instantaneous anion currents, outward- and inward-rectifying, were recorded in all noninfected cardiomyocytes. In addition, ~10% of cardiomyocytes expressed a large anion-preferable, time-dependent current activated at positive membrane potentials. Hypotonic (230?mOsm) treatment resulted in the disappearance of the time-dependent current but provoked a dramatic increase of the instantaneous outward-rectifying one. Both instantaneous currents were suppressed by intracellular Mg(2+). T. cruzi infection did not provoke new anion currents in the host cells but caused an increase of the density of intrinsic swelling-activated outward current, up to twice in heavily infected cells. The occurrence of a time-dependent current dramatically increased in infected cells in the presence of Mg(2+) in the intracellular solution, from ~10 to ~80%, without a significant change of the current density. Our findings represent one further, besides the known Plasmodium falciparum, example of an intracellular parasite which upregulates the anionic currents expressed in the host cell.     

Selective killing of the human malaria parasite Plasmodium falciparum by a benzylthiazolium dye

Malaria is an infectious disease caused by protozoan parasites of the genus Plasmodium. The most virulent form of the disease is caused by Plasmodium falciparum which infects hundreds of millions of people and is responsible for the deaths of 1-2 million individuals each year. An essential part of the parasitic process is the remodeling of the red blood cell membrane and its protein constituents to permit a higher flux of nutrients and waste products into or away from the intracellular parasite. Much of this increased permeability is due to a single type of broad specificity channel variously called the new permeation pathway (NPP), the nutrient channel, and the Plasmodial surface anion channel (PSAC). This channel is permeable to a range of low molecular weight solutes both charged and uncharged, with a strong preference for anions. Drugs such as furosemide that are known to block anion-selective channels inhibit PSAC. In this study, we have investigated a dye known as benzothiocarboxypurine, BCP, which had been studied as a possible diagnostic aid given its selective uptake by P. falciparum infected red cells. We found that the dye enters parasitized red cells via the furosemide-inhibitable PSAC, forms a brightly fluorescent complex with parasite nucleic acids, and is selectively toxic to infected cells. Our study describes an antimalarial agent that exploits the altered permeability of Plasmodium-infected red cells as a means to killing the parasite and highlights a chemical reagent that may prove useful in high throughput screening of compounds for inhibitors of the channel.     

Increased permeability of the malaria-infected erythrocyte to organic cations

The human malaria parasite, Plasmodium falciparum, induces in the plasma membrane of its host red blood cell new permeation pathways (NPP) that allow the influx of a variety of low molecular weight solutes. In this study we have demonstrated that the NPP confer upon the parasitised erythrocyte a substantial permeability to a range of monovalent organic (quaternary ammonium) cations, the largest having an estimated minimum cross-sectional diameter of 11-12 A. The rate of permeation of these cations showed a marked dependence on the nature of the anion present, increasing with the lyotropicity of the anion. There was no clear relationship between the permeation rate and either the size or the hydrophobicity of these solutes. However, the data were consistent with the rate of permeation being influenced by a combination of these two factors, with the pathways showing a marked preference for the relatively small and hydrophobic phenyltrimethylammonium ion over larger or less hydrophobic solutes. Large quaternary ammonium cations inhibited flux via the NPP, as did long-chain n-alkanols. For both classes of compound the inhibitory potency increased with the size and hydrophobicity of the solute. This study extends the range of solutes known to permeate the NPP of malaria-infected erythrocytes as well as providing some insight into the factors governing the rate of permeation.     

Protein targeting from malaria parasites to host erythrocytes

Malaria is caused by obligate intracellular parasites, which live in host erythrocytes and remodel these cells to provide optimally for their own needs. Plasmodium falciparum, responsible for malaria in humans, transports many proteins into erythrocytes which help the parasite survive in the host. The recent discovery of a host cell-targeting sequence present in both soluble and transmembrane P. falciparum proteins provoked a discussion on the potential mechanisms of parasite protein entry into infected erythrocytes which is summarized here.