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.     

Complex inheritance of the plasmodial surface anion channel in a Plasmodium falciparum genetic cross

Human erythrocytes infected with the malaria parasite Plasmodium falciparum have increased permeabilities to many solutes. The plasmodial surface anion channel (PSAC) may mediate these changes. Despite good understanding of the biochemical and biophysical properties, the genetic basis of PSAC activity remains unknown. Functional polymorphisms in laboratory isolates and two mutants generated by in vitro selection implicate a parasite-encoded channel, although parasite-induced modifications of endogenous channels have not been formally excluded. Here, we identified stable differences in furosemide efficacy against PSAC activity induced by HB3 and 3D7A parasites. This difference was apparent in both single PSAC patch-clamp recordings and in sorbitol-mediated osmotic lysis measurements, confirming that Cl^- and sorbitol are transported by a single-channel type. Examination of 19 progeny from a genetic cross between HB3 and 3D7A revealed complex inheritance with some cloned progeny exhibiting furosemide affinities outside the range of parental values. Isolates generated by selfing of the 3D7A clone also exhibited altered furosemide affinities, implicating changes in one or more alleles during meiosis or passage through a primate host. PSAC may be encoded by multiple parasite genes (e.g. a multi-gene family or multiple genes that encode distinct channel subunits) or a single polymorphic gene under strong selective pressure.     

Babesia and plasmodia increase host erythrocyte permeability through distinct mechanisms

Human erythrocytes infected with Plasmodium falciparum have markedly increased permeability to diverse solutes, many of which may be mediated by an unusual small conductance ion channel, the plasmodial surface anion channel (PSAC). Because these increases may be essential for parasite survival in the bloodstream, an important question is whether other intraerythrocytic parasites induce similar ion channels. Here, we examined this question using human erythrocytes infected with Babesia divergens, a distantly related apicomplexan parasite that can cause severe disease in immunocompromised humans. Osmotic lysis experiments after enrichment of infected erythrocytes with a new method revealed that these parasites also increase host permeability to various organic solutes. These permeability changes differed significantly from those induced by P. falciparum in transport rates, selectivity profiles and temperature dependence. Cell-attached and whole-cell patch-clamp experiments confirmed and extended these differences because neither PSAC-like channels nor significant increases in whole-cell anion conductance were seen after B. divergens infection. While both babesia and plasmodia increase host erythrocyte permeability to a diverse collection of organic solutes, they utilize fundamentally different mechanisms.     

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.     

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.     

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.     

Plasmodial surface anion channel-independent phloridzin resistance in Plasmodium falciparum

The plasmodial surface anion channel (PSAC) is an unusual ion channel induced on the human red blood cell membrane after infection with the malaria parasite, Plasmodium falciparum. Because PSAC is permeant to small metabolic precursors essential for parasite growth and is present on red blood cells infected with geographically divergent parasite isolates, it may be an ideal target for future antimalarial development. Here, we used chemically induced mutagenesis and known PSAC antagonists that inhibit in vitro parasite growth to examine whether resistance mutations in PSAC can be readily induced. Stable mutants resistant to phloridzin were generated and selected within 3 weeks after treatment with 1-methyl-3-nitro-1-nitrosoguanidine. These mutants were evaluated with osmotic lysis and electrophysiological transport assays, which indicate that PSAC inhibition by phloridzin is complex with at least two different modes of inhibition. Mutants resistant to the growth inhibitory effects of phloridzin expressed PSAC activity indistinguishable from that on sensitive parasites, indicating selection of resistance via mutations in one or more other parasite targets. Failure to induce mutations in PSAC activity is consistent with a highly constrained channel protein less susceptible to resistance mutations; whether this protein is parasite- or host-encoded remains to be determined.     

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.     

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.     

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.     

Synergistic Malaria Parasite Killing by Two Types of Plasmodial Surface Anion Channel Inhibitors

Malaria parasites increase their host erythrocyte’s permeability to a broad range of ions and organic solutes. The plasmodial surface anion channel (PSAC) mediates this uptake and is an established drug target. Development of therapies targeting this channel is limited by several problems including interactions between known inhibitors and permeating solutes that lead to incomplete channel block. Here, we designed and executed a high-throughput screen to identify a novel class of PSAC inhibitors that overcome this solute-inhibitor interaction. These new inhibitors differ from existing blockers and have distinct effects on channel-mediated transport, supporting a model of two separate routes for solute permeation though PSAC. Combinations of inhibitors specific for the two routes had strong synergistic action against in vitro parasite propagation, whereas combinations acting on a single route produced only additive effects. The magnitude of synergism depended on external nutrient concentrations, consistent with an essential role of the channel in parasite nutrient acquisition. The identified inhibitors will enable a better understanding of the channel’s structure-function and may be starting points for novel combination therapies that produce synergistic parasite killing.     

Expression of the Skate (<Emphasis Type="Italic">Raja erinacea</Emphasis>) AE1 Osmolyte Channel in <Emphasis Type="Italic">Xenopus laevis</Emphasis> Oocytes: Monovalent Cation Permeability

The aim of this study was to express the cloned skate anion exchanger 1 (skAE1) in Xenopus oocytes and determine whether the differences in monovalent cation permeabilities in hypotonically stimulated skate and trout erythrocytes could be due to differences in the presence or absence of intracellular channel regulators between the two species or in the intrinsic permeability properties of the channels themselves. The expressed protein (skAE1) was inserted into the oocyte cell membrane and facilitated both Cl⁻ exchange and taurine transport. Expression of skAE1 in oocytes showed similar monovalent cation permeabilities as previously reported for skate erythrocytes and different from both trout erythrocytes and trAE1 expressed in Xenopus oocytes. These results show that the skAE1 expressed in oocytes functions in a manner similar to that of the osmolyte channel in hypotonically activated skate erythrocytes and supports the hypothesis that differences in the monovalent cation permeabilities of the osmolyte channels in skate and trout RBCs resides in the differences in permeability properties of the channels between the two species.This revised version was published online in August 2005 with a corrected cover date.     

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.     

Plasmodium falciparum-activated chloride channels are defective in erythrocytes from cystic fibrosis patients

An inwardly rectifying anion channel in malaria-infected red blood cells has been proposed to function as the "new permeation pathway" for parasite nutrient acquisition. As the channel shares several properties with the cystic fibrosis transmembrane conductance regulator (CFTR), we tested their interrelationship by whole-cell current measurements in Plasmodium falciparum-infected and uninfected red blood cells from control and cystic fibrosis (CF) patients. A CFTR-like linear chloride conductance as well as a malaria parasite-induced and a shrinkage-activated endogenous inwardly rectifying chloride conductance with properties identical to the malaria-induced channel were all found to be defective in CF erythrocytes. Surprisingly, the absence of the inwardly rectifying chloride conductance in CF erythrocytes had no gross effect on in vitro parasite growth or new permeation pathway activity, supporting an argument against a close association between the Plasmodium-activated chloride channel and the new permeation pathway. The functional expression of CFTR in red blood cells opens new perspectives to exploit the erythrocyte as a readily available cell type in electrophysiological, diagnostic, and therapeutic studies of CF.     

Epigenetic switches in clag3 genes mediate blasticidin S resistance in malaria parasites

Malaria parasites induce changes in the permeability of the infected erythrocyte membrane to numerous solutes, including toxic compounds. In Plasmodium falciparum, this is mainly mediated by PSAC, a broad‐selectivity channel that requires the product of parasite clag3 genes for its activity. The two paralogous clag3 genes, clag3.1 and clag3.2, can be silenced by epigenetic mechanisms and show mutually exclusive expression. Here we show that resistance to the antibiotic blasticidin S (BSD) is associated with switches in the expression of these genes that result in altered solute uptake. Low concentrations of the drug selected parasites that switched from clag3.2 to clag3.1 expression, implying that expression of one or the other clag3 gene confers different transport efficiency to PSAC for some solutes. Selection with higher BSD concentrations resulted in simultaneous silencing of both clag3 genes, which severely compromises PSAC formation as demonstrated by blocked uptake of other PSAC substrates. Changes in the expression of clag3 genes were not accompanied by large genetic rearrangements or mutations at the clag3 loci or elsewhere in the genome. These resultsdemonstrate that malaria parasites can become resistant to toxic compounds such as drugs by epigenetic switches in the expression of genes necessary for the formation of solute channels.     

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.     

Diltiazem at high concentration increases the ionic permeability of biological membranes

Summary The effects of diltiazem, a drug which inhibits the calcium channels in cardiac muscle as well as the light-sensitive channels in photoreceptor cells, were studied on ionic fluxes in both membrane and intact cell preparations. Diltiazem nonselectively increased the ionic permeability to both anions and cations in photoreceptor rod outer segment and synaptic membrane vesicles as well as in intact erythrocytes. Under our conditions, the estimated threshold for the diltiazem effect varied between 12.5 and 200 m. In each case the concentration dependence exhibited the sigmoidal shape characteristic of positive cooperativity. The effect of diltiazem on ionic fluxes from phospholipid vesicles were strongly influenced by phospholipid composition and membrane charge. By contrast, diltiazem inhibited the efflux of⁸⁶Rb from photoreceptor cells of intact aspartate-isolated retina, an effect opposite to that of diltiazem on ionic permeabilities in photoreceptor membrane vesicle preparations.These data raise serious doubts on the specificity of diltiazem as a calcium channel blocker or as a cGMP channel blocker when used at concentrations higher than 10 m.     

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.     

Anion Selectivity of Slow Anion Channels in the Plasma Membrane of Guard Cells (Large Nitrate Permeability)

Closing of stomatal pores in the leaf epidermis of higher plants is mediated by long-term release of potassium and the anions chloride and malate from guard cells and by parallel metabolism of malate. Previous studies have shown that slowly activating anion channels in the plasma membrane of guard cells can provide a major pathway for anion efflux while also controlling K+ efflux during stomatal closing: Anion efflux produces depolarization of the guard cell plasma membrane that drives K+ efflux required for stomatal closing. The patch-clamp technique was applied to Vicia faba guard cells to determine the permeability of physiologically significant anions and halides through slow anion channels to assess the contribution of these anion channels to anion efflux during stomatal closing. Permeability ratio measurements showed that all tested anions were permeable with the selectivity sequence relative to Cl- of NO3- > Br- > F- ~ Cl- ~ I- > malate. Large malate concentrations in the cytosol (150 mM) produced a slow down-regulation of slow anion channel currents. Single anion channel currents were recorded that correlated with whole-cell anion currents. Single slow anion channels confirmed the large permeability ratio for nitrate over chloride ions. Furthermore, single-channel studies support previous indications of multiple conductance states of slow anion channels, suggesting cooperativity among anion channels. Anion conductances showed that slow anion channels can mediate physiological rates of Cl- and initial malate efflux required for mediation of stomatal closure. The large NO3- permeability as well as the significant permeabilities of all anions tested indicates that slow anion channels do not discriminate strongly among anions. Furthermore, these data suggest that slow anion channels can provide an efficient pathway for efflux of physiologically important anions from guard cells and possibly also from other higher plant cells that express slow anion channels.     

Calcium and the malaria parasite: parasite maturation and the loss of red cell deformability

In the studies reported here, we examined the role of calcium in the maturation of the human malaria parasite Plasmodium falciparum, and in the loss of red cell deformability associated with parasite maturation. P. falciparum alters the permeability of its host red cell, which normally maintains submicromolar cytoplasmic concentrations of calcium. Infection of the red cell and parasite maturation produce a 30-fold increase in calcium uptake. Both parasite maturation and the loss of red cell deformability are blocked by EGTA (by extracellular-free calcium concentrations less than or equal to 35 microM) and by other calcium antagonists. The loss of red cell deformability that occurs with parasite maturation is accompanied by alterations in the cytoskeletal proteins of parasitized red cells similar to those produced by the calcium ionophore A23187 (reductions in bands 2.1 [ankyrin], 4.1, and 5 [actin]). These results establish that parasite development and the loss of red cell deformability are calcium-dependent. They suggest that parasite-induced changes in the calcium permeability of the red cell activate endogenous transglutaminase activity by raising the free calcium concentration of the red cell cytoplasm.