Organic Osmolyte Permeabilities of the Malaria-induced Anion Conductances in Human Erythrocytes

The intraerythrocytic development of P. falciparum induces the so-called new permeability pathways (NPPs) that appear in the membrane of the parasitized erythrocyte. Via the NPP the host cell provides nutrients and vitamins (Saliba et al., 1998) and allows disposal of waste products such as lactic acid (Poole and Halestrap, 1993) and hemoglobin-derived amino acids (Lew et al., 2003). The NPP is thought to reflect ion channels with a high permeability for inorganic anions (Cl⁻, I⁻, SCN⁻), and also for a wide variety of anionic, cationic, and electroneutral organic molecules (Kirk et al., 1994; Ginsburg and Kirk, 1998) such as amino acids, nucleosides, sugars, and polyols (Kirk, 2001). As a consequence, infected erythrocytes swell and hemolyze in isosmotic sorbitol solution (Lambros and Vanderberg, 1979; Ginsburg et al., 1983). The NPP is inhibited by classical anion channel/transport inhibitors (Breuer et al., 1987; Kirk and Horner, 1995a). Most recent studies disclosed the activation of anion conductances in the host cell membrane visible by patch-clamp recording during the trophozoite stage of the blood cycle. On the basis of rectification behavior and difference in inhibitor sensitivity, two distinct anion conductances could be discriminated, i.e., an inwardly rectifying (Desai et al., 2000; Egee et al., 2002; Huber et al., 2002) and an outwardly rectifying conductance (Huber et al., 2002). The activation of these two conductances in infected human erythrocytes has recently been confirmed (Staines et al., 2003), but their functional significance for the malaria infection has not been defined. Since it is still on debate whether one or more channels/permeabilities contribute to the NPP, this study has been designed to elucidate whether the inwardly and outwardly rectifying channels display identical or different properties in permeability, drug sensitivity, regulation, and functional significance. A preliminary account of this work has been published in abstract form (Duranton et al., 2003a).

The P. falciparum strain FCR-3 was permanently maintained in culture using a modified protocol of Trager and Jensen (Trager and Jensen, 1976; Cranmer et al., 1997). In brief, RPMI medium 1640 (Biochrom) was supplemented with 25 mM HEPES/NaOH pH 7.4, 20 μg/ml gentamicin sulfate, 2 mM glutamine, 200 mM hypoxanthine, 0.5% Albumax II (GIBCO-BRL), and 2% of human serum. Washed human erythrocytes of blood group O⁺ (banked) were added to a hematocrit of 5%. Parasites were maintained at a parasitaemia of 2–5% in an atmosphere of 90% N₂/5% O₂/5% CO₂ at 37°C. For patch-clamp experiments parasites were taken directly from the asynchronous culture. For hemolysis and tracer flux experiments, trophozoite- and schizont-infected erythrocytes were enriched magnetically using MACS technology (Staalsoe et al., 1999). In brief, parasite cultures were centrifuged, resuspended in PBS/2% BSA/5 mM glucose and applied to a CS column in a SuperMACS (Miltenyi Biotech). The flow-through contained uninfected red blood cells and ring stages whereas the eluted fraction had a parasitaemia of up to 90% trophozoites/shizonts. All reagents used for culture and patch-clamp experiments were purchased from Sigma-Aldrich if not stated otherwise.

Analysis of parasite developmental stage: P. falciparum–infected erythrocytes were incubated 30 min in PBS buffer containing the fluorescent RNA/DNA dye SYTO 16 (50 μM, Molecular Probes). Fluorescence images were taken at 480/40 nm excitation and 535/50 nm emission wavelength (beamsplitter Q505LP, AHFAnalysentechnik AG) and images were compared with the transmission light micrographs.

Erythrocyte cell volume changes during whole-cell recordings: Erythrocyte volumes were compared for different bath osmolarities. The experiments were performed with a 140 mM NMDGCl pipette solution (see below) combined with a NaCl bath solution (see below) in which the osmolarity was increased and decreased by adding sorbitol (100 mM) and removing NaCl (50 mM), respectively.

For control whole-cell experiments, erythrocytes from healthy donors (donors gave informed consent) were diluted (blood dilution ∼1/1,000) in NaCl bath solution used for the patch-clamp experiments (see below).

P. falciparum–infected erythrocytes are chosen optically by the presence of a prominent (typically moving) light-dense intraerythrocytic structure (hemozoin-containing food vacuole of the mid/late trophozoite-stage; Fig. 1). Infected erythrocytes were recorded at room temperature (patch-clamp data acquired in the authors' laboratory in infected erythrocytes at 37°C or at room temperature did not statistically differ in whole-cell current amplitude or phenotype; unpublished data). Continuous superfusion (∼1 ml/min) was applied through a flow system inserted into the dish (bath volume ∼200 μl).

Contradictory data on whole-cell currents of P. falciparum–infected human erythrocytes have been published. Our previous study demonstrated inwardly and outwardly rectifying anion conductances (Huber et al., 2002), whereas three other groups, in sharp contrast, observed only an inwardly rectifying conductance in infected erythrocytes (Desai et al., 2000; Egee et al., 2002). To solve this conflict the groups of Roscoff and Oxford and ourselves worked together, comparing experimental protocols and culture conditions. As a result, we were able to demonstrate that serum (or albumax) of the culture medium induces in infected (but not in noninfected) cells the outwardly rectifying whole-cell current fraction (Staines et al., 2003). In our hands, superfusion of the recorded cell with serum (or albumax) -free solutions did not result in a run-down of the induced outward rectifying whole-cell current. Instead, inactivation of the outwardly rectifying current component required an extensive washing of the erythrocytes. Since the present study worked on nonwashed infected cells, the outwardly rectifying component was present in the whole-cell experiments.

The bath was grounded via a bridge filled with NaCl bath solution (see below). Borosilicate glass pipettes (8–14 MΩ pipette resistance; GC150 TF-10, Clark Medical Instruments) manufactured by a microprocessor-driven DMZ puller (Zeitz) were used in combination with a STM electrical micromanipulator (Lang Gmbh and Co. KG). Currents were recorded in fast whole-cell, voltage-clamp mode, and 3 kHz low-pass filtered by an EPC-9 amplifier (Heka) using Pulse software (Heka) and an ITC-16 Interface (Instrutech). After giga-Ohm seal formation, the membrane was ruptured by additional suction and/or brief electrical pulses (∼700 mV during 100–200 μs). Rupture of the membrane and entry in whole-cell recording configuration was indicated by a minute increase in capacitance and a simultaneous bleaching of the erythrocyte due to dialysis of hemoglobin by the pipette solution.

The liquid junction potentials ΔE between the pipette and the bath solutions and between the salt bridge and the bath solutions were estimated as described earlier (Barry and Lynch, 1991). Data were corrected for the estimated ΔE values. Whole-cell currents were evoked by 10 to 11 voltage pulses (400 ms each) from −30-mV holding potential to voltages between −100 and +80–100 mV. Original whole-cell current traces are depicted after 500 Hz low-pass filtering. Applied voltages refer to the cytoplasmic face of the membrane with respect to the extracellular space. Inward currents, defined as flow of positive charge from the extracellular to the cytoplasmic membrane face, are negative currents and depicted as downward deflections of the original current traces. Current values were analyzed by averaging the whole-cell currents between 350 and 375 ms of each square pulse.

Most cells were recorded with a pipette solution containing (in mM) 140 NMDG-Cl (pH 7.4), 10 HEPES/NMDG (pH 7.4), 5 MgCl₂, 1 Mg-ATP, 0.5 EGTA (=isotonic NMDG-Cl pipette solution). To swell or shrink the cells during whole-cell recording an isotonic bath solution was combined with a hypertonic or hypotonic NMDG-Cl pipette solution, respectively, containing (in mM): 140 NMDG-Cl (pH 7.4), 40 mannitol, 10 HEPES/NMDG (pH 7.4), 5 MgCl₂, 1 Mg-ATP, 0.5 EGTA (=hypertonic NMDG-Cl pipette solution) and 90 NMDG-Cl (pH 7.4), 10 HEPES/NMDG (pH 7.4), 5 MgCl₂, 1 Mg-ATP, 0.5 EGTA (=hypotonic NMDG-Cl pipette solution). Further pipette solutions were used to study the permeability of the inwardly rectifying conductance for monocarboxylates (in mM): 140 Na-l-lactate, 10 HEPES/NaOH (pH 7.4), 5 MgCl₂, 1 Mg-ATP, 0.5 EGTA.

Cells were superfused with standard bath solution (in mM): 140 NaCl, 10 HEPES/NaOH (pH 7.4), 1 CaCl₂, 1 MgCl₂. To determine the monocarboxylate permeability of the outwardly rectifying conductance, NaCl of the bath was isosmotically replaced by Na-l-lactate or -d-lactate: A Cl⁻-free bath solution containing (in mM) 145 Na-l(d)-gluconate, 10 HEPES/NaOH (pH 7.4) was used in further experiments. To study the effects of neutral carbohydrates on the whole-cell current part of bath NaCl (50 mM) was substituted by sorbitol, mannitol, or sucrose (100 mM each). In further records different concentrations of sucrose or mannitol were added to the NaCl or Na-l-lactate bath solution. To preshrink the cells in some experiments NaCl (25 mM) was added to the standard NaCl bath solution.

Enriched trophozoite-infected erythrocytes (parasitaemia >90%; hematocrit 5%) were spun down and 10 μl of the pellet were resuspended in 200 μl of isosmotic sorbitol solution (290 mM sorbitol/5 mM HEPES/NaOH pH 7.4), and hemolyzed for 10 min at 21°C in the presence and absence of NPPB or furosemide (from 0.01 to 100 μM). Further hemolysis experiments were performed using isosmotic solutions with isosmotic replacement of increasing concentrations of sorbitol by either NaCl or sucrose. After centrifugation the hemoglobin color of the supernatants (150 μl) was recorded qualitatively by image scanning. In addition, the hemoglobin concentration was determined quantitatively by photometry (absorbance at 546 nm after oxidation to cyanomet-hemoglobin).

Unidirectional flux of the polyol [¹⁴C] sorbitol (Biotrend) into enriched trophozoite/shizont–infected erythrocytes was measured according to the method described for rapid substrate uptakes (Kanaani and Ginsburg, 1991; Kirk and Horner, 1995b). Briefly, 150 μl of [¹⁴C] sorbitol (3.7 × 10⁴ Bq/ml; 1.4 × 10⁴ Bq/nmol specific actifity) containing incubation solutions were layered over 200 μl of dibutylphthalate oil. The incubation solution additionally contained 290 mM sorbitol and 5 mM HEPES/NaOH pH 7.4. In further solutions, 20 and 60 mM of sorbitol was iso-osmotically replaced by either 20 and 60 mM sucrose or 10 and 30 mM NaCl, respectively. The uptake (21°C) was started by adding the trophozoite-infected erythrocytes (5 μl, >90% parasitaemia; 80% hematocrit) to the [¹⁴C] sorbitol containing solutions and terminated at the appropriate time by rapid centrifugation (10,000 g, 20 s) of the cells through the oil. The [¹⁴C] sorbitol solution which remained on top of the oil was removed and the tubes were washed five times by submerging the cell- and oil-containing tubes in distilled water and subsequently discarding the aqueous phase. After removal of the oil, the cell pellet was lysed and deproteinised by successive addition of Triton X-100 (0.1%; 0.5 ml) and trichloroacetic acid (6%, 0.5 ml). After 10 min of incubation (21°C) precipitated protein was separated by centrifugation (10 min, 10,000 g) and the radioactivity of the supernatant was measured using a β-scintillation counter (Wallac 1406).

To study the lactate permeabilities of the erythrocyte membrane from normal and mid/late trophozoite stage-parasitized cells (Fig. 1), whole-cell currents were recorded upon replacing Cl⁻ in the bath solution by equiosmolar amounts of l-lactate. In noninfected erythrocytes, with NMDG-Cl pipette solution, whole-cell currents measured in the presence of NaCl or Na-l-lactate bath solutions were extremely low (Fig. 2, A and B). In sharp contrast, whole-cell currents of P. falciparum–infected erythrocytes (Fig. 2 C, top line) recorded with NMDG-Cl pipette solution were 50–100-fold larger. In contrast to the noninfected cells these currents were anion selective, as suggested by the reversal potential (V_rev)3 of the infection-induced currents which were close to E_Cl (Fig. 2 D). In infected cells, replacement of Cl⁻ in the bath by lactate induced only a moderate shift of the V_rev by about +20.0 ± 1.9 mV (Fig. 2 D) with the change of E_Cl (from 0 to +90 mV) and only an ∼60% inhibition of the outward current indicating a P_lactate/P_Cl = 0.3–0.4) for the overall current in infected erythrocytes at V_rev. The inward current (e.g., at −100 mV voltage), which was carried by the outflow of Cl⁻ from the pipette into the bath, did not change by the substitution of bath Cl⁻ with l-lactate, indicating that l-lactate did not block the conductance(s) (Fig. 2 D, circles and diamonds). In further experiments using Na-l-lactate in the pipette and NaCl in the bath (Fig. 2 C, bottom line) the whole-cell currents reversed at −22.8 ± 1.8 mV again between E_Cl (−66 mV) and E_lactate (+∞). Substitution of bath Cl⁻ by l-lactate decreased the outward current by ∼60% and shifted V_rev to −3.3 ± 1.7 mV, which was close to E_lactate (0 mV). With both pipette solutions, superfusion of the cells with Na-gluconate (a larger and less permeant anion than Cl⁻) inhibited the outward currents almost completely and shifted the V_revs toward high positive voltages (Fig. 2, D and E, squares), indicating a relatively low permeability for gluconate (P_gluconate/P_Cl ∼0.04 and P_gluconate/P_lactate ∼0.1).

To test the stereo specificity of the lactate permeability, infected cells were recorded with NMDG-Cl pipette solution and NaCl or Na-d-lactate bath solution. Replacement of the NaCl bath solution by Na-d-lactate induced a decrease of the outward current and a shift of the V_rev to +23.3 ± 3 mV (Fig. 2 F). Fig. 2 G shows that the mean reversal potential recorded either with Na-d- or Na-l-lactate bath solution did not differ (NMDG-Cl pipette solution), indicating that the lactate permeability did not discriminate between both lactate enantiomers.

To study the pharmacology of the lactate currents, infected cells were recorded with Na-l-lactate in the bath and pipette solutions at increasing concentrations of NPPB and furosemide, respectively. Both blockers inhibited first an outwardly rectifying current component and then at higher concentrations the remaining inwardly rectifying current fraction (Fig. 3, A and B). The IC₅₀s for outwardly and inwardly rectifying lactate currents were ∼0.1 μM and in the range of 30 μM for NPPB (Fig. 3, D and E, open symbols), and ∼1 and 100 μM for furosemide, respectively (closed symbols). With the inhibition of outward current by low concentrations of NPPB (1μM), V_rev of the remaining (inwardly rectifying) current shifted significantly by +25 ± 1 mV (n = 3) from E_lactate toward E_Cl (E_Cl = +23 mV), suggesting no or a very low relative permeability for lactate (P_lactate/P_Cl << 0.1) of the inwardly rectifier as compared with the outward rectifier (Fig. 3 C, squares). The NPPB-1 μM–sensitive outwardly rectifying current fraction reversed at +3 mV, indicating again a high relative lactate permeability (P_lactate/P_Cl ∼0.4; Fig. 3 C, circles).

To test whether neutral carbohydrates influence the anion conductances, a fraction (50 mM) of the NaCl in the bath was iso-osmotically replaced by sorbitol, mannitol, or sucrose (100 mM each) during continuous whole-cell recording (NMDG-Cl pipette solution; Fig. 4). Partial isosmotic replacement of NaCl by sucrose, mannitol, or sorbitol each decreased the current (Fig. 4 C). Most importantly, sucrose inhibited the outwardly rectifying conductance significantly stronger than replacement with sorbitol (Fig. 4, A and C). The current fraction ΔI = I_sorbitol − I_sucrose showed an I/V relation typical for the outwardly rectifier (Fig. 4 B), indicating that the Cl⁻ current inhibited by neutral carbohydrates was outwardly rectifying.

To test for regulation of both P. falciparum–induced conductances by cell-volume changes, infected erythrocytes were swollen and shrunken by using osmolarity gradients between the pipette and the bath solutions (see materials and methods). The osmolarity gradients induced changes in cell size of the whole-cell recorded cell as monitored by light microscopy (Fig. 5 A), indicating that osmolarity gradients indeed induced cell-volume changes of the recorded cell.

When infected cells were shrunken by hypotonic pipette solution, inward whole-cell currents inactivated time dependently within the first minutes and equilibrated within 4 min of recording (Fig. 5, C and E, triangles). This inactivation was not observed (within at least 10 min of recording), when cell-swelling was induced by a hypertonic pipette solution (Fig. 5, D and E, squares). In sharp contrast, the mean outward conductance recorded with hypotonic, isotonic, and hypertonic NMDG-Cl pipette solution remained virtually constant (Fig. 5 E). As a consequence, the cell volume–sensitive current fraction rectified inwardly (Fig. 5 F). Noninfected erythrocytes did not exhibit anion-selective whole-cell currents, even upon osmotic swelling by hypertonic NMDG-Cl pipette solution (isotonic NaCl bath solution; Fig. 5 B), indicating that the cell swelling–dependent inward current was induced by the infection.

In further experiments cells were first shrunken by addition of NaCl (25 mM) to the isotonic NaCl bath solution (isotonic NMDG-Cl pipette solution). Cell shrinkage induced a decrease of inward currents by −45 ± 12% (n = 6) due to the cell volume dependence of the inwardly rectifying conductance without modifying the outward currents. Further addition of increasing concentrations of sucrose, in sharp contrast, reversibly decreased the whole-cell outward currents (Fig. 5 G). Fig. 5 H shows the I/V relations under control conditions (open circles) and after application of sucrose (400 mM, open triangles). The inhibited current fraction ΔI = I_control − I_sucrose (Fig. 5 H, closed diamonds) was outwardly rectifying, suggesting that the outward rectifier was inhibited by sucrose. The dose-dependent inhibition by sucrose of the relative outward conductance is shown in Fig. 5 I.

To compare the influence of neutral carbohydrates on lactate and Cl⁻ currents further experiments were performed using Na-lactate in pipette and bath solution or NMDG-Cl in the pipette and NaCl in the bath solution. Addition of mannitol (200 mM) to the bath reversibly decreased the whole-cell currents (Fig. 6 A). Similar to the action of sucrose on the Cl⁻ current (see Fig. 5 G), mannitol inhibited an outwardly rectifying fraction (ΔI = I_control − I_mannitol) of the lactate current (Fig. 6 B). The relative inhibition exerted by mannitol on the lactate current was about two times higher than on the Cl⁻ current (Fig. 6 C), indicating that the inhibitory potency of the carbohydrate was dependent on the nature of the charge carrier.

Finally, the NPPB and furosemide inhibition of the hemolysis in isosmotic sorbitol solution was compared with those of both conductances (Fig. 7, A and B). NPPB and furosemide inhibited the sorbitol hemolysis of infected erythrocytes in a dose-dependent manner (Fig. 7 A) with IC₅₀s (∼1 μM for NPPB and ∼10 μM for furosemide; Fig. 7 A) similar to those for the outward rectifying lactate current (Fig. 3 D). In addition, the effect of sucrose on the infection-induced hemolysis was determined by isosmotic substitution of sorbitol with increasing concentrations of sucrose and—for control—of NaCl (Fig. 7 B). Replacement by sucrose but not by NaCl inhibited hemolysis of trophozoite-infected cells with an IC₅₀ of ∼10 mM, indicating (a) competition between sorbitol and sucrose within the permeability pore and (b) low permeability of sucrose.

To further test for the inhibitory effect of sucrose on the sorbitol pathway of infected erythrocytes, isotopic flux measurements of [¹⁴C] sorbitol were performed under the experimental conditions used for the hemolysis experiments. Fig. 7 C shows the time course of the [¹⁴C] sorbitol uptake in isoosmotic sorbitol solution. Within 2 min of incubation the uptake apparently saturated. Incubation (30 s) of the infected cells in the presence of 20 or 60 mM of sucrose (replacing the same amount of sorbitol) significantly decreased the uptake of [¹⁴C] sorbitol as illustrated in Fig. 7 D. In contrast, substitution of 20 and 60 mM of sorbitol by 10 and 30 mM of NaCl did not significantly decrease [¹⁴C] sorbitol uptake, respectively (Fig. 7 D).

The malaria parasite Plasmodium falciparum has been demonstrated to lower the concentration of impermeable osmolytes in the host cytosol by excess hemoglobin digestion and discharge of the amino acids via the NPP (Lew et al., 2003). In addition, the NPP has been proposed to dispose of lactate since the endogenous erythrocytes transport pathways are not sufficient to cope with the rate of lactic acid production by the parasite (Poole and Halestrap, 1993). Thus, by the release of organic osmolytes the NPP counteracts infection-induced swelling and avoids premature lysis of the host cell (Kirk, 2001; Staines et al., 2001, 2003; Lew et al., 2003).

In the present and two previous reports (Huber et al., 2002; Staines et al., 2003), inwardly and outwardly rectifying whole-cell anion conductances were identified in P. falciparum–infected human erythrocytes. Recent studies using similar whole-cell protocols (Desai et al., 2000; Duranton et al., 2002, 2003b; Egee et al., 2002; Huber et al., 2002; Lang et al., 2003) indicate that these conductances are not active spontaneously in the majority of noninfected cells, but activate upon oxidative stress, protein kinase A stimulation, or membrane stretch (Egee et al., 2002; Huber et al., 2002). Thus, these conductances at least in part are generated by endogenous erythrocyte proteins. In this study, no spontaneous activity of the inwardly and outwardly rectifying anion conductance was observed in noninfected cells. In addition, no activation of these conductances was elicited in noninfected cells by cell swelling or by the use of nonphysiological pipette and bath solutions, strongly suggesting that the observed conductances in parasitized cells were indeed induced by the infection but not by the experimental setup. In theory, the inwardly and the outwardly rectifying current phenotype of the infected erythrocyte might be generated by two individual channel proteins, by a multichannel complex, or by one molecule with two functional states. In any case, inward and outward rectifiers seem to be regulated differentially (serum, voltage, cell volume), pointing to different functions of both conductances for the erythrocyte-parasite interaction.

The inwardly and outwardly rectifying anion conductances of infected erythrocytes differed in lactate permeability (see Fig. 2). The highly lactate-permeable current fraction shows (a) NPPB and furosemide sensitivities, (b) time-dependent inactivation at hyperpolarizing voltage, and (c) outwardly rectifying current voltage relationship identical to those reported previously for the outwardly rectifying Cl⁻ currents (Huber et al., 2002). As demonstrated for the infection-induced NPP by tracer flux (Cranmer et al., 1995), this outwardly rectifying conductance did not differentiate between d- and l-lactate enantiomeres thus exhibiting no stereo specificity.

As a consequence of its rectification behavior, the outward rectifier mediates a much higher lactate influx into the cell at positive membrane potentials than lactate efflux at negative potentials. This voltage dependence, therefore, does not seem to be optimally suited for the disposal of lactate out of the cells. Nevertheless, the outward rectifier activated at high negative voltages and generated lactate inward currents in the range of −40 pA at −40 mV voltage (see the 1 μM NPPB-sensitive current fraction in Fig. 3 C, open circles). This current corresponds to the permeation of ∼50 fmol lactate per cell and second. This value is huge compared with the calculated lactate disposal of infected cells (0.003 fmol per cell and second; Poole and Halestrap, 1993), suggesting that the outwardly rectifying conductance can indeed transport physiologically relevant amounts of lactate.

The inwardly rectifying conductance, in sharp contrast to the outward rectifier, was much less lactate permeable. These data seem to be at variance to a previous study (Desai et al., 2000) reporting a high relative lactate permeability for an infection-induced inwardly rectifying anion conductance, but in this report the authors did not observe outwardly rectifying currents. The inward rectifier in this previous study (Desai et al., 2000) resembled the outward rectifier of the present study by the relative lactate permeability (0.4 and 0.43, respectively) and the high sensitivity to furosemide and NPPB (1 μM furosemide and 0.8 μM NPPB inhibited 30% and 41% of the inward rectifier in the previous study while 1 μM furosemide and 1 μM NPPB inhibited ∼55% and 80% of the outward rectifier in the present work). This might suggest that the inward rectifier of the previous study (Desai et al., 2000) and the outward rectifiers of the present study possibly represent modifications of the same conductance type. The inward rectifier of this previous study, however, differs substantially from the inward rectifier of the present study (relative lactate permeability <0.1; IC₅₀ for NPPB and furosemide >10 μM). The present study further discloses that the inwardly rectifying conductance is sensitive to cell volume: Cell shrinkage inactivates and cell swelling activates the inward rectifier. The outwardly rectifying conductance, in contrast, did not exhibit any apparent cell volume dependence.

In addition to cell volume regulation, the infection-induced NPP of the host cell membrane supplies the parasite with nutrients, molecules in general larger than lactate. For instance, the parasite needs pantothenate for coenzyme A synthesis (Saliba et al., 1998) and isoleucine for protein synthesis (which cannot be provided by hemoglobin digestion; Sherman, 1983). To test for the putative permeabilities of the two infection-induced anion conductances to large molecules, the present study analyzed the interference of neutral carbohydrates with the Cl⁻ and lactate currents of infected erythrocytes. As a result, neutral carbohydrates applied iso-osmotically or iso-ionically inhibited a current fraction with a current-voltage relation typical for the outwardly rectifier. This inhibition occurred instantaneously upon carbohydrate application, was concentration dependent, reversible, and not dependent on cell swelling or shrinkage. These data indicate that the effect of the carbohydrates is not the result of changes in ionic strength or tonicity of the bath solution or of increase in transmembrane osmotic pressure. The efficacy of sorbitol, mannitol, and sucrose was not identical, indicating that the inhibitory effect on the outwardly rectifying Cl⁻ current is dependent on the nature of the osmolyte. In addition, the inhibitory effect of any given carbohydrate species was dependent on the nature of the charge carrier: mannitol in the bath (200 mM) inhibited the lactate current by some 50% but the Cl⁻ current only by some 25%. This interplay between charge carrier and neutral carbohydrate strongly suggests a competition between neutral and the charged solute within the permeation pore rather than an allosteric effect of the carbohydrates on the channel protein. The outwardly rectifying lactate current and the sorbitol permeability (as measured by isosmotic hemolysis) were similarly sensitive to NPPB and furosemide. Finally, disaccharides reportedly block isosmotic sorbitol hemolysis of infected cells (Ginsburg et al., 1983) but, in contrast, failed to inhibit [¹⁴C] sorbitol uptake (Ginsburg et al., 1985). In this study, sucrose blocked the hemolysis in isosmotic sorbitol solution and exerted a moderate but significant inhibitory effect on the outwardly rectifying Cl⁻ current (IC₅₀ in the range of 400 mM). In contrast [¹⁴C] sorbitol uptake in sorbitol solution was inhibited by 20 mM sucrose. Moreover, the [¹⁴C] sorbitol uptake in isosmotic sorbitol solution reached a plateau within 1–2 min, whereas uptake saturation in NaCl reportedly is not complete at 30 min of incubation (Ginsburg et al., 1985). This suggests that the sorbitol permeability itself and the competition between sorbitol and sucrose at the pore are strongly depending on the ionic environment. E.g., suspending the infected cells in isosmotic sorbitol strongly depolarizes (<0 mV in NaCl and >100 mV in sorbitol) the erythrocyte membrane potential, enhancing the conductance of the outwardly rectifying anion channel.

The sucrose inhibition of the sorbitol hemolysis and uptake points to a competition between both substrates with a permeability of sorbitol >> sucrose. Permeability for sugars/polyols in the rank order of sorbitol > mannitol >> sucrose have been demonstrated for P. falciparum–infected (Ginsburg et al., 1985; Huber et al., 2002) and –oxidized erythrocytes (Huber et al., 2002) by isosmotic hemolysis experiments. This rank order is inverse to the carbohydrate inhibition of the outwardly rectifying current which occurs in the sequence of sucrose (> mannitol) > sorbitol. This inverse correlation suggests that (a) channels that are occupied by carbohydrates are not permeable for charged molecules and (b) slowly (or non) permeating carbohydrates are occupying the channel pore for a longer period and are therefore more potent inhibitors of the current than fast permeating carbohydrates. Together, all those observations indicate that the outwardly rectifier participates in the sorbitol permeation pathway.

In conclusion, P. falciparum–infected erythrocytes express two types of anion conductances at the mid-late trophozoite stage of parasite development. The inwardly rectifying anion conductance is activated by cell swelling. In contrast, the outwardly rectifying current is insensitive to cell volume and might be involved in transport of lactate, amino acids, and further organic osmolytes.

The authors wish to thank Dr. Mark Waidmann, Department of Transfusion Medicine, University of Tubingen for providing human erythrocytes.

The experiments have been supported by the “Schwerpunktprogramm Life inside cells” of the Deutsche Forschungsgemeinschaft (DFG, La 315/11-1), by the “Forschungsschwerpunktprogramm des Landes Baden-Württemberg Dynamik und Modulation zellulärer Infektionsprozesse” and by the Fortüne program (#838-1-1) of the University of Tübingen. C. Duranton and E. Shumilina have been supported by a grant of the Alexander von Humboldt foundation, and C. Sandu by a Marie Curie fellowship.

Olaf S. Andersen served as editor.