The Plasmodial Surface Anion Channel Is Functionally Conserved in Divergent Malaria Parasites

Parasite culture and osmotic lysis assays. P. knowlesi was cultured under in vitro conditions with RBCs collected from healthy rhesus monkeys (Macaca mulatta) and RPMI 1640 supplemented with 50 mg/liter hypoxanthine and 20% human serum. In contrast to P. falciparum's 44 to 48 h cycle in human RBCs, the erythrocytic cycle averaged 24 h. For both parasites, the kinetics of osmotic lysis in various solutions was followed as described previously (38). In brief, trophozoite-infected RBCs were enriched to 95 to 99% by percoll-sorbitol separation (1), washed in phosphate-buffered saline (PBS) (150 mM NaCl, 20 mM Na-phosphate, pH 7.5), and resuspended to a 0.25% hematocrit in buffer (20 mM Na-HEPES, 0.1 mg/ml bovine serum albumin, pH 7.5) with 280 mM of sorbitol, isoleucine, or alanine. Na⁺ permeability was evaluated in PBS with 0.1 mg/ml bovine serum albumin. The transmittance of 700 nm light, %T, through these cell suspensions was then continuously followed at 37°C; these measurements track changes in turbidity that result from osmotic swelling and lysis of cells. This method produces estimates of permeation rates through PSAC that quantitatively match estimates obtained with both radioisotope flux and patch-clamp (38).

Electrophysiology.Single-channel and whole-cell patch-clamp recordings of rhesus monkey RBCs were obtained with quartz glass pipettes pulled to tip diameters of <0.5 μm and resistances of 1 to 4 MΩ. Most experiments utilized symmetrical bath and pipette solutions of (in mM) 1,000 choline-Cl, 115 NaCl, 10 MgCl₂, 5 CaCl₂, and 20 Na-HEPES, pH 7.4. Some experiments replaced choline-Cl with equimolar NaCl and identified channel activity with indistinguishable properties. Recordings were filtered at 5 KHz with an eight-pole Bessel filter and digitized at 100 KHz. Seal resistances were typically >100 GΩ, as required for detection of these small-conductance channels (2).

Whole-cell patch-clamp to determine anion selectivity was performed similarly but with a bath solution of (in mM) 500 NaCl, 10 MgCl₂, 5 CaCl₂, 20 Na-HEPES, pH 7.4, and pipette solutions of either 500 NaClO₄ or 500 Na-lactate with 10 MgCl₂, 5 CaCl₂, and 20 Na-HEPES, pH 7.4. Under these conditions, the permeability coefficient of ClO₄⁻ or the lactate anion relative to Cl⁻ can be determined by measuring the reversal potential (E_rev), the membrane voltage that produces zero current.

Single-channel amplitudes, open probabilities, and dwell distributions were measured with home-written code as described previously (2, 6).

Radioisotope uptake. P. knowlesi-infected or uninfected rhesus monkey RBCs were washed and used to measure uptake of [³H]alanine (2 μCi/ml) at a 10% hematocrit in PBS supplemented with 1 mM unlabeled alanine at 22°C. At 5-min intervals, uptake was terminated by transfer of 330-μl aliquots to 1.5 ml of tracer-free PBS with 200 μM furosemide at 4°C and centrifugation at 14,000 × g through dibutyl phthalate. Each time point was measured in duplicate. Cell pellets were digested and counted as previously described (11). Uptake was corrected for extracellular trapping by subtracting the uptake measured at zero time in the presence of furosemide.

Broad increases in permeability after P. knowlesi infection.PSAC-mediated permeability changes after infection of human RBCs by P. falciparum can be readily identified by an increased osmotic fragility in isotonic solutions of permeant solutes (17). In these solutions, there is net uptake of the permeant solute via PSAC, cell swelling, and selective osmotic lysis of infected RBCs. Lysis of P. falciparum-infected human RBCs can be quantitatively and continuously tracked with a simple light-scattering assay that measures the turbidity of the cell suspension (38).

Here, we found that this assay can also be used to follow osmotic lysis of rhesus monkey RBCs with or without P. knowlesi infection. These kinetic measurements revealed marked increases in sorbitol, alanine, and isoleucine permeabilities after infection, which could be largely inhibited by 200 μM furosemide (Fig. 1A through C, upper panels), a well-studied antagonist of the P. falciparum PSAC (2). Uninfected rhesus RBCs exhibited negligible permeability to each of these solutes (Fig. 1A through C, lower panels). Both uninfected and infected RBCs were stable in isotonic NaCl solutions (Fig. 1D), consistent with sustained low Na⁺ permeability despite increases in organic solute permeability. We also confirmed these permeability changes with more conventional [³H]alanine radioisotope accumulation experiments (Fig. 1E). Both this list of solutes and estimates of their absolute permeabilities parallel previous measurements in the P. falciparum-human RBC system (5, 17, 38), suggesting PSAC-like channels on P. knowlesi-infected rhesus RBCs.

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FIG. 1.

Increased organic solute permeability after P. knowlesi infection. (A through D) Osmotic lysis of trophozoite-infected rhesus RBCs in buffered sorbitol, isoleucine, alanine, and NaCl solutions (black curves, upper panels). Each lower panel represents an identical experiment with uninfected rhesus monkey RBCs; their lack of measurable lysis reflects their low intrinsic permeability to these solutes. In all panels, red traces reflect the lysis time course in the presence of 200 μM furosemide. Transmittance of 700 nm light though cell suspensions was converted to percent lysis with measurements on identical but fully lysed suspensions of sorbitol-treated infected RBCs or detergent-treated uninfected RBCs. Notice that each of the P. knowlesi-induced permeability changes is furosemide-sensitive. (E) [³H]alanine uptake in uninfected RBCs (triangles) or infected RBCs (circles) with or without 200 μM furosemide (red or black symbols, respectively). Uptake was not corrected for parasitemia (6% in this experiment); such corrections would increase the difference between infected and uninfected cells by more than 15-fold. [³H]isoleucine exhibited similar increases in uptake (not shown).

Identification and characterization of the P. knowlesi PSAC.We used electrophysiological methods to probe the mechanistic basis of these increases. Although technically complicated by the somewhat smaller size of rhesus RBCs when compared to human RBCs (35), we successfully achieved high-resistance whole-cell patch-clamp recordings on these cells. In symmetrical bath and pipette solutions containing Cl⁻, P. knowlesi-infected RBCs exhibited large whole-cell currents that were absent from uninfected RBCs (Fig. 2). Infected RBC whole-cell measurements revealed a steep voltage dependence with significantly larger currents at negative voltages than at positive voltages despite equal but opposite driving forces. Furosemide abolished these currents, consistent with a broad-specificity channel that mediates the transport of both inorganic anions and the organic solutes evaluated for Fig. 1. In identical whole-cell experiments with uninfected rhesus monkey RBCs, the measured currents were significantly smaller and voltage independent, indicating low permeabilities similar to those of human RBCs (8). (With uninfected RBCs of both species, it is not possible to precisely calculate conductive Cl⁻ permeabilities from whole-cell measurements because of errors in measuring their small currents.)

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FIG. 2.

Whole-cell patch-clamp measurements. (A) Ensemble of current responses to V_m values of −100 to +100 mV in 10 mV increments for uninfected rhesus monkey RBC in the whole-cell configuration with 1.0 M choline-Cl in both bath and pipette. (B) Current responses of P. knowlesi-infected RBC to the same protocol before or after addition of saturating concentrations of furosemide (left and right groups of traces, respectively). Dashes on the left and right of traces represent the baseline current with zero applied voltage. Notice the currents at negative V_m are greater than those at corresponding positive V_m and that furosemide largely abolishes these currents. Scale bars represent 25 ms (horizontal) and 3 nA (vertical) in both panels A and B. (C) Comparison of whole-cell chord conductances between P. knowlesi-infected rhesus RBCs (black bar, mean ± standard error of the mean [SEM]) and P. falciparum-infected human RBCs (white bar). (D) Anion selectivity determined with bi-ionic experiments with 500 mM Na⁺Cl⁻ in the bath and either 500 mM Na⁺ClO₄⁻ or 500 mM Na⁺-lactate in the pipette. Black and white bars represent mean reversal potentials ± SEM for experiments with P. knowlesi-infected rhesus and P. falciparum-infected human RBCs, respectively. The similar reversal potentials indicate similar anion selectivity ratios for these parasite-induced currents and yield mean estimates of permeability ratios relative to Cl⁻ of 3.8 and 0.3 for ClO₄⁻ and the lactate anion, respectively.

We wondered how these P. knowlesi-induced currents compare quantitatively with those induced by P. falciparum trophozoites on human RBCs. Because both exhibit larger and more ohmic whole-cell currents at negative imposed membrane potentials (V_m), we tallied chord conductances between −100 and 0 mV for a number of cells in each system (20 and 21 RBCs for P. knowlesi and P. falciparum, respectively) (Fig. 2C). These chord conductances were similar despite the phylogenetic distance between both the two parasites. We did not perform statistical analyses of these chord conductances because of possible systematic differences in parasite maturity and in pipette access resistances.

We also compared the ion selectivity properties of these parasite-induced currents with bi-ionic experiments that created anion gradients across the RBC membrane while maintaining symmetrical cation concentrations. Under these conditions, anion channels exhibit altered current-voltage profiles with reversal potentials (E_rev) quantitatively linked to relative anion permeabilities (18). With Cl⁻ as the predominant extracellular anion, we found that intracellular ClO₄⁻ or lactate produced marked and opposing changes in E_rev (Fig. 2D), indicating significantly greater permeabilities for these anions than for Na⁺. Our whole-cell studies with both parasites indicate similar anion selectivities (ClO₄⁻ > Cl⁻ > lactate), consistent with one or more weak binding sites for anions as they cross the host RBC membrane (8).

We next investigated the various similarities between P. knowlesi- and P. falciparum-induced permeability changes with single-channel patch-clamp. In P. falciparum, these changes are adequately accounted for by PSAC, whose small conductance requires hypertonic salt solutions for unambiguous detection (2). Because P. knowlesi may also induce small-conductance anion channels, we performed cell-attached patch-clamp of infected rhesus monkey RBCs in 1,145 mM Cl⁻ solutions. Under these conditions, one or more functional copies of a ∼20-pS channel were observed in approximately 50% of patches on infected cells (Fig. 3A); this channel was never seen on uninfected rhesus monkey RBCs (23 cells). We measured this new channel's single-channel amplitudes, open probability, and net ion flux at a range of voltages (Fig. 3B through D) and found that its voltage dependence parallels that of the macroscopic whole-cell currents (Fig. 2B). We also measured the durations of open and closed events at a V_m of −100 mV in prolonged single-channel recordings (Fig. 4). These dwell time analyses indicated that the gating behavior of the P. knowlesi-induced channel is indistinguishable from that of the P. falciparum PSAC. Because gating, single-channel conductance, and voltage dependence constitute a functional signature, we concluded that the ion channels induced by these two parasites are members of a highly conserved family. We suggest that the members of the PSAC family be distinguished by using the parasite species name as a prefix.

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FIG. 3.

Single-channel recordings on P. knowlesi-infected rhesus RBCs. (A) Cell-attached recording from an infected RBC patch with one functional channel molecule. V_m values were as indicated to the right of each trace. Scale bars represent 150 ms (horizontal) and 2.0 pA (vertical). Seal resistance was 160 GΩ. Closed-channel levels are indicated with black dashes at both ends of each trace. Notice the fast-flickering transitions at negative V_m and the significantly fewer openings at positive voltages. (B) Single-channel current amplitudes (i) versus V_m for the P. knowlesi and P. falciparum channels (open and filled circles, respectively). Each symbol represents the mean ± SEM of multiple measurements. The solid line is the best fit of the measurements between V_m of −100 and +20 mV and corresponds to a 23-pS channel. (C) Open probability (P_o) versus V_m for the P. knowlesi channel, measured from 10 to 12 s of single-channel recording at each voltage. The solid line represents the best fit to a single exponential decay. (D) Time-averaged current (I) for a single P. knowlesi channel, calculated at each voltage as the product of measurements in panels B and C. The inward-rectifying behavior of this calculated current parallels whole-cell measurements in Fig. 2, suggesting that the P. knowlesi PSAC is the predominant conductive pathway in the infected rhesus RBC membrane.

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FIG. 4.

Open (A) and closed (B) dwell time distributions for single-channel recordings on P. knowlesi- and P. falciparum-infected cells (red and black traces, respectively). The number of events tallied in each histogram ranged from 9,700 to 35,000. The P. knowlesi and P. falciparum channels exhibit indistinguishable gating in the absence of inhibitors.

Comparative pharmacology of P. falciparum and P. knowlesi channels.Our osmotic lysis experiments suggest that the P. knowlesi PSAC is inhibited by furosemide (Fig. 1). The effects of this inhibitor on the P. falciparum PSAC have been extensively studied, revealing a single nonluminal binding site with a K_m of 2.7 μM on the channel extracellular face (2, 7). Strangely, the P. falciparum PSAC is also inhibited by dantrolene, a specific Ca²⁺ channel antagonist that does not inhibit human anion channels (Lisk and Desai, submitted). We wondered whether the P. knowlesi PSAC shares these pharmacological properties and used single-channel recordings to explore possible mechanisms. With low concentrations of each of these agents, we found clear inhibitory effects on the P. knowlesi PSAC (Fig. 5A) that resemble the inhibition with intermediate kinetics described for the P. falciparum PSAC, indicating another level of conservation for this family of channels.

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FIG. 5.

Single-channel pharmacology and a lower furosemide affinity for the P. knowlesi PSAC. (A) Traces reflect current flow through a single P. knowlesi PSAC at V_m of −100 mV without inhibitor or with 4 μM furosemide, 10 μM furosemide, or 10 μM dantrolene (top to bottom traces, respectively). Red dashes on each side of traces reflect the closed-channel level. Scale bars represent 100 ms (horizontal) and 2.0 pA (vertical). (B and C) Osmotic lysis time courses for P. falciparum-infected human and P. knowlesi-infected rhesus RBCs in the buffered sorbitol lysis solution with 0, 4, and 10 μM furosemide (left to right traces in each panel). Direct comparison of these two groups of unprocessed transmittance measurements (%T) is possible because both panels used identical hematocrits and a common blank. Notice that lysis kinetics are inhibited to a greater extent for the P. falciparum channel at both nonsaturating furosemide concentrations. (D) Furosemide dose-response for inhibition of the P. knowlesi PSAC. Sorbitol permeability coefficients (P_s) were calculated from up to eight lysis time courses at each concentration as described previously (38) and normalized to a value of 1.0 without inhibitor. The solid line represents the best fit to y = K_m/(K_m + x). (E) Closed dwell distributions from single-molecule recordings of the P. falciparum and P. knowlesi channels (black and red traces, respectively) in the presence of 10 μM furosemide. Each histogram tallies approximately 80,000 closing events. For each profile, the peak at a time of >10 ms corresponds to the population mean for furosemide-induced closings; this peak is a direct measure of furosemide unbinding rates from the inhibited channel. Furosemide unbinds more quickly from the P. knowlesi channel, accounting for its lower affinity there.

Are there any differences between these two PSAC channels that may have resulted from the phylogenetic distance between P. falciparum and P. knowlesi? We used the simple osmotic lysis assay to survey channel properties and found modest but reproducible differences in their levels of inhibition by furosemide. Paired experiments with 4 μM and 10 μM furosemide consistently produced less robust inhibition of sorbitol-induced osmotic lysis of P. knowlesi-infected rhesus RBCs (Fig. 5B and C). This difference was statistically significant at both concentrations (seven trials for each parasite at 4 μM and at 10 μM with P values of 0.004 and 0.007, respectively, in two-tailed Student's t tests), suggesting a lower furosemide affinity for the P. knowlesi PSAC. A furosemide dose response for inhibition of lysis revealed Michaelian kinetics with an estimated K_m of 5.1 ± 0.5 μM for the P. knowlesi PSAC (Fig. 5D), approximately twice that previously reported for the P. falciparum ortholog. We explored this difference further with single-channel recordings of the two channels in the presence of furosemide. Because the differences in the furosemide affinity are modest, visual examination of individual traces could not readily identify differences between the two channel types (not shown). Nevertheless, analysis of open and closed durations from single-channel recordings with furosemide should be able to identify even modest differences in affinity by quantifying a large number of channel-inhibitor interactions. As previously described for the P. falciparum PSAC (2), furosemide did not affect the durations of the P. knowlesi PSAC openings, consistent with inhibition at a nonluminal site (not shown). Analysis of closed durations, however, revealed a marked difference between these two related channels. The distribution of furosemide-induced closings exhibited a substantially shorter mean duration for the P. knowlesi PSAC than the P. falciparum PSAC (Fig. 5E). This shorter closed duration suggests that the P. knowlesi channel's lower affinity results from a faster rate constant for furosemide unbinding from related but not identical sites on the extracellular face of these channels.