Na(+) regulation in the malaria parasite Plasmodium falciparum involves the cation ATPase PfATP4 and is a target of the spiroindolone antimalarials

Abstract

The malaria parasite Plasmodium falciparum establishes in the host erythrocyte plasma membrane new permeability pathways that mediate nutrient uptake into the infected cell. These pathways simultaneously allow Na(+) influx, causing [Na(+)] in the infected erythrocyte cytosol to increase to high levels. The intraerythrocytic parasite itself maintains a low cytosolic [Na(+)] via unknown mechanisms. Here we present evidence that the intraerythrocytic parasite actively extrudes Na(+) against an inward gradient via PfATP4, a parasite plasma membrane protein with sequence similarities to Na(+)-ATPases of lower eukaryotes. Mutations in PfATP4 confer resistance to a potent class of antimalarials, the spiroindolones. Consistent with this, the spiroindolones cause a profound disruption in parasite Na(+) homeostasis, which is attenuated in parasites bearing resistance-conferring mutations in PfATP4. The mutant parasites also show some impairment of Na(+) regulation. Taken together, our results are consistent with PfATP4 being a Na(+) efflux ATPase and a target of the spiroindolones.

Graphical abstract

Figure 1

Effects of an Ionophore and Ion Transport Inhibitors on [Na⁺]_i in Saponin-Isolated, SBFI-loaded P. falciparum Trophozoites (A–E) [Na⁺]_i traces showing the effect of addition (at the point indicated by the closed triangle) of (A) gramicidin (5 μM), (B) EIPA (20 μM), (C) ouabain (2 mM), (D) furosemide (100 μM), and (E) orthovanadate (100 μM). For all additions except ouabain, the compounds were added as a concentrated stock. Cells were exposed to 2 mM ouabain by being sedimented by centrifugation then resuspended at the time point indicated in an equivalent saline containing the inhibitor. The traces shown in each case are representative of those obtained from at least three independent cell preparations. See also Figure S1.

Figure 2

PfATP4 and the Effect of the Spiroindolones on Ion Regulation in Saponin-Isolated P. falciparum Trophozoites (A) Amino acid alignment of residues 849–856 of PfATP4 with the equivalent regions in ENA Na⁺-ATPases from Saccharomyces cerevisiae (ScENA1; P13587), Leishmania donovani (LdCA1; AAC19126), Trypanosoma cruzi (TcENA1; XP_817442.1), and Entamoeba histolytica (Enthist1; XM_652464). Black shaded residues are completely conserved; gray shaded residues are functionally conserved. The alignment is based on a previous alignment of fungal, bryophyte, and protozoal ENA ATPases from Rodríguez-Navarro and Benito (2010) who highlighted the conservation of this eight amino acid motif (⁷⁴¹MIEALHRR in ScENA1) in ENA ATPases. The ⁸⁵⁴KRK triple-basic motif in PfATP4 (boxed) plays an important role in Na⁺ transport in ENA Na⁺-ATPases and is absent from PMCA, SERCA, and Na⁺/K⁺-ATPases (Rodríguez-Navarro and Benito, 2010). The triple-basic motif is not present in the same position in any of the other annotated P. falciparum P-type ATPases (including PfATPase1 [PFE0805w], PfATPase3 [PFE0195w], PfATP6 [PFA0310c], and two putative cation-transporting P-type ATPases [MAL13P1.246, PF07_0115]). Note that the PfATP4 sequence used (from PlasmoDB: PFL0590c) was the updated sequence reannotated to correct a missed nucleotide, thus removing a previously incorrectly annotated intron. (B) Chemical structures of the enantiomers NITD246/NITD247 and NITD138/NITD139. (C) Traces showing the effects of the four spiroindolones, each at a concentration of 50 nM, on [Na⁺]_i in SBFI-loaded 3D7 parasites suspended in standard saline. The spiroindolones were added at the time point indicated by the closed triangle. (D) Concentration dependence of the effect of each of the four spiroindolones on the initial rate of Na⁺ influx (● NITD246; ■ NITD247; ◇ NITD138; ▿NITD139). The initial rate of Na⁺ influx was estimated from traces such as those represented in (C) (see also Figure S2A) as described in Experimental Procedures. Each data point represents the mean Na⁺ influx rate averaged from at least three independent experiments and is shown ±SEM. For the purpose of the curve fitting, the maximum rate of Na⁺ influx (y^max in the sigmoidal curve described in Experimental Procedures) was set to 0.11 mM/s, the mean of the Na⁺ influx rates measured using the maximally effective concentrations of the three most potent inhibitors (NITD246, NITD139, and NITD247). (E) Summary of the IC₅₀ values for inhibition of parasite proliferation and for disruption of [Na⁺]_i regulation (i.e., the concentration of each inhibitor required to cause the [Na⁺]_i to increase from its normal resting value at half the maximal rate). The IC₅₀ values cited for inhibition of parasite proliferation are the mean ± SEM of those estimated in the number of independent experiments shown in parentheses (with each independent experiment performed in triplicate). The IC₅₀ values for disruption of [Na⁺]_i regulation are derived from the fitted curves shown in (D). (F) Traces showing the effects on pH_i of the addition of NITD246 (25 nM, at the point indicated by the black triangle) followed by the addition of concanamycin A (75 nM, at the point indicated by the open triangle) to BCECF-loaded parasites suspended in either standard saline (black trace) or Na⁺-free solution (in which Na⁺ was replaced with an equimolar concentration of choline⁺; gray trace). See also Figure S2C for a similar trace using orthovanadate instead of NITD246. (G) Trace showing the effect of the addition of NITD246 (50 nM, at the point indicated by the closed triangle) and CPA (2 μM, at the point indicated by the open triangle) on [Ca²⁺]_i in fura-2-loaded parasites suspended in standard saline supplemented with 1 μM Ca²⁺. All of the traces shown are, in each case, representative of those obtained from at least three independent cell preparations. See also Figure S2.

Figure 3

Response of Saponin-Isolated P. falciparum Trophozoites to an Imposed Intracellular Na⁺ Load (A) Trace showing the effect of removal of extracellular K⁺ on [Na⁺]_i in SBFI-loaded parasites. At the time point indicated by the open triangle, the cells (in standard saline) were washed twice by centrifugation and resuspension in a K⁺-free saline (in which K⁺ was replaced isosmotically with Na⁺). At the time point indicated by the closed triangle, 10 mM K⁺ (as KCl) was added to the suspension. The trace is representative of that obtained from at least twenty independent cell preparations. (B) Trace showing the effect of the same maneuvers (i.e., removal of extracellular K⁺ at the point indicated by the open triangle then restoration at the point indicated by the closed triangle) on the pH_i in BCECF-loaded parasite suspension. The trace is representative of that obtained from at least seven independent cell preparations. (C–E) Effect of ion transport inhibitors on the recovery of [Na⁺]_i from an imposed intracellular Na⁺ load. SBFI-loaded parasite suspensions were subjected to a Na⁺ load (by suspension in K⁺-free medium) as illustrated in (A), and the traces commenced with the addition to the suspension of 10 mM KCl either with (gray traces) or without (control, black traces) (C) EIPA (20 μM), (D) furosemide (100 μM), or (E) NITD246 (1 nM or 5 nM). The traces shown are each representative of those obtained from at least three independent cell preparations.

Figure 4

Na⁺-Dependence and Spiroindolone-Sensitivity of Membrane-Associated ATPase Activity in P. falciparum Infected Human Erythrocytes ATPase activity was estimated from the rate of production of P_i and measured using the PiColorLock Gold Phosphate Detection Kit following the addition of 0.25 mM ATP. Membrane preparations were suspended in either a high (100 mM) Na⁺ solution (black bars) or a low (0.5 mM) Na⁺ solution (in which Na⁺ was replaced with equimolar choline; white bars) in the absence or presence of 50 nM NITD246. ATPase activity is expressed as a percentage of that measured in high-Na⁺ medium in the absence of inhibitor (control). Asterisks indicating a statistically significant difference from the control (p < 0.05); NS denotes p > 0.05. The data are averaged from five independent experiments and are shown +SEM.

Figure 5

Comparisons of PfATP4 NITD609-R^Dd2 Clone #2 Parasites with their Dd2 Parent Line. The PfATP4 NITD609-R^Dd2 Clone #2 Parasites were Generated by Exposure of Dd2 P. falciparum Parasites to the Potent Spiroindolone NITD609 (A) Recovery of [Na⁺]_i following an intracellular Na⁺ load imposed by the removal and restoration of extracellular K⁺ (as illustrated in Figure 3). The traces are representative of those obtained from at least three independent cell preparations, and the smooth lines are the fitted curves (see Experimental Procedures). The grey trace is from PfATP4 NITD609-RDd2 clone #2 parasites, and the black trace is from the Dd2 parent line. (B) Inhibition of parasite proliferation by excess extracellular Na⁺, with “Excess [Na⁺]_o” denoting the increase in [Na⁺]_o above that normally present in the parasite culture medium (∼133 mM). (C) Inhibition of parasite proliferation by NITD246. (D) Disruption of parasite [Na⁺] regulation by NITD246 (estimated from the rate of increase of the [Na⁺]_i immediately following the addition of NITD246 to isolated SBFI-loaded parasites). (E) Inhibition of membrane-associated ATPase activity by NITD246 (measured as the rate of ATP hydrolysis in membranes isolated from parasitized erythrocytes). In (B)–(E) the data are averaged from at least three experiments and are shown ±SEM. In (B)–(E) the open symbols are from PfATP4 NITD609-RDd2 clone #2 parasites, and the closed symbols are from the Dd2 parent line. All data in (B)-(E) are averaged from at least three experiments and are shown ±SEM. The corresponding IC₅₀ values are given in Table 1.

Figure 6

Schematic Representation Showing the Proposed Role of PfATP4 in Na⁺ Homeostasis in the Intraerythrocytic P. falciparum Trophozoite-Stage Parasite (A) PfATP4 is postulated to function as an ENA Na⁺-ATPase, actively extruding Na⁺ from the intraerythrocytic parasite, countering the influx of Na⁺ (which occurs via unknown pathways), and maintaining a [Na⁺]_i (∼11 mM) more than 10-fold lower than [Na⁺]_o (125 mM in the experiments conducted here). The PfATP4-mediated efflux of Na⁺ is postulated to be accompanied by an influx of H⁺ ions, and this constitutes a significant acid load, which is countered by H⁺ extrusion via the parasite’s plasma membrane V-type H⁺-ATPase. (B) PfATP4 is inhibited by the spiroindolones (as well as by orthovanadate and CPA). Inhibition of PfATP4 results in an increase in [Na⁺]_i (Figure 2C) as Na⁺ moves into the cell, down its electrochemical gradient, via the Na⁺ influx pathways. At the same time there is an increase in pH_i (Figure 2F) attributable to the V-type H⁺-ATPase now operating in the absence of the PfATP4-mediated acid load. The alkalinisation seen following inhibition of PfATP4 is not seen for parasites washed and resuspended in Na⁺-free medium (Figure 2F), as under these conditions [Na⁺]_i is close to zero (Figure S1D), PfATP4 is nonfunctional, and there is therefore no PfATP4-mediated acid load.