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1.
Mol Biochem Parasitol ; 189(1-2): 1-4, 2013 May.
Article in English | MEDLINE | ID: mdl-23623918

ABSTRACT

The intraerythrocytic malaria parasite has, on its plasma membrane, a H(+)-extruding V-type H(+)-ATPase that plays a central role in maintaining the resting cytosolic pH at around 7.3. Previous studies have demonstrated the presence in the parasite of an unknown acidification mechanism that is revealed on inhibition of the V-type H(+)-ATPase. Here we show that this acidification is dependent on the presence of extracellular Na(+), and is associated with the activity of a plasma membrane Na(+)-ATPase that is inhibited by the novel antimalarial spiroindolone NITD246 and is postulated to export Na(+) ions in counter-transport with H(+) ions. The proposed import of H(+) by the Na(+)-extruding Na(+)-ATPase necessitates "abundant H(+) pumping" by the V-type H(+)-ATPase (Ginsburg H. Abundant proton pumping in Plasmodium falciparum, but why? Trends in Parasitology 2002;18:483-6) and has significant implications for the energy budget of the parasite.


Subject(s)
Acids/metabolism , Adenosine Triphosphatases/metabolism , Cation Transport Proteins/metabolism , Erythrocytes/metabolism , Erythrocytes/parasitology , Plasmodium falciparum/drug effects , Proton-Translocating ATPases/metabolism , Sodium/metabolism , Antimalarials/metabolism
2.
Cell Host Microbe ; 13(2): 227-37, 2013 Feb 13.
Article in English | MEDLINE | ID: mdl-23414762

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.


Subject(s)
Adenosine Triphosphatases/metabolism , Antimalarials/pharmacology , Cation Transport Proteins/metabolism , Plasmodium falciparum/enzymology , Sodium-Potassium-Exchanging ATPase/metabolism , Sodium/metabolism , Adenosine Triphosphatases/genetics , Cation Transport Proteins/genetics , Drug Resistance , Enzyme Activation , Enzyme Inhibitors/pharmacology , Erythrocyte Membrane/drug effects , Erythrocyte Membrane/metabolism , Erythrocytes/metabolism , Erythrocytes/parasitology , Homeostasis , Humans , Indoles/pharmacology , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mutation , Ouabain/pharmacology , Parasitic Sensitivity Tests , Plasmodium falciparum/drug effects , Plasmodium falciparum/genetics , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Spiro Compounds/pharmacology , Trophozoites/drug effects , Trophozoites/metabolism
3.
J Biol Chem ; 285(24): 18615-26, 2010 Jun 11.
Article in English | MEDLINE | ID: mdl-20332090

ABSTRACT

The intraerythrocytic malaria parasite exerts tight control over its ionic composition. In this study, a combination of fluorescent ion indicators and (36)Cl(-) flux measurements was used to investigate the transport of Cl(-) and the Cl(-)-dependent transport of "H(+)-equivalents" in mature (trophozoite stage) parasites, isolated from their host erythrocytes. Removal of extracellular Cl(-), resulting in an outward [Cl(-)] gradient, gave rise to a cytosolic alkalinization (i.e. a net efflux of H(+)-equivalents). This was reversed on restoration of extracellular Cl(-). The flux of H(+)-equivalents was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and, when measured in ATP-depleted parasites, showed a pronounced dependence on the pH of the parasite cytosol; the flux was low at cytosolic pH values < 7.2 but increased steeply with cytosolic pH at values > 7.2. (36)Cl(-) influx measurements revealed the presence of a Cl(-) uptake mechanism with characteristics similar to those of the Cl(-)-dependent H(+)-equivalent flux. The intracellular concentration of Cl(-) in the parasite was estimated to be approximately 48 mm in situ. The data are consistent with the intraerythrocytic parasite having in its plasma membrane a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive transporter that, under physiological conditions, imports Cl(-) together with H(+)-equivalents, resulting in an intracellular Cl(-) concentration well above that which would occur if Cl(-) ions were distributed passively in accordance with the parasite's large, inwardly negative membrane potential.


Subject(s)
Chlorides/chemistry , Erythrocytes/parasitology , Plasmodium falciparum/metabolism , Adenosine Triphosphate/metabolism , Animals , Biological Transport , Cytosol/metabolism , Erythrocyte Membrane/parasitology , Hydrogen-Ion Concentration , Ion Transport , Kinetics , Malaria/metabolism , Malaria/parasitology , Microscopy, Confocal/methods , Protons , Spectrometry, Fluorescence/methods
4.
Mol Biochem Parasitol ; 169(1): 63-5, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19766147

ABSTRACT

Despite evidence that the suspension of malaria cultures leads to improved parasite growth, the practice of culturing the parasite under static conditions remains widespread. Here, extending previous work, we have quantified the favourable effects of continuous agitation on three indices of culture growth: (i) parasite yield, (ii) culture synchrony after a synchronisation procedure, and (iii) the prevalence of multiple infections. In addition, we show that under continuous suspension, the time taken for genetically altered parasites to re-populate cultures post-transfection is dramatically reduced.


Subject(s)
Cell Culture Techniques/methods , Plasmodium falciparum/growth & development , Erythrocytes/parasitology , Humans , Malaria, Falciparum/parasitology , Plasmodium falciparum/genetics , Transfection
5.
Mol Biochem Parasitol ; 162(1): 96-9, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18675853

ABSTRACT

The intraerythrocytic malaria parasite, Plasmodium falciparum maintains an intracellular pH (pH(i)) of around 7.3. If subjected to an experimentally imposed acidification the parasite extrudes H(+), thereby undergoing a pH(i) recovery. In a recent study, Bennett et al. [Bennett TN, Patel J, Ferdig MT, Roepe PD. P. falciparum Na(+)/H(+) exchanger activity and quinine resistance. Mol Biochem Parasitol 2007;153:48-58] used the H(+) ionophore nigericin, in conjunction with an acidic medium, to acidify the parasite cytosol, and then used bovine serum albumin (BSA) to scavenge the nigericin from the parasite membrane. The ensuing Na(+)-dependent pH(i) recovery, seen following an increase in the extracellular pH, was attributed to a plasma membrane Na(+)/H(+) exchanger. This is at odds with previous reports that the primary H(+) extrusion mechanism in the parasite is a plasma membrane V-type H(+)-ATPase. Here we present evidence that the Na(+)-dependent efflux of H(+) from parasites acidified using nigericin/BSA is attributable to Na(+)/H(+) exchange via residual nigericin remaining in the parasite plasma membrane, rather than to endogenous transporter activity.


Subject(s)
Erythrocytes/parasitology , Hydrogen/metabolism , Plasmodium falciparum/physiology , Sodium-Hydrogen Exchangers/metabolism , Animals , Cell Membrane/metabolism , Culture Media , Humans , Hydrogen-Ion Concentration , Ionophores/metabolism , Ionophores/pharmacology , Nigericin/metabolism , Nigericin/pharmacology , Plasmodium falciparum/growth & development
6.
Nature ; 443(7111): 582-5, 2006 Oct 05.
Article in English | MEDLINE | ID: mdl-17006451

ABSTRACT

As the malaria parasite, Plasmodium falciparum, grows within its host erythrocyte it induces an increase in the permeability of the erythrocyte membrane to a range of low-molecular-mass solutes, including Na+ and K+ (ref. 1). This results in a progressive increase in the concentration of Na+ in the erythrocyte cytosol. The parasite cytosol has a relatively low Na+ concentration and there is therefore a large inward Na+ gradient across the parasite plasma membrane. Here we show that the parasite exploits the Na+ electrochemical gradient to energize the uptake of inorganic phosphate (P(i)), an essential nutrient. P(i) was taken up into the intracellular parasite by a Na+-dependent transporter, with a stoichiometry of 2Na+:1P(i) and with an apparent preference for the monovalent over the divalent form of P(i). A P(i) transporter (PfPiT) belonging to the PiT family was cloned from the parasite and localized to the parasite surface. Expression of PfPiT in Xenopus oocytes resulted in Na+-dependent P(i) uptake with characteristics similar to those observed for P(i) uptake in the parasite. This study provides new insight into the significance of the malaria-parasite-induced alteration of the ionic composition of its host cell.


Subject(s)
Malaria/parasitology , Phosphate Transport Proteins/metabolism , Phosphates/metabolism , Plasmodium falciparum/drug effects , Plasmodium falciparum/metabolism , Sodium/pharmacology , Animals , Biological Transport/drug effects , Erythrocytes/drug effects , Erythrocytes/parasitology , Hydrogen-Ion Concentration , Kinetics , Oocytes , Phylogeny , Saponins/pharmacology , Xenopus
7.
Biochem Biophys Res Commun ; 320(2): 311-7, 2004 Jul 23.
Article in English | MEDLINE | ID: mdl-15219828

ABSTRACT

The uptake by the intraerythrocytic malaria parasite of the phospholipid precursor choline was investigated in parasites 'isolated' from their host cells by saponin permeabilization of the erythrocyte membrane. Choline is transported across the parasite plasma membrane then phosphorylated and thereby trapped within the parasite. Choline influx was inhibited competitively by quinine. It increased with increasing extracellular pH, decreased on depolarization of the parasite plasma membrane with a protonophore or by increasing extracellular [K+], and increased in response to hyperpolarization of the membrane by decreasing extracellular [K+] or by addition of the K+ channel blocker Cs+. In ATP-depleted parasites choline was taken up but not phosphorylated. Under these conditions, imposition of an inwardly negative membrane potential using the K+ ionophore valinomycin resulted in the accumulation of choline to an intracellular concentration more than 15-fold higher than the extracellular concentration. Choline influx is therefore an electrogenic process, energized by the parasite plasma membrane potential.


Subject(s)
Choline/metabolism , Membrane Potentials , Plasmodium falciparum/metabolism , Animals , Biological Transport , Hydrogen-Ion Concentration , Kinetics , Phosphorylation , Plasmodium falciparum/physiology
9.
J Biol Chem ; 279(12): 11264-72, 2004 Mar 19.
Article in English | MEDLINE | ID: mdl-14630911

ABSTRACT

The membrane potential (Deltapsi) of the mature asexual form of the human malaria parasite, Plasmodium falciparum, isolated from its host erythrocyte using a saponin permeabilization technique, was investigated using both the radiolabeled Deltapsi indicator tetraphenylphosphonium ([(3)H]TPP(+)) and the fluorescent Deltapsi indicator DiBAC(4)(3) (bis-oxonol). For isolated parasites suspended in a high Na(+), low K(+) solution, Deltapsi was estimated from the measured distribution of [(3)H]TPP(+) to be -95 +/- 2 mV. Deltapsi was reduced by the specific V-type H(+) pump inhibitor bafilomycin A(1), by the H(+) ionophore CCCP, and by glucose deprivation. Acidification of the parasite cytosol (induced by the addition of lactate) resulted in a transient hyperpolarization, whereas a cytosolic alkalinization (induced by the addition of NH(4)(+)) resulted in a transient depolarization. A decrease in the extracellular pH resulted in a membrane depolarization, whereas an increase in the extracellular pH resulted in a membrane hyperpolarization. The parasite plasma membrane depolarized in response to an increase in the extracellular K(+) concentration and hyperpolarized in response to a decrease in the extracellular K(+) concentration and to the addition of the K(+) channel blockers Ba(2+) or Cs(+) to the suspending medium. The data are consistent with Deltapsi of the intraerythrocytic P. falciparum trophozoite being due to the electrogenic extrusion of H(+) via the V-type H(+) pump at the parasite surface. The current associated with the efflux of H(+) is countered, in part, by the influx of K(+) via Ba(2+)- and Cs(+)-sensitive K(+) channels in the parasite plasma membrane.


Subject(s)
Membrane Potentials , Plasmodium falciparum/physiology , Animals , Barbiturates/chemistry , Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology , Erythrocytes/parasitology , Fluorescent Dyes/chemistry , Glucose/metabolism , Ionophores , Isoxazoles/chemistry , Macrolides/pharmacology , Onium Compounds/chemistry , Organophosphorus Compounds/chemistry , Potassium Channels/drug effects
10.
J Biol Chem ; 278(8): 5605-12, 2003 Feb 21.
Article in English | MEDLINE | ID: mdl-12427765

ABSTRACT

As it grows within the human erythrocyte, the malaria parasite, Plasmodium falciparum, ingests the erythrocyte cytosol, depositing it via an endocytotic feeding mechanism in the "digestive vacuole," a specialized acidic organelle. The digestive vacuole is the site of hemoglobin degradation, the storage site for hemozoin (an inert biocrystal of toxic heme), the site of action of many antimalarial drugs, and the site of proteins known to be involved in antimalarial drug resistance. The acidic pH of this organelle is thought to play a critical role in its various functions; however, the mechanisms by which the pH within the vacuole is maintained are not well understood. In this study, we have used a combination of techniques to demonstrate the presence on the P. falciparum digestive vacuole membrane of two discrete H(+) pumping mechanisms, both capable of acidifying the vacuole interior. One is a V-type H(+)-ATPase, sensitive to concanamycin A and bafilomycin A(1). The other is a H(+)-pyrophosphatase, which was inhibited by NaF and showed a partial dependence on K(+). The operation of the H(+)-pyrophosphatase was dependent on the presence of a Mg(2+)-pyrophosphate complex, and kinetic experiments gave results consistent with free pyrophosphate acting as an inhibitor of the protein. The presence of the combination of a H(+)-ATPase and a H(+)-pyrophosphatase on the P. falciparum digestive vacuole is similar to the situation in the acidic tonoplasts (vacuoles) of plant cells.


Subject(s)
Digestive System/enzymology , Hydrogen-Ion Concentration , Plasmodium falciparum/enzymology , Pyrophosphatases/metabolism , Vacuolar Proton-Translocating ATPases/metabolism , Animals , Digestion , Erythrocytes/parasitology , Humans , Inorganic Pyrophosphatase , Kinetics , Malaria, Falciparum/parasitology
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