Feedback Inhibition of Pantothenate Kinase Regulates Pantothenol Uptake by the Malaria Parasite
2007; Elsevier BV; Volume: 282; Issue: 35 Linguagem: Inglês
10.1074/jbc.m704610200
ISSN1083-351X
AutoresAdele M. Lehane, Rosa V. Marchetti, Christina Spry, Donelly A. van Schalkwyk, Rong‐Wei Teng, Kiaran Kirk, Kevin J. Saliba,
Tópico(s)Vitamin K Research Studies
ResumoTo survive, the human malaria parasite Plasmodium falciparum must acquire pantothenate (vitamin B5) from the external medium. Pantothenol (provitamin B5) inhibits parasite growth by competing with pantothenate for pantothenate kinase, the first enzyme in the coenzyme A biosynthesis pathway. In this study we investigated pantothenol uptake by P. falciparum and in doing so gained insights into the regulation of the parasite's coenzyme A biosynthesis pathway. Pantothenol was shown to enter P. falciparum-infected erythrocytes via two routes, the furosemide-inhibited "new permeation pathways" induced by the parasite in the infected erythrocyte membrane (the sole access route for pantothenate) and a second, furosemide-insensitive pathway. Having entered the erythrocyte, pantothenol is taken up by the intracellular parasite via a mechanism showing functional characteristics distinct from those of the parasite's pantothenate uptake mechanism. On reaching the parasite cytosol, pantothenol is phosphorylated and thereby trapped by pantothenate kinase, shown here to be under feedback inhibition control by coenzyme A. Furosemide reduced this inherent feedback inhibition by competing with coenzyme A for binding to pantothenate kinase, thereby increasing pantothenol uptake. To survive, the human malaria parasite Plasmodium falciparum must acquire pantothenate (vitamin B5) from the external medium. Pantothenol (provitamin B5) inhibits parasite growth by competing with pantothenate for pantothenate kinase, the first enzyme in the coenzyme A biosynthesis pathway. In this study we investigated pantothenol uptake by P. falciparum and in doing so gained insights into the regulation of the parasite's coenzyme A biosynthesis pathway. Pantothenol was shown to enter P. falciparum-infected erythrocytes via two routes, the furosemide-inhibited "new permeation pathways" induced by the parasite in the infected erythrocyte membrane (the sole access route for pantothenate) and a second, furosemide-insensitive pathway. Having entered the erythrocyte, pantothenol is taken up by the intracellular parasite via a mechanism showing functional characteristics distinct from those of the parasite's pantothenate uptake mechanism. On reaching the parasite cytosol, pantothenol is phosphorylated and thereby trapped by pantothenate kinase, shown here to be under feedback inhibition control by coenzyme A. Furosemide reduced this inherent feedback inhibition by competing with coenzyme A for binding to pantothenate kinase, thereby increasing pantothenol uptake. Pantothenate (vitamin B5) is a precursor of coenzyme A (CoA), an obligate enzyme cofactor required in numerous metabolic processes. The five-step conversion of pantothenate to CoA is common to prokaryotes and eukaryotes, although the amino acid sequences of the enzymes involved vary considerably (1Leonardi R. Zhang Y.M. Rock C.O. Jackowski S. Prog. Lipid Res. 2005; 44: 125-153Crossref PubMed Scopus (427) Google Scholar). The first step in this pathway is the conversion of pantothenate to 4′-phosphopantothenate by pantothenate kinase (PanK). 2The abbreviations used are:PanKpantothenate kinasePfPanKP. falciparum PanKBCECF2′,7′-bis-(2-carboxyethyl)-5,6-carboxyfluoresceinNPPnew permeation pathwaysESIelectrospray ionizationMSmass spectrometrypCMBSp-chloromercuribenzene sulfonateNEMN-ethylmaleimide. Plants, fungi, and various bacteria synthesize pantothenate de novo, whereas animals and certain microbes lack this ability and must acquire exogenous pantothenate (1Leonardi R. Zhang Y.M. Rock C.O. Jackowski S. Prog. Lipid Res. 2005; 44: 125-153Crossref PubMed Scopus (427) Google Scholar). pantothenate kinase P. falciparum PanK 2′,7′-bis-(2-carboxyethyl)-5,6-carboxyfluorescein new permeation pathways electrospray ionization mass spectrometry p-chloromercuribenzene sulfonate N-ethylmaleimide. The human malaria parasite Plasmodium falciparum cannot synthesize pantothenate and is completely dependent on its uptake from the external medium (2Divo A.A. Geary T.G. Davis N.L. Jensen J.B. J. Protozool. 1985; 32: 59-64Crossref PubMed Scopus (175) Google Scholar, 3Saliba K.J. Ferru I. Kirk K. Antimicrob. Agents Chemother. 2005; 49: 632-637Crossref PubMed Scopus (57) Google Scholar). The membranes of uninfected human erythrocytes are largely impermeant to pantothenate (4Saliba K.J. Horner H.A. Kirk K. J. Biol. Chem. 1998; 273: 10190-10195Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), but the vitamin enters infected erythrocytes via the new permeation pathways (NPP) induced in the erythrocyte membrane after P. falciparum infection (4Saliba K.J. Horner H.A. Kirk K. J. Biol. Chem. 1998; 273: 10190-10195Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Upon entering the host erythrocyte, pantothenate is taken up by the parasite via a low affinity H+-coupled transporter then, once in the parasite cytosol, is phosphorylated by the parasite's pantothenate kinase (PfPanK) as the first step in its conversion to CoA (4Saliba K.J. Horner H.A. Kirk K. J. Biol. Chem. 1998; 273: 10190-10195Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 5Saliba K.J. Kirk K. J. Biol. Chem. 2001; 276: 18115-18121Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). There have been several reports of antibacterial (6Madinaveitia J. Martin A.R. Rose F.L. Swain G. Biochem. J. 1945; 39: 85-91Crossref PubMed Google Scholar, 7Snell E.E. Shive W. J. Biol. Chem. 1945; 158: 551-559Abstract Full Text PDF Google Scholar, 8Clifton G. Bryant S.R. Skinner C.G. Arch. Biochem. Biophys. 1970; 137: 523-528Crossref PubMed Scopus (50) Google Scholar) and antiplasmodial (9Trager W. Trans. N. Y. Acad. Sci. 1966; 28: 1094-1108Crossref PubMed Scopus (25) Google Scholar, 10Trager W. J. Protozool. 1971; 18: 232-239Crossref PubMed Scopus (16) Google Scholar) pantothenate analogs. Recent recognition of the sequence dissimilarity between prokaryotic and eukaryotic CoA biosynthesis enzymes (11Calder R.B. Williams R.S. Ramaswamy G. Rock C.O. Campbell E. Unkles S.E. Kinghorn J.R. Jackowski S. J. Biol. Chem. 1999; 274: 2014-2020Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) has renewed interest in the CoA biosynthesis pathway as a target for the development of novel antibacterial agents (12Strauss E. Begley T.P. J. Biol. Chem. 2002; 277: 48205-48209Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 13Choudhry A.E. Mandichak T.L. Broskey J.P. Egolf R.W. Kinsland C. Begley T.P. Seefeld M.A. Ku T.W. Brown J.R. Zalacain M. Ratnam K. Antimicrob. Agents Chemother. 2003; 47: 2051-2055Crossref PubMed Scopus (80) Google Scholar, 14Virga K.G. Zhang Y.M. Leonardi R. Ivey R.A. Hevener K. Park H.W. Jackowski S. Rock C.O. Lee R.E. Bioorg. Med. Chem. 2005; 14: 1007-1020Crossref PubMed Scopus (56) Google Scholar). Similarly, recent evidence indicates that malaria parasite proteins required for pantothenate transport and metabolism hold promise as much-needed chemotherapeutic targets (15Kirk K. Saliba K.J. Curr. Drug Targets. 2007; 8: 75-88Crossref PubMed Scopus (59) Google Scholar). The P. falciparum pantothenate transporter and PfPanK (neither of which have been characterized at a molecular level) differ from studied mammalian counterparts in their biochemical characteristics (e.g. substrate affinity) (5Saliba K.J. Kirk K. J. Biol. Chem. 2001; 276: 18115-18121Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The value of this pathway as an antimalarial target was validated by the discovery that the widely used provitamin pantothenol (which differs from pantothenate only in the replacement of the terminal carboxyl group with a hydroxyl group) as well as a range of other pantothenate analogs (16Saliba K.J. Kirk K. Mol. Biochem. Parasitol. 2005; 141: 129-131Crossref PubMed Scopus (25) Google Scholar, 17Spry C. Chai C.L.L. Kirk K. Saliba K.J. Antimicrob. Agents Chemother. 2005; 49: 4649-4657Crossref PubMed Scopus (49) Google Scholar) inhibit the growth of P. falciparum in vitro via a mechanism that involves competitive inhibition of pantothenate phosphorylation by PfPanK (3Saliba K.J. Ferru I. Kirk K. Antimicrob. Agents Chemother. 2005; 49: 632-637Crossref PubMed Scopus (57) Google Scholar). In this study we have investigated the mechanism of pantothenol uptake by P. falciparum-infected human erythrocytes and by parasites functionally isolated from their host cells by a saponin permeabilization technique. The results demonstrate that, like pantothenate, pantothenol gains access into infected erythrocytes via the NPP, although an additional pathway accounts for a minor component of its uptake. Once inside the erythrocyte, pantothenol is taken up across the parasite plasma membrane via a mechanism(s) that is functionally distinct from that which mediates the uptake of pantothenate. On entering the parasite, pantothenol is, like pantothenate, phosphorylated by PfPanK, which we show here to be inhibited by CoA. Furosemide was found to alleviate this CoA-mediated negative feedback inhibition of PfPanK and thereby increases the uptake of pantothenol by the parasite despite reducing its initial rate of uptake into P. falciparum-infected erythrocytes by inhibiting the NPP. Reagents—[14C]Pantothenol (50 mCi/mmol) and [14C]pantothenate (55 mCi/mmol) were purchased from American Radiolabeled Chemicals. [14C]Choline (55 mCi/mmol) and [3H]hypoxanthine (14.7 Ci/mmol) were purchased from Amersham Biosciences. Parasite Culture and Isolation—Experiments were performed using the 3D7 or FAF6 strains of P. falciparum. The parasites were cultured in Group O, Rh+ erythrocytes and synchronized with sorbitol as described elsewhere (18Allen R.J.W. Kirk K. J. Biol. Chem. 2004; 279: 11264-11272Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Parasites were "isolated" from their host erythrocytes by saponin treatment (4Saliba K.J. Horner H.A. Kirk K. J. Biol. Chem. 1998; 273: 10190-10195Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), which permeabilizes the cholesterol-containing erythrocyte plasma membrane and parasitophorous vacuole membrane (19Ansorge I. Benting J. Bhakdi S. Lingelbach K. Biochem. J. 1996; 315: 307-314Crossref PubMed Scopus (162) Google Scholar) while leaving the parasite plasma membrane intact (20Saliba K.J. Kirk K. J. Biol. Chem. 1999; 274: 33213-33219Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). [14C]Pantothenol and [14C]Pantothenate Uptake across the Erythrocyte Membrane—The uptake of [14C]pantothenol or [14C]pantothenate into intact trophozoite-infected and uninfected erythrocytes was measured essentially as described previously for [14C]pantothenate (4Saliba K.J. Horner H.A. Kirk K. J. Biol. Chem. 1998; 273: 10190-10195Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Uptake was commenced by adding [14C]pantothenol (2 μm, 0.10 μCi/ml in final reaction) or [14C]pantothenate (2 μm, 0.11 μCi/ml) to the cell suspension and terminated by centrifuging (15,800 × g, 2 min) 200-μl aliquots of the cell suspension (∼7 × 107 cells/ml for infected erythrocytes and between 7 × 107 and 3 × 109 cells/ml for uninfected erythrocytes) through 300 μl of dibutyl phthalate, thus separating the cells from the supernatant solution containing the radiolabeled compound. Trophozoite-infected erythrocytes were separated from uninfected cells using the Miltenyi Biotec VarioMACS magnet (21Paul F. Roath S. Melville D. Warhurst D.C. Osisanya J.O. Lancet. 1981; 2: 70-71Abstract PubMed Scopus (118) Google Scholar, 22Staalsoe T. Giha H.A. Dodoo D. Theander T.G. Hviid L. Cytometry. 1999; 35: 329-336Crossref PubMed Google Scholar), giving a parasitemia of 95–97%. A cell volume of 75 fl was assumed for both infected and uninfected erythrocytes (4Saliba K.J. Horner H.A. Kirk K. J. Biol. Chem. 1998; 273: 10190-10195Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). [14C]Pantothenol, [14C]Pantothenate, and [14C]Choline Uptake into Isolated Parasites— The uptake of radiolabeled compounds by isolated trophozoites suspended in HEPES-buffered saline (125 mm NaCl, 5 mm KCl, 1 mm MgCl2,20mm glucose, 25 mm HEPES, pH 7.1 unless stated otherwise) at 37 °C was measured essentially as described previously (23Lehane A.M. Saliba K.J. Allen R.J.W. Kirk K. Biochem. Biophys. Res. Commun. 2004; 320: 311-317Crossref PubMed Scopus (45) Google Scholar). In experiments with ATP-depleted parasites, glucose was omitted from the saline (and the NaCl concentration was increased to 135 mm so as to maintain the osmolarity of the saline), and parasites were preincubated in this solution for 15–30 min at 37 °C. In the experiments giving rise to Fig. 5C, an inwardly negative membrane potential was imposed on ATP-depleted parasites as described previously (23Lehane A.M. Saliba K.J. Allen R.J.W. Kirk K. Biochem. Biophys. Res. Commun. 2004; 320: 311-317Crossref PubMed Scopus (45) Google Scholar). In all experiments the concentration of the extracellular radiolabeled compound was 2 μm, and the cells were suspended at a density of ∼108 cells/ml. An isolated parasite volume of 28 fl was assumed (4Saliba K.J. Horner H.A. Kirk K. J. Biol. Chem. 1998; 273: 10190-10195Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Furosemide, phloretin, N-ethylmaleimide (NEM), carbonyl cyanide m-chlorophenylhydrazone, and valinomycin were added to the parasites (at the same time as the addition of radiolabel) as stock solutions in Me2SO, and an equal volume of Me2SO was added to the relevant controls (the maximum Me2SO concentration in the reaction solution was 0.4%, v/v). p-Chloromercuribenzene sulfonate (pCMBS) was dissolved in water. [14C]Pantothenol and [14C]Pantothenate Phosphorylation— The amount of [14C]pantothenol and [14C]pantothenate phosphorylated by PfPanK in parasite lysate (prepared from ∼7 × 108 cells/ml) was measured under different conditions at predetermined time points by precipitating phosphorylated compounds from solution using the Somogyi reagent (ZnSO4 and Ba(OH)2), as described previously (4Saliba K.J. Horner H.A. Kirk K. J. Biol. Chem. 1998; 273: 10190-10195Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). The results of key experiments were confirmed with an alternate assay that relies on the separation of the phosphorylated from the unphosphorylated [14C]substrate by binding the phosphorylated [14C]substrate to DE81 filter disks followed by scintillation counting, as described previously (14Virga K.G. Zhang Y.M. Leonardi R. Ivey R.A. Hevener K. Park H.W. Jackowski S. Rock C.O. Lee R.E. Bioorg. Med. Chem. 2005; 14: 1007-1020Crossref PubMed Scopus (56) Google Scholar, 24Ivey R.A. Zhang Y.M. Virga K.G. Hevener K. Lee R.E. Rock C.O. Jackowski S. Park H.W. J. Biol. Chem. 2004; 279: 35622-35629Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) with the following two modifications; the [14C]substrate concentration was 0.1 Ci/ml (2 μm), and the filter disks were washed 3 times, each time with 3 ml of an acetic acid:ethanol:water (1:95:4, v/v/v) mixture. In those experiments in which CoA and furosemide were added to the parasite lysate, they were added at the same time as the radiolabeled compound, and the relevant solvent (water and Me2SO) controls were included in each case. Biosynthesis, Purification, and Analysis of 4′-Phosphopantothenol—A mixture of pantothenol, ATP, MgCl2, and furosemide in 50 mm KH2PO4 buffer, pH 7.4, was at time 0 combined with lysate prepared aseptically from ∼3 × 108 P. falciparum parasites, yielding a final volume of 1.8 ml and final concentrations as follows: pantothenol (1 mm), ATP (15 mm), MgCl2 (15 mm), and furosemide (100 μm). Furosemide was included in the reaction to alleviate feedback inhibition of PanK by CoA or CoA thioesters that may have been present in the parasite lysate (see "Results"). The mixture was incubated for 48 h at 37 °C, and the reaction was terminated by transferring the mixture to 95 °C for 5 min. Precipitated protein was pelleted by centrifugation at 16,000 × g for 5 min. A reaction mixture identical to the one described above but also containing 0.5 μCi/ml [14C]pantothenol was incubated at 37 °C for 48 h and processed in the same manner. The supernatant from this reaction was spotted on a plastic-backed 0.2-mm-thick silica gel 60 plate (Merck), and the plate was developed to 14 cm from the base line in a solvent of ethanol, 28% NH4OH (6:4, v/v). Horizontal strips of the thin-layer chromatography (TLC) plate (0.5 cm in width) were transferred to scintillation vials, to which 100 μl of water followed by 3 ml of scintillation fluid was added. The vials were vortexed, and the radioactivity on each section of the plate was determined by scintillation counting. Scintillation counting revealed the production of a 14C-labeled pantothenol metabolite with an Rf (0.61) distinct from that of pantothenol (Rf = 0.88). The product of the unlabeled reaction could thereby be purified from unreacted pantothenol and other components in the supernatant by TLC. The reaction supernatant was applied in a band to a TLC plate, and the plate was developed to 14 cm from the base line in ethanol, 28% NH4OH (6:4, v/v). The region bracketing the product (Rf = 0.61) was scraped from the plate, and the product was extracted from the silica gel by suspending the silica in water (5 times) and each time centrifuging at 14,000 × g for 2 min. The water-soluble extracts were combined, concentrated in vacuo, and subjected to 1H NMR and ESI-MS analysis. NMR spectra were recorded on a Bruker Avance 800 NMR spectrometer operating at 800.13 MHz. The chemical shifts (δ) are reported as the shift in ppm from trimethylsilyl-2,2,3,3-tetradeuteropropionic acid (0.00 ppm). ESI-MS analysis was performed by the Research School of Chemistry Mass Spectrometry Facility, The Australian National University. Low resolution mass spectra were recorded on a Micromass-Waters LC-ZMD single quadrupole liquid chromatograph-mass spectrometer, and high resolution mass spectra were recorded on a Bruker Apex III 4.7T Fourier transform ion cyclotron resonance mass spectrometer using negative ion detection. The product of the reaction was confirmed as 4′-phosphopantothenol by both 1H NMR and ESI-MS. 1H NMR (800 MHz, D2O): δ 4.11 (s, 1H), 3.77 (dd, 1H), 3.65 (t, 2H), 3.47 (dd, 1H), 3.32 (t, 2H), 1.79 (m, 2H), 1.01 (s, 3H), 0.89 (s, 3H). MS (ESI) m/z: 284.2 ([M-H]–). High resolution mass spectrometry: calculated, 284.0899 for C9H19NO7P; found, 284.0912. Measurement of Cytosolic pH— The pH of the parasite cytosol was monitored using 2′,7′-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) as described elsewhere (20Saliba K.J. Kirk K. J. Biol. Chem. 1999; 274: 33213-33219Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Statistics—Statistical comparisons were made using Student's two-tailed t tests for paired or unpaired samples, as appropriate. Pantothenol Is Taken Up by Infected and Uninfected Erythrocytes—[14C]Pantothenol entry into human erythrocytes infected with P. falciparum trophozoites was initially rapid, reaching a distribution ratio ([pantothenol]i/[pantothenol]o) of 1 within 1 min, but slowing thereafter, reaching a distribution ratio of 2.2 ± 0.2 (mean ± S.E.) by 20 min (Fig. 1A, open circles). Furosemide (100 μm), an effective inhibitor of the NPP, reduced the initial rate of [14C]pantothenol uptake from a value ≥19 ± 3 μmol/(1012 cells·h) to 2.7 ± 0.3 μmol/(1012 cells·h) (p = 0.027), consistent with the NPP being the major but not the sole route for the uptake of pantothenol. However, furosemide significantly increased (approximately doubled; p = 0.036) the total accumulation seen over the 20-min time course (Fig. 1A, filled circles). To determine whether the component of [14C]pantothenol uptake that was not inhibited by furosemide could be attributed to an endogenous erythrocyte pathway, we investigated [14C]pantothenol uptake by uninfected erythrocytes. Compared with [14C]pantothenate, which enters uninfected erythrocytes very slowly (Fig. 1B, open squares), [14C]pantothenol crossed the uninfected erythrocyte membrane rapidly (Fig. 1B, open circles), with an initial rate of 4.2 ± 0.4 μmol/(1012 cells·h). Furosemide did not significantly affect this initial rate (4.2 ± 0.8 μmol/(1012 cells·h)) or [14C]pantothenol accumulation after 20 min (p = 0.9 and 0.5, respectively; Fig. 1B, filled circles). Similarly, the addition of 10 mm pantothenol to the external medium had no significant effect on [14C]pantothenol uptake (not shown), consistent with pantothenol crossing the uninfected erythrocyte membrane either by simple diffusion through the membrane bilayer or through a low affinity transport system. An Arrhenius plot of the transport of pantothenol across the uninfected erythrocyte membrane (Fig. 1B, inset) was nonlinear and gave activation energies of 64 ± 11 kJ/mol at temperatures >25 °C and 106 ± 4 kJ/mol at temperatures 1 might be explained either by an active transport process or by the metabolism of pantothenol within the parasite. Because pantothenol has been shown to act as a competitive inhibitor of the phosphorylation of pantothenate (3Saliba K.J. Ferru I. Kirk K. Antimicrob. Agents Chemother. 2005; 49: 632-637Crossref PubMed Scopus (57) Google Scholar), we investigated whether pantothenol is itself phosphorylated by PfPanK using lysate prepared from isolated parasites. 1H NMR and ESI-MS analyses revealed that incubation of pantothenol with parasite lysate resulted in the generation of 4′-phosphopantothenol (see "Experimental Procedures" for details). We then compared the rate of pantothenol phosphorylation to that of pantothenate phosphorylation by parasite lysates. In paired experiments 62 ± 2% of [14C]pantothenol was phosphorylated after 40 min (Fig. 3, open circles) compared with 84 ± 1% of the same concentration of [14C]pantothenate (Fig. 3, open squares). The initial phosphorylation rates were 6.1 ± 0.1 mmol/ (1012 cells·h) and 10 ± 2 mmol/(1012 cells·h) for pantothenol and pantothenate, respectively. Similar results were obtained with an alternate assay for measuring pantothenate/pantothenol phosphorylation (Fig. 3, inset). Thus, pantothenol is phosphorylated by PfPanK but at a slower rate than pantothenate. Pantothenol uptake by the parasite, therefore, reflects a combination of transport and metabolism.FIGURE 3[14C]Pantothenol and [14C]pantothenate phosphorylation by PfPanK. Time courses for the phosphorylation of [14C]pantothenol (0.35 μm; open circles) and [14C]pantothenate (0.35 μm; open squares) by PfPanK in parasite lysate, as determined using the Somogyi reagent. The data are from a single experiment (which included duplicates, shown ± range/2) and are representative of those obtained in two separate experiments. Inset, the same experiment as in main figure, but the amount of [14C]pantothenol and [14C]pantothenate phosphorylation was determined by binding the phosphorylated substrate to DE81 filter disks followed by scintillation counting. The data are averaged from three separate experiments and shown ± S.E. In both experiments, where not shown, error bars fall within the symbol.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Furosemide Increases Pantothenol Uptake by Isolated Parasites—The observation that furosemide increased pantothenol uptake by infected erythrocytes (Fig. 1A) but not by uninfected erythrocytes (Fig. 1B) is consistent with furosemide increasing pantothenol uptake by infected erythrocytes by acting on the sequestration of pantothenol within the parasite. This was tested directly. Furosemide greatly increased [14C]pantothenol uptake by isolated parasites (Fig. 4A), increasing the distribution ratio after 5 min from 3.6 ± 0.3 to 13 ± 2(p = 0.01) and after 20 min from 7.9 ± 1.5 to 25 ± 4(p = 0.048). By contrast, furosemide did not significantly increase the uptake of [14C]pantothenate after 5 min (p = 0.9; Fig. 4A, inset). The effect of furosemide on the transport of pantothenol and pantothenate across the parasite plasma membrane was investigated in parasites in which the contribution of the phosphorylation reaction to the measured uptake was inhibited by the inclusion of 500 μm unlabeled pantothenate in the solution. This concentration of pantothenate is sufficient to saturate PfPanK (which has a Km of 0.3 μm) (5Saliba K.J. Kirk K. J. Biol. Chem. 2001; 276: 18115-18121Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) but should have little effect on the pantothenate transport mechanism (which has a Km of 23 mm) (5Saliba K.J. Kirk K. J. Biol. Chem. 2001; 276: 18115-18121Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Under these conditions both compounds equilibrated across the parasite plasma membrane, with pantothenol entering the parasite more rapidly than pantothenate (p = 0.03). The initial rates of pantothenate and pantothenol uptake were 4.2 ± 0.1 μmol/(1012 cells·h) and ≥16 ± 2 μmol/(1012 cells·h), respectively (Fig. 4B, open bars and filled bars, respectively). Furosemide did not significantly affect the transport rates of either compound (p > 0.06; Fig. 4B). Furthermore, ATP depletion did not affect the transport of pantothenol (p = 0.07; Fig. 4B) but caused a slight slowing of the initial rate of pantothenate transport to 2.5 ± 0.1 μmol/(1012 cells·h) (p = 0.01; Fig. 4B). Pantothenol Uptake by Isolated Parasites Is pH-dependent— The transport of pantothenate across the parasite plasma membrane is H+-dependent (5Saliba K.J. Kirk K. J. Biol. Chem. 2001; 276: 18115-18121Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The pH dependence of [14C]pantothenol uptake by isolated parasites was investigated over the extracellular pH (pHo) range 6.1 to 8.1. The corresponding intracellular pH (pHi) range is 7.07–7.72 (23Lehane A.M. Saliba K.J. Allen R.J.W. Kirk K. Biochem. Biophys. Res. Commun. 2004; 320: 311-317Crossref PubMed Scopus (45) Google Scholar). [14C]Pantothenol uptake showed a marked pH dependence, increasing with increasing pHo (Fig. 5A). There was a significant difference (p = 0.01) between the initial rates at pH 6.1 (0.7 ± 0.3 μmol/(1012 cells·h)) and 8.1 (5.0 ± 1.0 μmol/(1012 cells·h)). This is the opposite pH dependence from that seen with pantothenate (5Saliba K.J. Kirk K. J. Biol. Chem. 2001; 276: 18115-18121Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), consistent with a fundamentally different uptake mechanism being involved. The effect of pH on pantothenol uptake may reflect a pH dependence of the transport step or of the subsequent phosphorylation of the compound within the parasite. To determine whether pantothenol transport was associated with the movement of H+ equivalents, pHi was measured using the fluorescent pH indicator BCECF. The addition of 40 mm pantothenol to suspensions of BCECF-loaded isolated parasites did not affect pHi (Fig. 5B), consistent with pantothenol transport not being H+-coupled. By contrast, the addition of 40 mm pantothenate caused a decrease in pHi (Fig. 5B), as reported previously (5Saliba K.J. Kirk K. J. Biol. Chem. 2001; 276: 18115-18121Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). It is also possible that changes in pH alter the conformation of pantothenol, affecting its ability to diffuse (or be transported) across the lipid bilayer. To investigate this possibility we used the Pallas for Windows software to predict the LogD (octanol/water distribution coefficient) over a wide pH range. The software predicted a LogD of –0.93 over the pH range of 0–11, only increasing when the pH was increased further. pH is, therefore, unlikely to alter the membrane permeability properties of pantothenol. A decrease in pHo results in a depolarization of the parasite plasma membrane from its normal value of approximately –95 mV (18Allen R.J.W. Kirk K. J. Biol. Chem. 2004; 279: 11264-11272Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). An increase in the uptake of choline by the parasite at higher pHo values h
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