An Acid-activated Nucleobase Transporter from Leishmania major

Parasitic protozoa are unable to synthesize purines de novo and must import preformed purine nucleobases or nucleosides from their hosts. Leishmania major expresses two purine nucleobase transporters, LmaNT3 and LmaNT4. Previous studies revealed that at neutral pH, LmaNT3 is a broad specificity, high affinity nucleobase transporter, whereas LmaNT4 mediates the uptake of only adenine. Because LmaNT4 is required for optimal viability of the amastigote stage of the parasite that lives within acidified phagolysomal vesicles of mammalian macrophages, the function of this permease was examined under acidic pH conditions. At acidic pH, LmaNT4 acquires the ability to transport adenine, hypoxanthine, guanine, and xanthine with Km values in the micromolar range, indicating that this transporter is activated at low pH. Thus, LmaNT4 is an acid-activated purine nucleobase transporter that functions optimally under the physiological conditions the parasite is exposed to in the macrophage phagolysosome. In contrast, LmaNT3 functions optimally at neutral pH. Two-electrode voltage clamp experiments performed on LmaNT3 and LmaNT4 expressed in Xenopus oocytes revealed substrate-induced inward directed currents at acidic pH, and application of substrates induced acidification of the oocyte cytosol. These observations imply that LmaNT3 and LmaNT4 are nucleobase/proton symporters. Parasitic protozoa are unable to synthesize purines de novo and must import preformed purine nucleobases or nucleosides from their hosts. Leishmania major expresses two purine nucleobase transporters, LmaNT3 and LmaNT4. Previous studies revealed that at neutral pH, LmaNT3 is a broad specificity, high affinity nucleobase transporter, whereas LmaNT4 mediates the uptake of only adenine. Because LmaNT4 is required for optimal viability of the amastigote stage of the parasite that lives within acidified phagolysomal vesicles of mammalian macrophages, the function of this permease was examined under acidic pH conditions. At acidic pH, LmaNT4 acquires the ability to transport adenine, hypoxanthine, guanine, and xanthine with Km values in the micromolar range, indicating that this transporter is activated at low pH. Thus, LmaNT4 is an acid-activated purine nucleobase transporter that functions optimally under the physiological conditions the parasite is exposed to in the macrophage phagolysosome. In contrast, LmaNT3 functions optimally at neutral pH. Two-electrode voltage clamp experiments performed on LmaNT3 and LmaNT4 expressed in Xenopus oocytes revealed substrate-induced inward directed currents at acidic pH, and application of substrates induced acidification of the oocyte cytosol. These observations imply that LmaNT3 and LmaNT4 are nucleobase/proton symporters. Leishmania species are parasitic protozoa that infect humans and animals and cause a spectrum of diseases in tropical and subtropical regions of the world (1Herwaldt B.L. Lancet. 1999; 354: 1191-1199Abstract Full Text Full Text PDF PubMed Scopus (1363) Google Scholar). The life cycle consists of three principal stages: flagellated promastigotes that colonize the midgut of the sand fly vector, metacyclic promastigotes that live in the mouth parts of the sand fly and are infectious to mammalian hosts, and non-flagellated amastigotes that reside within acidified phagolysosomal vesicles of vertebrate host macrophages. Like all other parasitic protozoa examined, Leishmania species are unable to synthesize the purine ring de novo and must import purines from both the insect and mammalian hosts (2Carter N.S. Rager N. Ullman B. Marr J.J. Nilsen T. Komuniecki R. Molecular and Medical Parasitology. Academic Press, London2003: 197-223Google Scholar). Salvage of these essential nutrients is initiated by uptake of nucleobases and nucleosides across the parasite plasma membrane by permeases of the ENT (equilibrative nucleoside transporter) family (SLC29 family). The Leishmania major genome (3Ivens A.C. Peacock C.S. Worthey E.A. Murphy L. Aggarwal G. Berriman M. Sisk E. Rajandream M.A. Adlem E. Aert R. Anupama A. Apostolou Z. Attipoe P. Bason N. Bauser C. Beck A. Beverley S.M. Bianchettin G. Borzym K. Bothe G. Bruschi C.V. Collins M. Cadag E. Ciarloni L. Clayton C. Coulson R.M. Cronin A. Cruz A.K. Davies R.M. De Gaudenzi J. Dobson D.E. Duesterhoeft A. Fazelina G. Fosker N. Frasch A.C. Fraser A. Fuchs M. Gabel C. Goble A. Goffeau A. Harris D. Hertz-Fowler C. Hilbert H. Horn D. Huang Y. Klages S. Knights A. Kube M. Larke N. Litvin L. Lord A. Louie T. Marra M. Masuy D. Matthews K. Michaeli S. Mottram J.C. Müller-Auer S. Munden H. Nelson S. Norbertczak H. Oliver K. O'neil S. Pentony M. Pohl T.M. Price C. Purnelle B. Quail M.A. Rabbinowitsch E. Reinhardt R. Rieger M. Rinta J. Robben J. Robertson L. Ruiz J.C. Rutter S. Saunders D. Schäfer M. Schein J. Schwartz D.C. Seeger K. Seyler A. Sharp S. Shin H. Sivam D. Squares R. Squares S. Tosato V. Vogt C. Volckaert G. Wambutt R. Warren T. Wedler H. Woodward J. Zhou S. Zimmermann W. Smith D.F. Blackwell J.M. Stuart K.D. Barrell B. Myler P.J. Science. 2005; 309: 436-442Crossref PubMed Scopus (1134) Google Scholar) encodes five members of this family, NT1.1, NT1.2, and NT2, which are nucleoside transporters (4Vasudevan G. Carter N.S. Drew M.E. Beverley S.M. Sanchez M.A. Seyfang A. Ullman B. Landfear S.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9873-9878Crossref PubMed Scopus (108) Google Scholar, 5Carter N.S. Drew M.E. Sanchez M. Vasudevan G. Landfear S.M. Ullman B. J. Biol. Chem. 2000; 275: 20935-20941Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), and NT3 and NT4, which are purine nucleobase permeases (6Sanchez M.A. Tryon R. Pierce S. Vasudevan G. Landfear S.M. Mol. Membr. Biol. 2004; 21: 11-18Crossref PubMed Scopus (36) Google Scholar, 7Ortiz D. Sanchez M.A. Pierce S. Herrmann T. Kimblin N. Archie Bouwer H.G. Landfear S.M. Mol. Microbiol. 2007; 64: 1228-1243Crossref PubMed Scopus (37) Google Scholar). Previous functional characterization of LmaNT3 (Lma indicates that the transporter is from L. major) revealed that it mediates the uptake of hypoxanthine, adenine, guanine, and xanthine with Km values in the low micromolar range (6Sanchez M.A. Tryon R. Pierce S. Vasudevan G. Landfear S.M. Mol. Membr. Biol. 2004; 21: 11-18Crossref PubMed Scopus (36) Google Scholar). In contrast, at neutral pH, LmaNT4 promotes the uptake of only adenine. Nonetheless, studies with the Δlmant4-null mutant (7Ortiz D. Sanchez M.A. Pierce S. Herrmann T. Kimblin N. Archie Bouwer H.G. Landfear S.M. Mol. Microbiol. 2007; 64: 1228-1243Crossref PubMed Scopus (37) Google Scholar) revealed that LmaNT4 was essential for optimal viability of L. major amastigotes in primary murine macrophages. In contrast, deletion of the LmaNT3 genes in the Δlmant3-null mutant was without effect on amastigote viability inside macrophages. Because amastigotes are exposed to a pH of ∼5 in the macrophage phagolysosome (8Antoine J.C. Prina E. Jouanne C. Bongrand P. Infect Immun. 1990; 58: 779-787Crossref PubMed Google Scholar), one potential explanation for the compromised viability of Δlmant4 amastigotes is that LmaNT4 operates optimally at acidic pH. In this case, deletion of the LmaNT4 genes would reduce the ability of amastigotes to salvage purines within the phagolysosome and would compromise their viability. If, in contrast, the LmaNT3 permease functions optimally at neutral pH, amastigotes could tolerate the loss of this transporter more easily, explaining the unaltered viability of Δlmant3 amastigotes. To test this possibility, we have studied the function of both LmaNT4 and LmaNT3 at both acidic and neutral pH. Indeed, LmaNT4, but not LmaNT3, is an acid-activated nucleobase transporter, implying that it functions optimally under the physiological conditions of the macrophage phagolysosome. L. major Friedlin VI (MHOM/IL/80Friedlin) parasites were cultured at 26 °C in Medium 199 containing 15% fetal bovine serum. Generation of the Δlmant-null mutant and complementation with the LmaNT3 and LmaNT4 genes have been described previously (7Ortiz D. Sanchez M.A. Pierce S. Herrmann T. Kimblin N. Archie Bouwer H.G. Landfear S.M. Mol. Microbiol. 2007; 64: 1228-1243Crossref PubMed Scopus (37) Google Scholar). Uptake of 3H-labeled nucleobases into parasites and Xenopus laevis oocytes were performed as described previously (7Ortiz D. Sanchez M.A. Pierce S. Herrmann T. Kimblin N. Archie Bouwer H.G. Landfear S.M. Mol. Microbiol. 2007; 64: 1228-1243Crossref PubMed Scopus (37) Google Scholar). The buffer for measurement of nucleobase uptake in parasites at different pH values was prepared by mixing solutions of 0.1 m Na2HPO4 and 0.1 m NaH2PO4 to obtain the desired pH value. Concentrated parasites in phosphate-buffered saline, pH 7.4, were diluted ∼5-fold into the sodium phosphate buffer and assayed immediately for uptake using radiolabeled ligand resuspended in the same buffer. Substrate saturation curves were fitted to the Michaelis-Menten equation by non-linear regression using Prism 4.0b (GraphPad Software, Inc.). cRNA transcripts were synthesized from linearized inserts containing the LmaNT3 and LmaNT4 open reading frames subcloned into the pL2.5 vector (9Arriza J.L. Kavanaugh M.P. Fairman W.A. Wu Y.N. Murdoch G.H. North R.A. Amara S.G. J. Biol. Chem. 1993; 268: 15329-15332Abstract Full Text PDF PubMed Google Scholar) using the mMESSAGE mMACHINE T7 ultra kit (Ambion). Defolliculated X. laevis oocytes were microinjected with 23 nl (∼10 ng) cRNA and maintained in ND96 buffer (96 mm NaCl, 2.0 mm KCl, 1.8 mm CaCl2, and 5 mm HEPES, pH 7.5) for 3 days at 16 °C. Water-injected oocytes were used as controls. Two-electrode voltage clamp experiments were performed in ND96 buffer at room temperature using GeneClamp 500B (Axon Instruments, Inc.). For measurements performed at pH 5.5, ND96 buffer was supplemented with 40 mm MES 3The abbreviations used are: MES4-morpholineethanesulfonic acidBCECF2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. buffer adjusted to that pH with HCl. Charge-flux ratios were determined by integrating the charge induced by 25 μm [3H]hypoxanthine or [3H]adenine over 5 min and by measuring the radioactivity accumulated by the same isolated oocytes over that time. Background uptake by uninjected oocytes was subtracted. 4-morpholineethanesulfonic acid 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. For fluorescence measurements, oocytes were preloaded with 30 μm BCECF for 30 min in ND96 buffer at room temperature. Fluorescence was measured using a Leica upright microscope equipped with a 20× objective, an Oregon Green Filter cube (exciter, HQ495/30X; dichroic, Q515LP; and emitter, HQ545/50m), and a photodiode (pin-020A, UCT Sensors, Inc.) coupled to an Axopatch 200B patch clamp amplifier (Axon Instruments, Inc.). The objective was focused on the animal pole, and fluorescence was monitored with Clampex 8.2 (Axon Instruments, Inc.). Functional characterization of LmaNT3 in Xenopus oocytes (6Sanchez M.A. Tryon R. Pierce S. Vasudevan G. Landfear S.M. Mol. Membr. Biol. 2004; 21: 11-18Crossref PubMed Scopus (36) Google Scholar) established that it transports the purine nucleobases hypoxanthine, adenine, guanine, and xanthine with Km values in the low micromolar range. Furthermore, this transporter is the principal nucleobase permease in promastigotes because deletion of the LmaNT3 genes in the Δlmant3-null mutant abrogated uptake of hypoxanthine and guanine, greatly reduced uptake of xanthine, but left residual transport of adenine (7Ortiz D. Sanchez M.A. Pierce S. Herrmann T. Kimblin N. Archie Bouwer H.G. Landfear S.M. Mol. Microbiol. 2007; 64: 1228-1243Crossref PubMed Scopus (37) Google Scholar) that is probably due to the intact LmNT4 transporter gene. Consequently, the Δlmant3-null mutant provided an opportune genetic background in which nucleobase transporters could be expressed from episomal expression vectors and functionally characterized. Subtraction of background uptake of any nucleobase by the Δlmant3-null mutant from the uptake measured for the null mutant complemented with LmaNT4, Δlmant3[pNT4], provided a quantitative measurement of uptake due to LmaNT4 alone. These experiments revealed that LmaNT4 transports adenine with low affinity, being non-saturable at 1 mm adenine but does not transport hypoxanthine, guanine, or xanthine at pH 7.4 (7Ortiz D. Sanchez M.A. Pierce S. Herrmann T. Kimblin N. Archie Bouwer H.G. Landfear S.M. Mol. Microbiol. 2007; 64: 1228-1243Crossref PubMed Scopus (37) Google Scholar). To determine whether LmaNT4 might be a nucleobase permease that is optimized to function in the acid environment that amastigotes experience in the macrophage phagolysosome (8Antoine J.C. Prina E. Jouanne C. Bongrand P. Infect Immun. 1990; 58: 779-787Crossref PubMed Google Scholar), uptake of 25 μm purine nucleobases by Δlmant3[pNT4] parasites was measured over a range of pH values from 5 to 8 (Fig. 1). Because the concentration of nucleobases in plasma and other extracellular fluids is typically in the low micromolar range (10Traut T.W. Mol. Cell. Biochem. 1994; 140: 1-22Crossref PubMed Scopus (1270) Google Scholar), uptake properties at this concentration are likely to be relevant to the roles of these permeases during an infection of a mammalian host. Indeed, LmaNT4 exhibited a pH maximum between 5 and 6 for transport of each nucleobase. In contrast, uptake by LmaNT3, measured using the Δlmant3[pNT3] cell line (Fig. 1), revealed maximum uptake activity at pH 7 for uptake of each of the purine nucleobases, except for hypoxanthine, whose uptake was not very responsive to changes in pH. To determine the apparent affinity for uptake of nucleobases, substrate response curves for adenine (Fig. 2A) and hypoxanthine (Fig. 2B) were obtained for LmaNT3 and LmaNT4 at pH 7.6, 6.6, and 5.5. LmaNT3 exhibited Km values in the low micromolar range for both substrates at all three pH values (Table 1). LmaNT4 revealed non-saturable transport for adenine at pH 7.6, but the apparent affinity increased with decreasing pH to exhibit a Km value of ∼40 μm at pH 5.5. For hypoxanthine, transport was non-saturable at pH 7.5 and 6.5, but transport began to saturate at pH 5.5 (estimated Km ∼ 400 μm).TABLE 1Estimation ofKm and Vmaxvalues for uptake of adenine and hypoxanthine by LmaNT3 (NT3) and LmaNT4 (NT4). The data inFig. 2 were fitted to the Michaelis-Menten equation by non-linear regression using Prism 4.0b software (GraphPad Software, Inc.). The numerical values forKm and Vmaxare reported as best fit values and “asymptotic” S.E. ((17)). ND indicates that kinetic values were not determined due to non-saturable substrate dependence curves. TheVmax(pmol/107cell/min)/Km(μm) values for data whereKm and Vmaxwere not determined and for hypoxanthine uptake by NT4 at pH 5.5 whereKm and Vmaxvalues exhibited high S.E. were calculated from the slope of the initial linear portion of theVversus[S] curve, where [S] ≪Km and the slope = Vmax/Km.AdenineHypoxanthineKmVmaxVmax/KmKmVmaxVmax/Kmμmpmol/107cell/minμmpmol/107cell/minNT3 pH 7.642 ± 10102 ± 72.438 ± 12134 ± 113.5 pH 6.624 ± 770 ± 52.931 ± 592 ± 42.9 pH 5.516 ± 360 ± 23.742 ± 16118 ± 132.8NT4 pH 7.6NDND0.35NDND0.15 pH 6.6NDND0.40NDND0.22 pH 5.539 ± 694 ± 52.4420 ± 370119 ± 640.43 in a These experiments that at 25 μm LmaNT3 activity is optimal at neutral pH (Fig. 1). In contrast, transport of purine nucleobases by LmaNT4 is activated by acidic pH (Fig. 1), and apparent substrate affinity is increased at acidic pH values (Fig. 2 and 1). LmaNT4 functions as an adenine transporter at neutral pH but acquires the ability to transport all purine nucleobases at acidic pH. In the a of transporter were calculated for LmaNT3 and LmaNT4 both adenine and hypoxanthine at the three pH values (Table 1). For LmaNT3, transporter was not pH. In contrast, LmaNT4 exhibited an and in transporter as the pH was from to 5.5. the that transport by LmaNT4 is activated by acidic pH conditions. is a hypoxanthine that is in the of S. Marr J.J. N. J. PubMed Scopus Google Scholar) and is by parasite nucleobase permeases LmaNT3 (7Ortiz D. Sanchez M.A. Pierce S. Herrmann T. Kimblin N. Archie Bouwer H.G. Landfear S.M. Mol. Microbiol. 2007; 64: 1228-1243Crossref PubMed Scopus (37) Google Scholar). To determine whether LmaNT4 could a for uptake in the acidified in which amastigotes we measured the uptake of by both nucleobase transporters at pH 5.5 and (Fig. and At a 1 μm concentration of (Fig. LmaNT3 the uptake of at both pH In contrast, LmaNT4 not transport of 1 μm at pH the in Δlmant3-null that the host for expression of LmaNT4. LmaNT4 uptake at pH 5.5. LmaNT3 and LmaNT4 uptake to at pH 5.5, at at that import in amastigotes be by both nucleobase LmaNT3 and LmaNT4 are expressed in both promastigotes and amastigotes (7Ortiz D. Sanchez M.A. Pierce S. Herrmann T. Kimblin N. Archie Bouwer H.G. Landfear S.M. Mol. Microbiol. 2007; 64: 1228-1243Crossref PubMed Scopus (37) Google Scholar), implying that both permeases are likely to be expressed in amastigotes. measurements of permease in amastigotes would the of of uptake revealed that at high (Fig. both permeases the uptake of this at both pH Nonetheless, LmaNT4 transports the more LmaNT3 at pH 5.5, whereas both transporters exhibit of transport at pH To uptake by LmaNT4 of at pH 5.5, curves were obtained for promastigotes of L. the previously Δlmant4-null mutant in which both of the LmaNT4 have been by (7Ortiz D. Sanchez M.A. Pierce S. Herrmann T. Kimblin N. Archie Bouwer H.G. Landfear S.M. Mol. Microbiol. 2007; 64: 1228-1243Crossref PubMed Scopus (37) Google Scholar), and the complemented null in which the LmaNT4 open reading is expressed from an episomal expression vector (7Ortiz D. Sanchez M.A. Pierce S. Herrmann T. Kimblin N. Archie Bouwer H.G. Landfear S.M. Mol. Microbiol. 2007; 64: 1228-1243Crossref PubMed Scopus (37) Google Scholar). revealed in at pH 5.5 the for of the Δlmant4-null mutant was that for the or the complemented null mutant μm and In contrast, in was at pH for the with the Δlmant4-null that LmaNT4 a in uptake of at acidic pH, as to the expression of this To by in it was to as the purine (Fig. and to other purines from the as hypoxanthine or adenine, which have been to with in Leishmania species B. PubMed Scopus Google Scholar). For this curves were not in with the three used as would likely be abrogated by the of low of purine nucleobases within the host The and nucleoside transporters from Leishmania are that exhibit substrate-induced currents expressed in Xenopus oocytes A. G. Carter N.S. Ullman B. Landfear S.M. Kavanaugh M.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). To determine whether LmaNT3 and LmaNT4 might be and to potential as a function of extracellular pH, both nucleobase permeases were expressed in Xenopus Two-electrode voltage clamp experiments performed at pH 5.5 in the of 1 mm adenine using oocytes LmaNT3 (Fig. and LmaNT4 (Fig. revealed inward directed currents that increased with inward (Fig. and for both In contrast, permease exhibited currents monitored at pH (Fig. and or at pH 7.5 not currents were Furthermore, currents were application of the substrate adenine (Fig. and and were To determine whether the currents inward of oocytes were with the application of 1 mm adenine at pH 5.5, a fluorescence for oocytes LmaNT3 (Fig. or LmaNT4 (Fig. indicating acidification of the oocyte cytosol. At pH acidification was for These that inward of at pH 5.5, where substrate-induced currents are but not at pH where currents are To determine the of nucleobase oocytes LmaNT3 and LmaNT4 were at a membrane potential of and with 25 μm [3H]adenine or [3H]hypoxanthine for 5 min at pH 5.5. The substrate-induced over this was measurements, the same oocytes were and the of nucleobase was determined by of to nucleobase measured in oocytes in each case, revealed a for of ± for LmaNT3 and 7.4 ± for LmaNT4 and of ± for LmaNT3 and ± for LmaNT4. The principal of the studies is that the transport activity of the LmaNT4 nucleobase permease is activated at acidic pH. measured at 25 μm this transporter mediates the uptake of only adenine at neutral pH but acquires the ability to hypoxanthine, guanine, and xanthine as at pH (Fig. 1). The pH is at in to increased apparent affinity of LmaNT4 for nucleobases at acidic pH values with (Table 1). Thus, adenine transport by LmaNT4 was non-saturable to μm substrate at pH but exhibited a Km value of ∼40 μm at pH 5.5 (Fig. 2 and 1). uptake of hypoxanthine by LmaNT4 not saturate to 400 μm substrate at pH or but exhibited an Km value of μm at pH 5.5. In to LmaNT4, the nucleobase permease LmaNT3 optimal transport activity for all purine nucleobases at neutral pH (Fig. 1), and apparent affinity for substrates was not (Table 1). These observations a functional between the two nucleobase permeases of L. major and that LmaNT4 is optimized to function in the low pH environment of the phagolysosome to which the infectious amastigote are is with the (7Ortiz D. Sanchez M.A. Pierce S. Herrmann T. Kimblin N. Archie Bouwer H.G. Landfear S.M. Mol. Microbiol. 2007; 64: 1228-1243Crossref PubMed Scopus (37) Google Scholar) that deletion of the LmaNT4 genes the of amastigotes within murine macrophages, whereas deletion of the LmaNT3 genes does not amastigote viability. are data the of purine nucleobases within nucleobases and nucleosides in plasma and extracellular fluids are typically in the range of μm (10Traut T.W. Mol. Cell. Biochem. 1994; 140: 1-22Crossref PubMed Scopus (1270) Google Scholar). that amastigotes are probably exposed to purine nucleobases in the low micromolar range. the in Km values of LmaNT4 as a function of decreasing pH this permease a more for purines under the physiological conditions in which amastigotes likely In contrast, LmaNT4 probably a in purine uptake in promastigotes that reside in conditions to S. P. 1998; PubMed Scopus Google Scholar). is with the that LmaNT3 is the principal nucleobase transporter of promastigotes and that the Δlmant3-null mutant very low of purine nucleobase with the of residual adenine transport (7Ortiz D. Sanchez M.A. Pierce S. Herrmann T. Kimblin N. Archie Bouwer H.G. Landfear S.M. Mol. Microbiol. 2007; 64: 1228-1243Crossref PubMed Scopus (37) Google Scholar). is that the nucleoside transporters S. K. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) and K. H. S. L. J. R. PubMed Scopus Google Scholar, H. with Scholar), a at acidic pH, transport at pH 5.5 but not at pH been that the permease an in uptake under conditions in the where pH as low as Thus, at three members of the ENT family in as as protozoa and humans exhibit at acidic pH The of inward directed substrate-induced currents by LmaNT3 and LmaNT4 (Fig. that these permeases likely function as under low pH conditions. These currents increased with membrane as for Furthermore, application of substrate to oocytes these permeases in acidification of the indicating an inward of The measured of for could have different Thus, be of the that is coupled to nucleobase and that is substrate-induced but not a of for would a to purine nucleobases inside the were not coupled to as currents were by substrate in containing or in which was by not The currents by LmaNT3 and LmaNT4 were only at pH 5.5 and not at pH 7.5 or Thus, the transport of both permeases are These observations that is likely to for both permeases under the acidic conditions of the phagolysosome. In contrast, the high affinity transport by LmaNT3 and affinity transport by LmaNT4 at neutral pH to be from LmaNT3 function as a at low pH and as a transporter at neutral pH. In the same in to LmaNT4. the reduced transport of LmaNT4 at neutral pH and at physiological of nucleobases that transport by this permease not be of