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• W1988703123 abstract "Equilibrative nucleoside transporters play essential roles in nutrient uptake, cardiovascular and renal function, and purine analog drug chemotherapies. Limited structural information is available for this family of transporters; however, residues in transmembrane domains 1, 2, 4, and 5 appear to be important for ligand and inhibitor binding. In order to identify regions of the transporter that are important for ligand specificity, a genetic selection for mutants of the inosine-guanosine-specific Crithidia fasciculata nucleoside transporter 2 (CfNT2) that had gained the ability to transport adenosine was carried out in the yeast Saccharomyces cerevisiae. Nearly all positive clones from the genetic selection carried mutations at lysine 155 in transmembrane domain 4, highlighting lysine 155 as a pivotal residue governing the ligand specificity of CfNT2. Mutation of lysine 155 to asparagine conferred affinity for adenosine on the mutant transporter at the expense of inosine and guanosine affinity due to weakened contacts to the purine ring of the ligand. Following systematic cysteine-scanning mutagenesis, thiol-specific modification of several positions within transmembrane domain 4 was found to interfere with inosine transport capability, indicating that this helix lines the water-filled ligand translocation channel. Additionally, the pattern of modification of transmembrane domain 4 suggested that it may deviate from helicity in the vicinity of residue 155. Position 155 was also protected from modification in the presence of ligand, suggesting that lysine 155 is in or near the ligand binding site. Transmembrane domain 4 and particularly lysine 155 appear to play key roles in ligand discrimination and translocation by CfNT2. Equilibrative nucleoside transporters play essential roles in nutrient uptake, cardiovascular and renal function, and purine analog drug chemotherapies. Limited structural information is available for this family of transporters; however, residues in transmembrane domains 1, 2, 4, and 5 appear to be important for ligand and inhibitor binding. In order to identify regions of the transporter that are important for ligand specificity, a genetic selection for mutants of the inosine-guanosine-specific Crithidia fasciculata nucleoside transporter 2 (CfNT2) that had gained the ability to transport adenosine was carried out in the yeast Saccharomyces cerevisiae. Nearly all positive clones from the genetic selection carried mutations at lysine 155 in transmembrane domain 4, highlighting lysine 155 as a pivotal residue governing the ligand specificity of CfNT2. Mutation of lysine 155 to asparagine conferred affinity for adenosine on the mutant transporter at the expense of inosine and guanosine affinity due to weakened contacts to the purine ring of the ligand. Following systematic cysteine-scanning mutagenesis, thiol-specific modification of several positions within transmembrane domain 4 was found to interfere with inosine transport capability, indicating that this helix lines the water-filled ligand translocation channel. Additionally, the pattern of modification of transmembrane domain 4 suggested that it may deviate from helicity in the vicinity of residue 155. Position 155 was also protected from modification in the presence of ligand, suggesting that lysine 155 is in or near the ligand binding site. Transmembrane domain 4 and particularly lysine 155 appear to play key roles in ligand discrimination and translocation by CfNT2. Both hydrophilic nutrients and nutrient analog drugs gain access to target cells via membrane-spanning protein transporters. One family of such proteins, the equilibrative nucleoside transporters (ENTs 2The abbreviations used are: ENTequilibrative nucleoside transporterTMtransmembrane domainSCAMsubstituted cysteine accessibility methodSDsynthetic defined mediumMTSEA2-aminoethyl methanethiosulfonateMTSESsodium (2-sulfonatoethyl)methanethiosulfonateORFopen reading frameMITmyo-inositol transporterEC50effective concentration of drug inhibiting growth by 50%PBSphosphate-buffered salineHAhemagglutinin. ; SLC29), transports purine and pyrimidine nucleobases and nucleosides into eukaryotic cells (1.Hyde R.J. Cass C.E. Young J.D. Baldwin S.A. Mol. Membr. Biol. 2001; 18: 53-63Crossref PubMed Google Scholar). In parasitic protozoa, such as Leishmania, Plasmodium, and trypanosomes, ENTs serve an essential function, because purines cannot be synthesized de novo by these organisms and must be obtained from the environment (2.Downie M.J. Kirk K. Mamoun C.B. Eukaryot. Cell. 2008; 7: 1231-1237Crossref PubMed Scopus (88) Google Scholar). Mammalian ENTs are of particular interest in renal and cardiovascular function (adenosine transport (3.Elwi A.N. Damaraju V.L. Baldwin S.A. Young J.D. Sawyer M.B. Cass C.E. Biochem. Cell Biol. 2006; 84: 844-858Crossref PubMed Google Scholar, 4.Young J.D. Yao S.Y. Sun L. Cass C.E. Baldwin S.A. Xenobiotica. 2008; 38: 995-1021Crossref PubMed Scopus (159) Google Scholar)) and in the pharmacogenomics of nucleoside analog drug uptake in antiparasitic, antiviral, and anticancer chemotherapies (5.Molina-Arcas M. Trigueros-Motos L. Casado F.J. Pastor-Anglada M. Nucleosides Nucleotides Nucleic Acids. 2008; 27: 769-778Crossref PubMed Scopus (35) Google Scholar). Although many ENT genes and cDNAs from diverse organisms have been cloned and biochemically characterized in recent years, and insight into the structure of the ENT family is emerging from recent threading (6.Arastu-Kapur S. Arendt C.S. Purnat T. Carter N.S. Ullman B. J. Biol. Chem. 2005; 280: 2213-2219Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 7.Papageorgiou I. De Koning H.P. Soteriadou K. Diallinas G. Int. J. Parasitol. 2008; 38: 641-653Crossref PubMed Scopus (15) Google Scholar) and ab initio (8.Valdés R. Arastu-Kapur S. Landfear S.M. Shinde U. J. Biol. Chem. 2009; 284: 19067-19076Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) structural models, a detailed understanding of how ENTs recognize their ligands has remained elusive. equilibrative nucleoside transporter transmembrane domain substituted cysteine accessibility method synthetic defined medium 2-aminoethyl methanethiosulfonate sodium (2-sulfonatoethyl)methanethiosulfonate open reading frame myo-inositol transporter effective concentration of drug inhibiting growth by 50% phosphate-buffered saline hemagglutinin. All ENTs studied to date are predicted to be composed of 11 transmembrane-spanning segments (TMs), with a large loop between TM6 and -7 that, like the N terminus, is intracellular, whereas the C terminus projects extracellularly (Fig. 1) (9.Arastu-Kapur S. Ford E. Ullman B. Carter N.S. J. Biol. Chem. 2003; 278: 33327-33333Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 10.Sundaram M. Yao S.Y. Ingram J.C. Berry Z.A. Abidi F. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 2001; 276: 45270-45275Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). All extracellular loops except the first are believed to be quite short, suggesting that ligand discrimination and binding probably depend on amino acid residues within the TMs that project into a water-filled channel. An ab initio structural model of LdNT1.1 predicts that all TMs except TM3, TM6, and TM9 are arranged about this central ligand translocation channel (8.Valdés R. Arastu-Kapur S. Landfear S.M. Shinde U. J. Biol. Chem. 2009; 284: 19067-19076Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Additionally, mutational analysis by many groups indicates that TMs in the N-terminal half of ENTs (TM1, -2, -4, and -5) (Fig. 1) contribute to ligand affinity and specificity because mutations have been identified in these TMs that confer gain-of-function (increased affinity for particular ligands or inhibitors) or selective loss of affinity for a subset of ligands (Fig. 1). Many other point mutations have been described that lead to partial or complete loss of ENT function (8.Valdés R. Arastu-Kapur S. Landfear S.M. Shinde U. J. Biol. Chem. 2009; 284: 19067-19076Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 9.Arastu-Kapur S. Ford E. Ullman B. Carter N.S. J. Biol. Chem. 2003; 278: 33327-33333Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 11.Endres C.J. Unadkat J.D. Mol. Pharmacol. 2005; 67: 837-844Crossref PubMed Scopus (35) Google Scholar, 12.Galazka J. Carter N.S. Bekhouche S. Arastu-Kapur S. Ullman B. Int. J. Biochem. Cell Biol. 2006; 38: 1221-1229Crossref PubMed Scopus (11) Google Scholar, 13.SenGupta D.J. Lum P.Y. Lai Y. Shubochkina E. Bakken A.H. Schneider G. Unadkat J.D. Biochemistry. 2002; 41: 1512-1519Crossref PubMed Scopus (75) Google Scholar, 14.Visser F. Sun L. Damaraju V. Tackaberry T. Peng Y. Robins M.J. Baldwin S.A. Young J.D. Cass C.E. J. Biol. Chem. 2007; 282: 14148-14157Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 15.Valdés R. Liu W. Ullman B. Landfear S.M. J. Biol. Chem. 2006; 281: 22647-22655Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 16.SenGupta D.J. Unadkat J.D. Biochem. Pharmacol. 2004; 67: 453-458Crossref PubMed Scopus (43) Google Scholar). However, the mutated residues have not been shown to specifically affect ligand binding rather than transporter structure or conformational changes required for ligand translocation. The most intriguing candidate for a ligand binding residue to date is Lys153 in TM4 of LdNT1.1, which when mutated to arginine confers a novel inosine affinity on this adenosine transporter (15.Valdés R. Liu W. Ullman B. Landfear S.M. J. Biol. Chem. 2006; 281: 22647-22655Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). This residue was not shown to be located in the ligand binding site, however. Here we show that transmembrane domain 4 lines the ligand translocation channel and helps to define the ligand binding site of an equilibrative nucleoside transporter, valuable information in the structural characterization of this ubiquitous and pharmacologically important protein family. We have identified Lys155 in TM4 (see Fig. 1, black circle) of the Crithidia fasciculata inosine-guanosine transporter CfNT2 (17.Liu W. Arendt C.S. Gessford S.K. Ntaba D. Carter N.S. Ullman B. Mol. Biochem. Parasitol. 2005; 140: 1-12Crossref PubMed Scopus (10) Google Scholar) as a key residue influencing CfNT2 ligand specificity using an unbiased genetic selection for change-of-specificity mutations. Substitutions of asparagine and alanine at this position conferred adenosine transport capability on this transporter and reduced affinity for inosine and guanosine by weakening contacts between the transporter and the purine ring of the ligand, indicating a relaxing of specificity and a phenotype somewhat distinct from that of the orthologous LdNT1.1-K153R mutant (15.Valdés R. Liu W. Ullman B. Landfear S.M. J. Biol. Chem. 2006; 281: 22647-22655Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Cysteine-scanning mutagenesis and thiol-specific modification (SCAM) of CfNT2 TM4 established that this TM lines the ligand translocation channel, which is consistent with the ab initio ENT model (8.Valdés R. Arastu-Kapur S. Landfear S.M. Shinde U. J. Biol. Chem. 2009; 284: 19067-19076Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). SCAM also demonstrated that Lys155 is water-exposed, lies near the center of TM4 in CfNT2, and is protected from modification by the presence of substrate, intimating that it is located in or near the ligand binding site. Interestingly, the SCAM pattern of TM4 was not strictly helical between residues 152 and 156, opening up the possibility that TM4 is a “broken” helix that contacts ligand at this flexible linker, similar to some sodium-coupled transporters (SLC6), such as LeuT (18.Yamashita A. Singh S.K. Kawate T. Jin Y. Gouaux E. Nature. 2005; 437: 215-223Crossref PubMed Scopus (1351) Google Scholar). Escherichia coli DH5α and TOP10 strains were used throughout (Invitrogen), and standard methods for recombinant DNA work were employed (19.Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Wiley Interscience, New York2002Google Scholar). The purine auxotrophic Saccharomyces cerevisiae strain YPH499 (MATa ura3-52 lys2-801 ade2-101 trp1-Δ63 leu2Δ1) was constructed by Sikorski and Hieter (20.Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). Synthetic defined (SD) media were prepared according to standard methods. Microbiological media and phosphate-buffered saline (PBS) tablets were obtained from Fisher and MP Biomedicals (Irvine, CA). Yeast were transformed by either the rapid or the high efficiency lithium acetate method (21.Gietz R.D. Woods R.A. Methods Enzymol. 2002; 350: 87-96Crossref PubMed Scopus (2077) Google Scholar). The Leishmania donovani Δldnt1/Δldnt2 line, which lacks all purine nucleoside uptake capability (22.Liu W. Boitz J.M. Galazka J. Arendt C.S. Carter N.S. Ullman B. Mol. Biochem. Parasitol. 2006; 150: 300-307Crossref PubMed Scopus (27) Google Scholar), was used for expression and biochemical characterization of cfnt2 mutant proteins. Parasites were transfected with plasmid DNA according to the method of Robinson and Beverley (23.Robinson K.A. Beverley S.M. Mol. Biochem. Parasitol. 2003; 128: 217-228Crossref PubMed Scopus (224) Google Scholar). Parasites were maintained in modified M199 medium as described by Goyard et al. (24.Goyard S. Segawa H. Gordon J. Showalter M. Duncan R. Turco S.J. Beverley S.M. Mol. Biochem. Parasitol. 2003; 130: 31-42Crossref PubMed Scopus (146) Google Scholar) supplemented with 5% fetal bovine serum (Invitrogen), 50 μg/ml hygromycin (Roche Applied Science), 50 μg/ml phleomycin (RPI Research, Mt. Prospect, IL), and 25 μg/ml G418 (BioWhittaker, Walkersville, MD) at 26 °C using adenine as a purine source. Hygromycin and phleomycin were the drugs employed in the selection of L. donovani Δldnt1/Δldnt2 (22.Liu W. Boitz J.M. Galazka J. Arendt C.S. Carter N.S. Ullman B. Mol. Biochem. Parasitol. 2006; 150: 300-307Crossref PubMed Scopus (27) Google Scholar). Oligonucleotides were obtained from Invitrogen. 2-Aminoethyl methanethiosulfonate (MTSEA), sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES), and isoguanosine were purchased from Toronto Research Chemicals, Inc. (North York, Ontario, Canada), 7-deazaguanosine was from ChemGenes (Wilmington, MA), and 2′,3′-dideoxyinosine was from ICN. [3H]Adenosine (30 Ci/mmol) and [3H]inosine (15 Ci/mmol) were obtained from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Virtually all other chemicals were purchased from Sigma and were of the highest grade available. All expression of CfNT2 derivatives and CfNT1 in yeast was driven from the high copy pRS426-Cu yeast-E. coli shuttle vector (25.Labbé S. Thiele D.J. Methods Enzymol. 1999; 306: 145-153Crossref PubMed Scopus (109) Google Scholar), which allows copper-inducible expression of the inserted gene and contains a URA3-selectable marker. The construction of the pRS426-Cu-CfNT2 and pRS426-Cu-CfNT1 expression vectors is described by Liu et al. (17.Liu W. Arendt C.S. Gessford S.K. Ntaba D. Carter N.S. Ullman B. Mol. Biochem. Parasitol. 2005; 140: 1-12Crossref PubMed Scopus (10) Google Scholar). Leishmanial expression of CfNT2 and its cfnt2 mutant variants was from the pALTneo vector (26.Laban A. Tobin J.F. Curotto de Lafaille M.A. Wirth D.F. Nature. 1990; 343: 572-574Crossref PubMed Scopus (125) Google Scholar) with an N-terminal HA tag, pALTneo-HA, as described (27.Arendt C.S. Ri K. Yates P.A. Ullman B. Anal. Biochem. 2007; 365: 185-193Crossref PubMed Scopus (13) Google Scholar). The construction of the cysteineless cfnt2 gene (cfnt2ΔCys) is described by Arendt et al. (27.Arendt C.S. Ri K. Yates P.A. Ullman B. Anal. Biochem. 2007; 365: 185-193Crossref PubMed Scopus (13) Google Scholar). All point mutants were constructed by site-directed mutagenesis of pALTneo-HA-CfNT2 or pALTneo-HA-cfnt2ΔCys performed according to the QuikChange mutagenesis protocol (Stratagene, La Jolla, CA) with primer design based on the method of Zheng et al. (28.Zheng L. Baumann U. Reymond J.L. Nucleic Acids Res. 2004; 32: e115Crossref PubMed Scopus (840) Google Scholar). Libraries of mutant cfnt2 molecules carried in the pRS426-Cu vector were constructed by mutagenic PCR followed by in vivo recombination and reconstitution of the expression vector within the yeast strain YPH499. The open reading frames of CfNT2 and cfnt2-G86V were subcloned into the E. coli vector pBluescript II KS(+) to provide PCR templates devoid of yeast sequences. Mutagenic PCR was conducted using the Diversify PCR random mutagenesis kit (Clontech) under conditions that were expected to produce 2.0 base changes per open reading frame (ORF), using pBluescript-CfNT2 (first and second rounds) or pBluescript-cfnt2-G86V (second round) as template. Two independent reaction mixtures were pooled, precipitated with ethanol in the presence of Pellet Paint (Invitrogen), and resuspended in a small volume of TE (10 mm Tris-HCl, 1 mm EDTA, pH 8) buffer to a concentration of ∼0.25 mg/ml. Within the pRS426-Cu-CfNT2 yeast expression vector, BglII and SphI restriction sites were engineered into the CfNT2 ORF ∼30 bp from the 5′- and 3′-ends, respectively. Digestion with BglII and SphI followed by gel purification yielded linear vector DNA containing 30 bp of 5′ and 3′ CfNT2 sequence, enough to allow homologous recombination with mutagenized PCR products and formation of a circular expression vector following co-transformation into yeast. YPH499 cells were transformed by the high efficiency lithium acetate method (21.Gietz R.D. Woods R.A. Methods Enzymol. 2002; 350: 87-96Crossref PubMed Scopus (2077) Google Scholar) using 0.25–1.0 μg of PCR-derived mutagenesis library material to 1 μg of linear vector fragment. Cells were immediately plated on SD ura− ade−, 200 μm adenosine, 100 μm CuSO4 (first round of selection) or SD ura− ade−, 100 μm adenosine, 100 μm guanosine, 100 μm CuSO4 (second round of selection). An aliquot of cells was plated on SD ura− to allow quantitation of transformation yield. Initial positive clones were restreaked on the same medium originally used for the selection to verify colony formation. Testing of some positive clones on SD ade− medium showed that growth on adenosine-containing medium was not due to reversion of the ade2 locus. Additionally, plating on 5-fluoroorotic acid to select against the URA3-containing plasmid, followed by streaking on SD ade−, 200 μm adenosine, 100 μm CuSO4 revealed that growth on adenosine was dependent on the presence of the CfNT2 plasmid. pRS426-Cu-cfnt2 plasmids from promising clones were rescued by the smash and grab method (29.Hoffman C.S. Winston F. Gene. 1987; 57: 267-272Crossref PubMed Scopus (2043) Google Scholar), followed by transformation of electrocompetent E. coli. Clones were sequenced to identify mutations, and plasmids of interest were retransformed into YPH499 for further study. All data presented were obtained with retransformed yeast strains. Log phase parasites (∼1 × 107 cells/ml) were washed twice with ice-cold PBS plus 10 mm glucose, brought to a density of 4 × 108 cells/ml, and incubated on ice with 1 mg/ml EZ-Link Sulfo-NHS-LC-Biotin (sulfosuccinimidyl-6-(biotinamido)hexanoate; Pierce and Thermo Fisher Scientific) in PBS plus 10 mm glucose for 1 h with occasional mixing. Cells were then washed three times with ice-cold 50 mm glycine in PBS and lysed in 1 ml of lysis buffer (20 mm Tris (pH 8.0), 100 mm NaCl, 10% glycerol, 1% Nonidet P-40, and protease inhibitors) for 1 h on a rotator at 4 °C. After centrifugation for 10 min at 16,000 × g at 4 °C, the supernatant was collected and incubated with washed streptavidin-agarose beads (Pierce) for 1 h at 4 °C. Beads were washed three times with 1 ml of lysis buffer and resuspended in a small volume of 2× Laemmli loading buffer. After separation of proteins by 10% SDS-PAGE, transfer to a polyvinylidene fluoride membrane, and blocking in 5% nonfat dry milk in PBS plus 1% Tween (PBST), the membrane was probed with anti-HA monoclonal antibodies (16B12, Covance (Princeton, NJ)) and by goat anti-mouse IgG1-HRP (Roche Applied Science) in 1% milk, PBST, followed by chemiluminescent detection (Western Lightning-ECL, PerkinElmer Life Sciences). For detection of biotin-conjugated myo-inositol transporter (MIT) as a loading control, blots were reprobed in 1% milk, PBST with anti-L. donovani MIT polyclonal antiserum (a gift of Dr. S. Landfear, Oregon Health and Science University) and goat anti-rabbit horseradish peroxidase (Pierce). Densitometry was performed on developed films using AlphaEaseFC (version 4.0.0, Alpha Innotech Corp., San Leandro, CA). Relative intensities of HA-cfnt2 spots compared with the HA-CfNT2 spot on each film were estimated after correcting for differences in intensities of MIT (loading control). Log phase parasites (∼1 × 107 cells/ml) were washed three times in ice-cold PBS plus 10 mm glucose and resuspended in the same buffer at a density of 2–4 × 108/ml. Cells were allowed to warm to room temperature for 15 min, and uptake of [3H]inosine or [3H]adenosine was measured by the oil-stop method (30.Aronow B. Kaur K. McCartan K. Ullman B. Mol. Biochem. Parasitol. 1987; 22: 29-37Crossref PubMed Scopus (104) Google Scholar). Briefly, 100 μl of the cell suspension were mixed with 100 μl of a 2× solution of ligand above a 200-μl cushion composed of a 33:7 mixture of 550 oil (Dow Corning, Midland, MI) to light paraffin oil (catalog number O-119, Fisher). At the desired intervals, cells were centrifuged through the oil cushion at 16,000 × g for 1 min. Following freezing of the sample on dry ice/ethanol, the bottoms of the tubes were clipped into scintillation tubes, and the cells were solubilized in 3 ml of Cytoscint (MP Biomedicals). Scintillation counting was performed after 24–48 h of solubilization and thorough mixing of the contents. For Km determinations, linear rates of uptake for Δldnt1/Δldnt2 pALTneo and Δldnt1/Δldnt2 pALTneo-HA-cfnt2-K155N cells were determined in parallel at six concentrations of [3H]inosine or [3H]adenosine over 2 min. Rates of uptake by vector-transfected cells were subtracted from cfnt2 rates at each concentration, and the corrected rates were fitted to the Michaelis-Menten equation and plotted in Prism 4.0 (Graphpad Software, Inc., La Jolla, CA). For competition assays, duplicate tubes containing 5 μm [3H]inosine plus a 500 μm concentration of a potential inhibitor and buffer-matched duplicate control tubes without inhibitor were assayed in parallel at a single time point within the linear range of uptake by the oil-stop method (10 s for Δldnt1/Δldnt2 pALTneo-HA-CfNT2 and 60 s for Δldnt1/Δldnt2 pALTneo-HA-cfnt2-K155N). Samples containing 5 mm unlabeled inosine were used to determine transporter-independent background uptake. Following scintillation counting, corrected uptake levels of parallel samples with and without inhibitor were compared with determined percentage inhibition as follows, (uptake with inhibitor − transporter-independent uptake)/(uptake without inhibitor − transporter-independent uptake). Relative inosine and adenosine uptake rates by various cfnt2-K155 mutant transporters, as summarized in Table 1, were measured and analyzed as follows. Uptake of 5 μm [3H]inosine into Δldnt1/Δldnt2 pALTneo-HA-cfnt2-K155 cells was determined in duplicate over a 4-min time course for each cell line, and rates were determined (pmol/min/107 cells) by linear regression analysis in Prism 4.0. The rates of 5 μm [3H]inosine uptake in the presence of 1 mm unlabeled inosine by each cell line were measured in parallel and subtracted from the rate of uptake in the absence of cold competitor. This transporter-mediated uptake was compared qualitatively among cell lines. Adenosine uptake by cfnt2-K155 cells was low, and the rate of 5 μm [3H]adenosine uptake was not significantly inhibited by the presence of 1 mm unlabeled adenosine. Therefore, linear rates of 5 μm [3H]adenosine uptake over 5 min were measured in duplicate for each cfnt2-K155 cell line and compared with the rate of uptake by vector-transfected cells (consistently 0.2–0.3 pmol/min/107 cells). Those cell lines that consistently had a linear rate of adenosine uptake more than 2 times higher than that of the vector-transfected cells scored at least one “+” on the qualitative scale. Those with a variable response from experiment to experiment were scored as “+/−”, and those that failed to show significant uptake above the vector background were scored as “−”.TABLE 1Adenosine and inosine uptake and analog toxicity by a panel of cfnt2-K155 mutants in L. donovani Δldnt1/Δldnt2Cell lineAdenosine, uptakeaUptake of 5 μm [3H]adenosine relative to vector cells (set to “−”), n = 2.Inosine uptakebUptake of 5 μm [3H]inosine relative to uptake in the presence of 1 mm unlabeled inosine, n = 2.Tubercidin EC50cEC50 values are expressed in μm (n = 3–5).Formycin B EC50cEC50 values are expressed in μm (n = 3–5).Vector−−89.1 ± 1318.9 ± 4.9CfNT1++++−0.0139 ± 0.00461.18 ± 0.14CfNT2−++++3.23 ± 1.20.0163 ± 0.0064cfnt2-K155N+++0.733 ± 0.230.363 ± 0.11cfnt2-K155A+++0.610 ± 0.200.0485 ± 0.018cfnt2-K155Q+/−+0.773 ± 0.300.0828 ± 0.032cfnt2-K155R++++5.74 ± 1.30.185 ± 0.085cfnt2-K155T−+1.62 ± 0.610.102 ± 0.037cfnt2-K155E−−1.90 ± 0.410.322 ± 0.11cfnt2-K155M−−3.01 ± 1.20.102 ± 0.043cfnt2-K155Y−−6.67 ± 0.912.41 ± 1.0cfnt2-K155L−−110. ± 1633.6 ± 5.4a Uptake of 5 μm [3H]adenosine relative to vector cells (set to “−”), n = 2.b Uptake of 5 μm [3H]inosine relative to uptake in the presence of 1 mm unlabeled inosine, n = 2.c EC50 values are expressed in μm (n = 3–5). Open table in a new tab For SCAM assays, aliquots of cells were treated at room temperature with 2.5 mm MTSEA or 10 mm MTSES in PBS plus 10 mm glucose for 10 min and used immediately for uptake experiments as described above. Triplicate measurements of uptake of 5 μm [3H]inosine were taken at a single time point within the linear range of uptake (1.5–5 min, depending on the particular mutant). Background uptake of 5 μm [3H]inosine by untreated cells in the presence of 5 mm unlabeled inosine was subtracted from all other values. In experiments in which protection by ligand was examined, experimental samples were supplemented with 1 mm inosine (from a 50 mm stock in PBS) 2 min prior to MTSES or MTSEA treatment. After 10 min of incubation, unreacted reagent and unlabeled ligand were removed by three washes with 1.5 ml of ice-cold PBS plus 10 mm glucose. Cell density was adjusted to 3–4 × 108/ml, and uptake of 1 μm [3H]inosine and 1 μm [3H]inosine plus 1 m unlabeled inosine was measured in triplicate at a single time point within the linear range of uptake as described above. In a 96-well plate, cells at 1 × 105 cells/ml were mixed with tubercidin or formycin B that had been diluted 2-fold over 11–15 wells. After ∼6 days of growth at 26 °C, Alamar Blue (Invitrogen) was added, and absorbance at 570 and 600 nm was measured at several time points. The percentage reduction of Alamar Blue was calculated as recommended by the manufacturer, and the time point that gave ∼100% reduction at low concentrations of drug (between 1.5 and 4.5 h) was used for calculations. The effective concentration of drug inhibiting growth by 50% (EC50) was determined by fitting the log-plotted data to a sigmoidal dose-response curve in Prism 4.0. Chemical modeling was performed with ADF® version 2008.01 from Scientific Computing and Modeling (Amsterdam) (31.te Velde G. Bickelhaupt F.M. Baerends E.J. Fonseca Guerra C. van Gisbergen S.J. Snijders J.G. Ziegler T. J. Comput. Chem. 2001; 22: 931-967Crossref Scopus (8227) Google Scholar, 32.Fonseca Guerra C. Snijders J.G. te Velde G. Baerends E.J. Theor. Chem. Acc. 1998; 99: 391-403Google Scholar) on a Mac Pro 8 core computer. Molecular geometries were optimized using density functional theory with a BLYP exchange correlation functional (33.Becke A.D. Phys. Rev. A. 1988; 38: 3098-3100Crossref PubMed Scopus (45332) Google Scholar, 34.Lee C. Yang W. Parr R.G. Phys. Rev. B Condens. Matter. 1988; 37: 785-789Crossref PubMed Scopus (87058) Google Scholar), triple-zeta single polarization atomic basis, and COSMO (35.Klamt A. J. Phys. Chem. 1995; 99: 2224-2235Crossref Scopus (3005) Google Scholar) simulation of a water solvent. Bader atomic charge analysis (36.Rodriguez J.I. Bader R.F. Ayers P.W. Michel C. Gotz A.W. Bo C. Chem. Phys. Lett. 2009; 472: 149-152Crossref Scopus (124) Google Scholar, 37.Rodríguez J.I. Köster A.M. Ayers P.W. Santos-Valle A. Vela A. Merino G. J. Comput. Chem. 2009; 30: 1082-1092Crossref PubMed Scopus (59) Google Scholar) was then performed using B3LYP hybrid exchange correlation (38.Becke A.D. J. Chem. Phys. 1993; 98: 5648Crossref Scopus (89888) Google Scholar), triple-zeta double polarization atomic basis, and COSMO simulation of water. Mutation of ligand-binding residues can result in loss of ligand affinity and therefore loss of transporter function; however, many other types of mutations can also give a loss-of-function phenotype. Therefore, a selection for rare mutants that led to a gain of function, namely a change in ligand specificity, was used to identify regions of the C. fasciculata inosine/guanosine transporter CfNT2 that were specifically required for ligand discrimination rather than ligand translocation and/or proper protein folding. The genetic selection was performed using the yeast S. cerevisiae, an organism that lacks endogenous purine nucleoside transport activity (6.Arastu-Kapur S. Arendt C.S. Purnat T. Carter N.S. Ullman B. J. Biol. Chem. 2005; 280: 2213-2219Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 39.Mäser P." @default.
• W1988703123 created "2016-06-24" @default.
• W1988703123 creator A5034619742 @default.
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• W1988703123 title "Role of Transmembrane Domain 4 in Ligand Permeation by Crithidia fasciculata Equilibrative Nucleoside Transporter 2 (CfNT2)" @default.
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