Mutation of Residue 33 of Human Equilibrative Nucleoside Transporters 1 and 2 Alters Sensitivity to Inhibition of Transport by Dilazep and Dipyridamole
2002; Elsevier BV; Volume: 277; Issue: 1 Linguagem: Inglês
10.1074/jbc.m105324200
ISSN1083-351X
AutoresFrank Visser, Mark F. Vickers, Amy M.L. Ng, Stephen A. Baldwin, James D. Young, Carol E. Cass,
Tópico(s)HIV/AIDS drug development and treatment
ResumoHuman equilibrative nucleoside transporters (hENT) 1 and 2 differ in that hENT1 is inhibited by nanomolar concentrations of dipyridamole and dilazep, whereas hENT2 is 2 and 3 orders of magnitude less sensitive, respectively. When a yeast expression plasmid containing the hENT1 cDNA was randomly mutated and screened by phenotypic complementation in Saccharomyces cerevisiae to identify mutants with reduced sensitivity to dilazep, clones with a point mutation that converted Met33to Ile (hENT1-M33I) were obtained. Characterization of the mutant protein in S. cerevisiae and Xenopus laevisoocytes revealed that the mutant had less than one-tenth the sensitivity to dilazep and dipyridamole than wild type hENT1, with no change in nitrobenzylmercaptopurine ribonucleoside (NBMPR) sensitivity or apparent uridine affinity. To determine whether the reciprocal mutation in hENT2 (Ile33 to Met) also altered sensitivity to dilazep and dipyridamole, hENT2-I33M was created by site-directed mutagenesis. Although the resulting mutant (hENT2-I33M) displayed >10-fold higher dilazep and dipyridamole sensitivity and >8-fold higher uridine affinity compared with wild type hENT2, it retained insensitivity to NBMPR. These data established that mutation of residue 33 (Met versus Ile) of hENT1 and hENT2 altered the dilazep and dipyridamole sensitivities in both proteins, suggesting that a common region of inhibitor interaction has been identified. Human equilibrative nucleoside transporters (hENT) 1 and 2 differ in that hENT1 is inhibited by nanomolar concentrations of dipyridamole and dilazep, whereas hENT2 is 2 and 3 orders of magnitude less sensitive, respectively. When a yeast expression plasmid containing the hENT1 cDNA was randomly mutated and screened by phenotypic complementation in Saccharomyces cerevisiae to identify mutants with reduced sensitivity to dilazep, clones with a point mutation that converted Met33to Ile (hENT1-M33I) were obtained. Characterization of the mutant protein in S. cerevisiae and Xenopus laevisoocytes revealed that the mutant had less than one-tenth the sensitivity to dilazep and dipyridamole than wild type hENT1, with no change in nitrobenzylmercaptopurine ribonucleoside (NBMPR) sensitivity or apparent uridine affinity. To determine whether the reciprocal mutation in hENT2 (Ile33 to Met) also altered sensitivity to dilazep and dipyridamole, hENT2-I33M was created by site-directed mutagenesis. Although the resulting mutant (hENT2-I33M) displayed >10-fold higher dilazep and dipyridamole sensitivity and >8-fold higher uridine affinity compared with wild type hENT2, it retained insensitivity to NBMPR. These data established that mutation of residue 33 (Met versus Ile) of hENT1 and hENT2 altered the dilazep and dipyridamole sensitivities in both proteins, suggesting that a common region of inhibitor interaction has been identified. concentrative nucleoside transporter equilibrative nucleoside transporter nitrobenzylmercaptopurine ribonucleoside (6-[(4-nitrobenzyl)thiol]-9-β-d-ribofuranosyl purine) transmembrane human mouse rat complete minimal medium glucose methotrexate phosphoglycerate kinase sulfanilamide Cellular uptake and release of nucleosides and nucleoside analog drugs is mediated by integral membrane nucleoside transporter proteins (1Vickers M.F. Young J.D. Baldwin S.A. Cass C.E. Emerging Therapeutic Targets. 2000; 4: 515-539Crossref Scopus (14) Google Scholar, 2Cass C.E. Young J.D. Baldwin S.A. Cabrita M.A. Graham K.A. Griffiths M. Jennings L.L. Mackey J.R. Ng A.M.L. Ritzel M.W.L. Vickers M.F. Yao S.Y.M. Amidon G.L. Sadee W. Membrane Transporters as Drug Targets. 12. Kluwer Academic/Plenum Publishers, 1999: 313-352Google Scholar, 3Griffith D.A. Jarvis S.M. Biochim. Biophys. Acta. 1996; 1286: 153-181Crossref PubMed Scopus (454) Google Scholar, 4Baldwin S.A. Mackey J.R. Cass C.E. Young J.D. Mol. Med. Today. 1999; 5: 216-224Abstract Full Text PDF PubMed Scopus (305) Google Scholar). These proteins are involved in salvage of extracellular nucleosides for nucleotide biosynthesis in mammalian cells, especially those that lack de novo synthesis pathways such as enterocytes and hemopoietic cells. They are critical for the cellular uptake of cytotoxic nucleoside analogs used in the treatment of human hematologic malignancies, solid tumors, and viral diseases (5Cass C.E. Georgopapadakou N.H. Drug Transport in Antimicrobial and Anticancer Chemotherapy. Marcel Deker, New York1995: 403-451Google Scholar, 6Mackey J.R. Baldwin S.A. Young J.D. Cass C.E. Drug Resistance Updates. 1998; 1: 310-324Crossref PubMed Scopus (141) Google Scholar). Nucleoside transporters also affect the cell surface concentration of adenosine, which is a signaling molecule that binds to G protein-coupled cell surface adenosine receptors, affecting physiological processes such as coronary vasodilation, renal vasoconstriction, neuromodulation, platelet aggregation, and lipolysis (7Jennings L.L. Cass C.E. Ritzel M.W.L. Yao S.Y.M. Young J.D. Griffiths M. Baldwin S.A. Drug Dev. Res. 1998; 45: 277-287Crossref Scopus (14) Google Scholar, 8Belardinelli L. Linden J. Berne R.M. Prog. Cardiovasc. Dis. 1989; 32: 73-97Crossref PubMed Scopus (723) Google Scholar). Mammalian nucleoside transporters are classified into two structurally and functionally distinct families: the concentrative nucleoside transporters (CNTs)1 and the equilibrative nucleoside transporters (ENTs). CNTs mediate Na+-dependent transport against the nucleoside concentration gradient and are found primarily in specialized cells such as intestinal and renal epithelia. Three CNT isoforms, a pyrimidine-nucleoside preferring (CNT1), a purine-nucleoside and uridine preferring (CNT2), and a broadly selective (CNT3) protein, have been identified by molecular cloning from mammalian tissues (9Ritzel M.W. Yao S.Y. Huang M.Y. Elliott J.F. Cass C.E. Young J.D. Am. J. Physiol. 1997; 272: C707-C714Crossref PubMed Google Scholar, 10Ritzel M.W. Yao S.Y. Ng A.M. Mackey J.R. Cass C.E. Young J.D. Mol. Membr. Biol. 1998; 15: 203-211Crossref PubMed Scopus (179) Google Scholar, 11Ritzel M.W.L. Ng A.M. Yao S.Y.M. Graham K. Loewen S.K. Smith K.M. Ritzel R.G. Mowles D.A. Carpenter P. Chen X. Karpinski E. Hyde R.J. Baldwin S.A. Cass C.E. Young J.D. J. Biol. Chem. 2001; 276: 2914-2927Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 12Yao S.Y. Ng A.M. Ritzel M.W. Gati W.P. Cass C.E. Young J.D. Mol. Pharmacol. 1996; 50: 1529-1535PubMed Google Scholar, 13Che M. Ortiz D.F. Arias I.M. J. Biol. Chem. 1995; 270: 13596-13599Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 14Huang Q.Q. Harvey C.M. Paterson A.R. Cass C.E. Young J.D. J. Biol. Chem. 1993; 268: 20613-20619Abstract Full Text PDF PubMed Google Scholar). Mammalian ENTs are responsible for facilitated diffusion of nucleosides across cell membranes and have a broad tissue distribution. Two ENT isoforms have been identified by molecular cloning and functional expression from mammalian tissues and mediate nucleoside transport processes that are functionally distinguished by their differential sensitivity to inhibition by NBMPR (1Vickers M.F. Young J.D. Baldwin S.A. Cass C.E. Emerging Therapeutic Targets. 2000; 4: 515-539Crossref Scopus (14) Google Scholar, 2Cass C.E. Young J.D. Baldwin S.A. Cabrita M.A. Graham K.A. Griffiths M. Jennings L.L. Mackey J.R. Ng A.M.L. Ritzel M.W.L. Vickers M.F. Yao S.Y.M. Amidon G.L. Sadee W. Membrane Transporters as Drug Targets. 12. Kluwer Academic/Plenum Publishers, 1999: 313-352Google Scholar, 3Griffith D.A. Jarvis S.M. Biochim. Biophys. Acta. 1996; 1286: 153-181Crossref PubMed Scopus (454) Google Scholar, 4Baldwin S.A. Mackey J.R. Cass C.E. Young J.D. Mol. Med. Today. 1999; 5: 216-224Abstract Full Text PDF PubMed Scopus (305) Google Scholar). NBMPR-sensitive nucleoside transport processes that bind NBMPR with high affinity, (Kd = 0.1–1 nm), have been assigned the functional designation es (equilibrativesensitive) and are mediated by ENT1 proteins. NBMPR-insensitive nucleoside transport processes are resistant to inhibition by micromolar concentrations of NBMPR, are functionally designated as ei (equilibrativeinsensitive), and are mediated by ENT2 proteins. ENTs are pharmacological targets for the coronary vasodilators dilazep, dipyridamole, and draflazine, which have been shown to inhibit transport and NBMPR binding (3Griffith D.A. Jarvis S.M. Biochim. Biophys. Acta. 1996; 1286: 153-181Crossref PubMed Scopus (454) Google Scholar, 15Van Belle H. Cardiovasc. Res. 1993; 27: 68-76Crossref PubMed Scopus (114) Google Scholar, 16Hammond J.R. Nauyn. Schmiedebergs Arch. Pharmacol. 2000; 361: 373-382Crossref PubMed Scopus (48) Google Scholar, 17Jarvis S.M. Mol. Pharmacol. 1986; 30: 659-665PubMed Google Scholar). Adenosine interacts with G protein-coupled cell surface receptors of endothelial and smooth muscle cells to induce vasodilation. Transporter-mediated adenosine uptake is the major means by which this interaction is terminated, a mechanism that is blocked by coronary vasodilator binding to the human ENT isoforms hENT1 and hENT2 (7Jennings L.L. Cass C.E. Ritzel M.W.L. Yao S.Y.M. Young J.D. Griffiths M. Baldwin S.A. Drug Dev. Res. 1998; 45: 277-287Crossref Scopus (14) Google Scholar). hENT2 shares 50% amino acid identity with hENT1 and is 2 and 3 orders of magnitude less sensitive, respectively, to inhibition by dipyridamole and dilazep than hENT1, whereas both rat isoforms (rENT1 and rENT2) are completely insensitive to these inhibitors (18Yao S.Y. Ng A.M. Muzyka W.R. Griffiths M. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 1997; 272: 28423-28430Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 19Ward J.L. Sherali A. Mo Z.P. Tse C.M. J. Biol. Chem. 2000; 275: 8375-8381Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). Human and rat ENT1 and ENT2 proteins share a common membrane architecture, recently confirmed by hydropathy analysis and glycosylation-scanning mutagenesis (20Sundaram M. Yao S.Y.M. 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 (125) Google Scholar), with 11 transmembrane (TM) segments, a large glycosylated loop between TM segments 1 and 2, and a large intracellular loop between TM segments 6 and 7. In a previous study, chimeric recombinant proteins were created between hENT1 and rENT1 to identify the structural domains of hENT1 that are responsible for interaction with dilazep and dipyridamole (21Sundaram M. Yao S.Y.M. Ng A.M.L. Griffiths M. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 1998; 273: 21519-21525Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The inhibitor sensitivities of the chimeras suggested that TM segments 3–6 contain the major site(s) of interaction with secondary contributions from TM segments 1–2, providing the first insight into the regions of hENT1 that are important for interaction with dilazep and dipyridamole. The individual amino acid residues responsible for interaction with dilazep and dipyridamole have not yet been identified. The goal of the current study was to identify amino acid residues involved in dilazep and dipyridamole interaction by using a phenotypic complementation assay to screen a library of randomly mutated yeast expression plasmids containing the hENT1 cDNA (pYPhENT1) for functional thymidine transport-competent mutants with reduced sensitivity to dilazep. The complementation assay is based on the ability of recombinant hENT1 produced in Saccharomyces cerevisiae to import thymidine under conditions of dTMP starvation, thereby allowing growth, which is inhibited by the addition of dilazep to the assay medium (22Hogue D.L. Ellison M.J. Young J.D. Cass C.E. J. Biol. Chem. 1996; 271: 9801-9808Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 23Vickers M.F. Mani R.S. Sundaram M. Hogue D.L. Young J.D. Baldwin S.A. Cass C.E. Biochem. J. 1999; 339: 21-32Crossref PubMed Google Scholar, 24Hogue D.L. Ellison M.J. Vickers M. Cass C.E. Biochem. Biophys. Res. Commun. 1997; 238: 811-816Crossref PubMed Scopus (9) Google Scholar). hENT1 cDNAs were isolated from the resulting mutant clones and sequenced, revealing a mutation in codon 33 that converted Met33 to Ile (M33I). When mutant and wild type recombinant hENT1 were produced in S. cerevisiae and Xenopus laevis oocytes to quantitate dilazep and dipyridamole sensitivity, a significant decrease in sensitivity was observed for the mutated protein. The corresponding residue in hENT2 (Ile33) was therefore converted to a Met by site-directed mutagenesis, and the sensitivity of the resulting mutant to dilazep and dipyridamole was assessed. The results suggested that residue 33 in the first TM segment (Met versus Ile) contributes importantly to the ability of dilazep and dipyridamole to interact with hENT1 and hENT2. KY114 (MATα, gal, ura3-52, trp1, lys2, ade2, hisd2000) was the parental yeast strain used to generate KTK, which produces recombinant Herpes simplexthymidine kinase (22Hogue D.L. Ellison M.J. Young J.D. Cass C.E. J. Biol. Chem. 1996; 271: 9801-9808Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), and fui1::TRP1, which contains a disruption in the gene encoding the endogenous uridine permease (FUI1) (25Vickers M.F. Yao S.Y. Baldwin S.A. Young J.D. Cass C.E. J. Biol. Chem. 2000; 275: 25931-25938Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Other strains were generated by transformation of the yeast/Escherichia coli shuttle vector pYPGE15 (26Brunelli J.P. Pall M.L. Yeast. 1993; 9: 1309-1318Crossref PubMed Scopus (95) Google Scholar) into KTK and fui1::TRP1 using a standard lithium acetate method (27Gietz D. St. Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 201425Crossref PubMed Scopus (2930) Google Scholar). cDNA inserts were under the transcriptional control of the constitutive PGK promoter. Yeast strains were maintained in complete minimal medium (CMM) containing 0.67% yeast nitrogen base (Difco, Detroit, MI), amino acids (as required to maintain auxotrophic selection), and 2% glucose (CMM/GLU). Agar plates contained CMM with various supplements and 2% agar (Difco). Plasmids were propagated inE. coli strain TOP10F′ (Invitrogen) and maintained in Luria broth with ampicillin (100 μg/ml). ForS. cerevisiae expression, the hENT1 open reading frame was amplified by PCR methodology using the primers (restriction sites underlined) 5′-XbaIes (5′-CCC TCT AGA ATG ACA ACC AGT CAC CAG CCT C-3′) and 3′-KpnIes (5′-CCC GGT ACC TCA CAC AAT TGC CCG GAA CAG G-3′) and inserted into the yeast expression vector pYPGE15 to generate pYPhENT1. For X. laevis oocytes expression, the hENT1-M33I cDNA was cloned into pBluescript II KS (+) (Stratagene) to generate pKS (+)-hENT1-M33I as previously described for the generation of pKS (+)-hENT1 (21Sundaram M. Yao S.Y.M. Ng A.M.L. Griffiths M. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 1998; 273: 21519-21525Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 28Griffiths M. Beaumont N. Yao S.Y. Sundaram M. Boumah C.E. Davies A. Kwong F.Y. Coe I. Cass C.E. Young J.D. Baldwin S.A. Nat. Med. 1997; 3: 89-93Crossref PubMed Scopus (361) Google Scholar). The hENT2 open reading frame was amplified by PCR using the primers (restriction sites underlined) 5′-XbaIei(5′-CCC TCT AGA ATG GCC CGA GGA GAC GCC-3′) and 3′-KpnIei (5′-CCC GGT ACC TCA GAG CAG CGC CTT GAA G-3′) and inserted into pYPGE15 to generate pYPhENT2. The hENT2 point mutant resulting in the I33M change in amino acid sequence was generated using megaprimer PCR methodology (29Sarkar G. Sommer S.S. BioTechniques. 1990; 8: 404-407PubMed Google Scholar). All reactions were performed using Pwo polymerase (Roche Molecular Biochemicals), and the resulting PCR products were verified by DNA sequencing using an ABI PRISM 310 sequence detection system (PerkinElmer Life Sciences). Double-stranded plasmid DNA (10 μg) was precipitated with ethanol/sodium acetate and resuspended in 500 μl of freshly prepared hydroxylamine solution (90 mg of NaOH, 350 mg of hydroxylamine HCl, pH ∼6.5, in 5 ml of H2O). The DNA was incubated for 16 h at 37 °C, and the reactions were terminated by the addition of 15 μl of 4 m NaCl, 50 μl of 1 mg/ml bovine serum albumin followed by precipitation with 1 ml of 95% ethanol. The DNA was resuspended in 100 μl of TE buffer (10 mm Tris, 1 mm EDTA, pH 8.0) and precipitated again with 15 μl of 4.0 m NaCl, 250 μl of 95% ethanol. The resuspension-precipitation procedure was repeated three times in total with a final resuspension in 20 μl of TE buffer. The complementation assay was based on the ability of recombinant hENT1 produced in yeast to salvage exogenously supplied thymidine under conditions of dTMP starvation (23Vickers M.F. Mani R.S. Sundaram M. Hogue D.L. Young J.D. Baldwin S.A. Cass C.E. Biochem. J. 1999; 339: 21-32Crossref PubMed Google Scholar). In brief, KTK cells transformed with pYPhENT1 using a lithium acetate procedure (27Gietz D. St. Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 201425Crossref PubMed Scopus (2930) Google Scholar) were plated directly onto CMM/GLU plates containing methotrexate (MTX) at 50 μg/ml and sulfanilamide (SAA) at 6 mg/ml (CMM/GLU/MTX/SAA). Colonies formed with an efficiency of ∼105 transformants/μg of DNA after incubation at 30 °C for 3.5 days in the presence of 10 μm thymidine, and complementation was prevented when 10 μm dilazep was also present. Hydroxylamine-treated pYPhENT1 (20 μg) was transformed into KTK cells, which were then plated onto CMM/GLU/MTX/SAA with 10 μm thymidine and 10 μm dilazep and incubated at 30 °C for 3.5 days. Colonies with apparent resistance to dilazep inhibition of complementation were isolated, grown in 5 ml of liquid CMM/GLU for 2 days, and restreaked onto CMM/GLU/MTX/SAA plates with 10 μm thymidine and 10 μm dilazep. The mutant hENT1 cDNAs were amplified from the yeast colonies by PCR, subcloned back into nonmutated pYPGE15, and sequenced. The plasmids pYPhENT1, pYPhENT1-M33I, pYPhENT2, and pYPhENT2-I33M were transformed into fui1::TRP1 yeast, a strain that lacks the endogenous uridine permease FUI1 (25Vickers M.F. Yao S.Y. Baldwin S.A. Young J.D. Cass C.E. J. Biol. Chem. 2000; 275: 25931-25938Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The transport of [3H]uridine (Moravek Biochemicals, Brea, CA) by logarithmically proliferating yeast was measured as described previously using the “oil stop” method (30Harley E.R. Paterson A.R. Cass C.E. Cancer Res. 1982; 42: 1289-1295PubMed Google Scholar,31Hogue D.L. Hodgson K.C. Cass C.E. Biochem. Cell Biol. 1990; 68: 199-209Crossref PubMed Scopus (18) Google Scholar) with the following modifications. Yeast were grown in CMM/GLU to anA600 of 0.7–1.5, washed once with fresh medium, and resuspended to an A600 of 2.0 in fresh medium. All transport assays were performed at room temperature and pH 7.0. 1-ml portions of yeast culture were distributed into 15-ml plastic centrifuge tubes to which 5–10-μl portions of stock dilazep, dipyridamole, or NBMPR (Sigma) solution or solvent alone (H2O, ethanol, or dimethyl sulfoxide) were added to achieve the desired final concentration. To allow for steady-state equilibration, the yeast were incubated in the presence of inhibitor for 30 min before addition of radiolabeled permeant (32Morrison J.F. Walsh C.T. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 201-301PubMed Google Scholar, 33Gati W.P. Paterson A.R.P. Mol. Pharmacol. 1989; 36: 134-141PubMed Google Scholar, 34Shi M. Young J.D. Biochem. J. 1986; 240: 879-883Crossref PubMed Scopus (17) Google Scholar, 35Jarvis S.M. Janmohamed S.N. Young J.D. Biochem. J. 1983; 216: 661-667Crossref PubMed Scopus (40) Google Scholar). Transport reactions were initiated by the rapid addition of a small volume of [3H]uridine to a final concentration of 2 μm. Transport reactions were terminated at graded time intervals by pipetting triplicate 200-μl portions of yeast suspension into 1.5-ml microcentrifuge tubes containing 200 μl of transport oil; the tubes were immediately centrifuged at 12,000 × gfor 2 min. The supernatants were removed by aspiration, the resulting pellets were solubilized with 5% Triton X-100 for 24 h, and the radioactive content was determined by liquid scintillation counting. In vitro synthesized transcripts were prepared from pKS(+)-hENT1 and pKS(+)-hENT1-M33I (SP6 MEGAscript Kit Ambion, Austin, TX) in water and injected into isolated mature stage VI oocytes from X. laevis as described previously (14Huang Q.Q. Harvey C.M. Paterson A.R. Cass C.E. Young J.D. J. Biol. Chem. 1993; 268: 20613-20619Abstract Full Text PDF PubMed Google Scholar). Mock-injected oocytes were injected with water alone. Transport assays were performed as described previously (21Sundaram M. Yao S.Y.M. Ng A.M.L. Griffiths M. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 1998; 273: 21519-21525Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 28Griffiths M. Beaumont N. Yao S.Y. Sundaram M. Boumah C.E. Davies A. Kwong F.Y. Coe I. Cass C.E. Young J.D. Baldwin S.A. Nat. Med. 1997; 3: 89-93Crossref PubMed Scopus (361) Google Scholar) on groups of 10 oocytes at 20 °C using [14C]uridine (Amersham Life Biosciences) (1 μCi/ml) in 200 μl of transport buffer containing 100 mmNaCl, 2 mm KCl, 1 mm CaCl2, 1 mm MgCl2, and 10 mm HEPES, pH 7.5. The initial rates of uridine uptake (10 μm) were determined using incubation periods of 5 min. Time courses for influx of [3H]uridine were measured into fui1::TRP1, a uridine transport-defective strain of yeast (25Vickers M.F. Yao S.Y. Baldwin S.A. Young J.D. Cass C.E. J. Biol. Chem. 2000; 275: 25931-25938Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), that contained pYPhENT1 or pYPhENT2 to determine incubation times that provided significant signal-to-noise ratios while also maintaining the initial rates of uptake (Fig.1). The time course for nonmediated uridine influx was obtained by assessing uridine uptake into pYPGE15-containing yeast and yielded a rate of 0.11 ± 0.01 pmol/mg protein/s. Time courses for uridine uptake into pYPhENT1- and pYPhENT2-containing yeast for the first 10 s (Fig. 1,inset) gave rates of 1.03 ± 0.40 and 1.63 ± 0.45 pmol/mg protein/s, respectively. Uptake time courses over 40 min were linear for both pYPhENT1- and pYPhENT2-containing yeast and yielded rates, respectively, of 0.93 ± 0.02 and 1.4 ± 0.02 pmol/mg protein/s. Uptake rates over the first 10 s were not different from the rates calculated from 40-min time courses, indicating that initial rates representing uridine transport were maintained over long periods of time. The extended linear time courses were likely due to efficient substrate “trapping” by conversion of uridine to UMP by uridine kinase, thereby minimizing backflow of [3H]uridine from the small intracellular compartment to the much larger extracellular volume. Uridine transport rates were determined for all subsequent experiments using incubation times of 10 or 20 min. MTX and SAA prevent the conversion of dUMP to dTMP by yeast thymidylate synthase and thus cause depletion of intracellular dTMP pools and inhibition of growth (22Hogue D.L. Ellison M.J. Young J.D. Cass C.E. J. Biol. Chem. 1996; 271: 9801-9808Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). KTK yeast producing recombinant hENT1 and H. simplexthymidine kinase can salvage thymidine via transporter-mediated uptake when low concentrations (e.g. 10 μm) are present in the growth medium, thereby allowing yeast to circumvent MTX/SAA-imposed growth arrest. Because thymidine salvage can be blocked by the inclusion of 10 μm dilazep in the complementation growth medium (23Vickers M.F. Mani R.S. Sundaram M. Hogue D.L. Young J.D. Baldwin S.A. Cass C.E. Biochem. J. 1999; 339: 21-32Crossref PubMed Google Scholar), this inhibition of thymidine rescue was used to screen a hENT1 random mutant library for functional proteins with reduced affinity for dilazep. pYPhENT1 was treated in vitrowith the mutagen hydroxylamine, transformed into KTK yeast, and screened for dilazep resistance. Dilazep-resistant yeast colonies were isolated, and the hENT1 cDNA was amplified and subcloned into nonmutated pYPGE15. Twenty-one resistant mutant cDNA clones were sequenced and shown to be identical, with a point mutation in codon 33 that converted Met to Ile. Recombinant human and mouse ENT1 proteins are highly sensitive to transport inhibition by dipyridamole, whereas recombinant human and mouse ENT2 proteins are much less sensitive (18Yao S.Y. Ng A.M. Muzyka W.R. Griffiths M. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 1997; 272: 28423-28430Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar,36Kiss A. Farah K. Kim J. Garriock R.J. Drysdale T.A. Hammond J.R. Biochem. J. 2000; 352: 363-372Crossref PubMed Scopus (82) Google Scholar). For example, the reported IC50 values for mENT1 and mENT2 produced in X. laevis oocytes were 75 and 2204 nm, respectively, which corresponds to a 29.4-fold difference (36Kiss A. Farah K. Kim J. Garriock R.J. Drysdale T.A. Hammond J.R. Biochem. J. 2000; 352: 363-372Crossref PubMed Scopus (82) Google Scholar). A transport-deficient cultured cell line stably transfected with recombinant hENT1 or hENT2 exhibited a 70-fold difference between the two proteins in sensitivity to dipyridamole with IC50 values of 5 and 356 nm, respectively (19Ward J.L. Sherali A. Mo Z.P. Tse C.M. J. Biol. Chem. 2000; 275: 8375-8381Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). The rat ENT isoforms (rENT1 and rENT2) are completely insensitive to dipyridamole and dilazep transport inhibition when produced in X. laevis oocytes (18Yao S.Y. Ng A.M. Muzyka W.R. Griffiths M. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 1997; 272: 28423-28430Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Multiple sequence alignment of the predicted amino acid sequences for the human, mouse, and rat ENT1 and ENT2 proteins revealed that the identity of the amino acid at residue 33 was consistent with the dilazep and dipyridamole sensitivity of the recombinant transporters (Fig. 2). Residue 33 is a Met in human and mouse ENT1, the most inhibitor-sensitive transporters, whereas it is an Ile in rat ENT1 and human, mouse, and rat ENT2 proteins, all of which exhibit transport activity that is insensitive to inhibition by dilazep and dipyridamole (18Yao S.Y. Ng A.M. Muzyka W.R. Griffiths M. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 1997; 272: 28423-28430Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 28Griffiths M. Beaumont N. Yao S.Y. Sundaram M. Boumah C.E. Davies A. Kwong F.Y. Coe I. Cass C.E. Young J.D. Baldwin S.A. Nat. Med. 1997; 3: 89-93Crossref PubMed Scopus (361) Google Scholar, 36Kiss A. Farah K. Kim J. Garriock R.J. Drysdale T.A. Hammond J.R. Biochem. J. 2000; 352: 363-372Crossref PubMed Scopus (82) Google Scholar, 37Griffiths M. Yao S.Y. Abidi F. Phillips S.E. Cass C.E. Young J.D. Baldwin S.A. Biochem. J. 1997; 328: 739-743Crossref PubMed Scopus (229) Google Scholar). The predicted topology model for hENT1 suggests that position 33 is the last residue in the first TM segment and may therefore be solvent-accessible and/or in the plane of the extracellular bilayer/solvent interface (20Sundaram M. Yao S.Y.M. 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 (125) Google Scholar, 28Griffiths M. Beaumont N. Yao S.Y. Sundaram M. Boumah C.E. Davies A. Kwong F.Y. Coe I. Cass C.E. Young J.D. Baldwin S.A. Nat. Med. 1997; 3: 89-93Crossref PubMed Scopus (361) Google Scholar). Uridine transport was measured in fui1::TRP1 yeast containing pYPhENT1 or pYPhENT1-M33I in the presence or absence of a single high concentration of dilazep, dipyridamole, or NBMPR (Fig.3A). hENT1-mediated uridine transport was inhibited ≥80% by 0.1 μm dilazep and 0.3 μm dipyridamole, whereas hENT1-M33I was capable of transport at 60% of the maximal rate in the presence of both inhibitors. These results suggested that hENT1-M33I was substantially less sensitive to dilazep and dipyridamole than wild type hENT1. In contrast, uridine transport was completely inhibited by 0.1 μm NBMPR in yeast with either recombinant protein, suggesting that residue 33 was not involved in the binding of NBMPR. Although hENT2 can be inhibited by high concentrations of dilazep and dipyridamole, it is 2 and 3 orders of magnitude less sensitive, respectively, to these compounds than hENT1 (19Ward J.L. Sherali A. Mo Z.P. Tse C.M. J. Biol. Chem. 2000; 275: 8375-8381Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). To investigate the role of residue 33 in inhibitor sensitivity of hENT2, Ile33was converted to Met using site-directed mutagenesis, and the effects of dilazep, dipyridamole and NBMPR on uridine transport were determined in fui1::TRP1 yeast containing either pYPhENT2 or pYPhENT2-I33M (Fig. 3B). Dilazep (10 μm) and dipyridamole (1 μm) had no effect on hENT2-mediated uridine transport, whereas both strongly inhibited hENT2-I33M-mediated transport. In contrast, uridine transport in yeast with either mutant or wild type hENT2 remained insensitive to NBMPR, a result that was consistent with the lack of an effect of the opposite conversion on NBMPR sensitivity of hENT1. These data, together with the data from Fig. 3A, indicated that residue 33 plays a key role in dilazep and dipyridamole inhibition of transport of both hENT1 and hENT2 and is not involved in NBMPR inhibition of transport. The effect of mutating residue 33 (Metversus Ile) of hENT1 and hENT2 on the kinetics of uridine transport was assessed by determining the concentration dependence of initial ra
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