Mutation of Trp29 of human equilibrative nucleoside transporter 1 alters affinity for coronary vasodilator drugs and nucleoside selectivity

2008 ◽  
Vol 414 (2) ◽  
pp. 291-300 ◽  
Author(s):  
Robert J. Paproski ◽  
Frank Visser ◽  
Jing Zhang ◽  
Tracey Tackaberry ◽  
Vijaya Damaraju ◽  
...  
Keyword(s):  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Transport Activity ◽  
Sequence Alignments ◽  
Equilibrative Nucleoside Transporter ◽  
Vasodilator Drugs ◽  
Selective Loss ◽  
Coronary Vasodilator ◽  
Ic50 Values ◽  
Adenosine Transport

hENT1 (human equilibrative nucleoside transporter 1) is inhibited by nanomolar concentrations of various structurally distinct coronary vasodilator drugs, including dipyridamole, dilazep, draflazine, soluflazine and NBMPR (nitrobenzylmercaptopurine ribonucleoside). When a library of randomly mutated hENT1 cDNAs was screened using a yeast-based functional complementation assay for resistance to dilazep, a clone containing the W29G mutation was identified. Multiple sequence alignments revealed that this residue was highly conserved. Mutations at Trp29 were generated and tested for adenosine transport activity and inhibitor sensitivity. Trp29 mutations significantly reduced the apparent Vmax and/or increased the apparent Km values for adenosine transport. Trp29 mutations increased the IC50 values for hENT1 inhibition by dipyridamole, dilazep, NBMPR, soluflazine and draflazine. NBMPR and soluflazine displayed remarkably similar trends, with large aromatic substitutions at residue 29 resulting in the lowest IC50 values, suggesting that both drugs could interact via ring-stacking interactions with Trp29. The W29T mutant displayed a selective loss of pyrimidine nucleoside transport activity, which contrasts with the previously identified L442I mutant that displayed a selective loss of purine nucleoside transport. W29T, L442I and the double mutant W29T/L442I were characterized kinetically for nucleoside transport activity. A helical wheel projection of TM (transmembrane segment) 1 suggests that Trp29 is positioned close to Met33, implicated previously in nucleoside and inhibitor recognition, and that both residues line the permeant translocation pathway. The data also suggest that Trp29 forms part of, or lies close to, the binding sites for dipyridamole, dilazep, NBMPR, soluflazine and draflazine.

scholarly journals Mutation of leucine-92 selectively reduces the apparent affinity of inosine, guanosine, NBMPR [S6-(4-nitrobenzyl)-mercaptopurine riboside] and dilazep for the human equilibrative nucleoside transporter, hENT1

2004 ◽  
Vol 380 (1) ◽  
pp. 131-137 ◽  
Author(s):  
Christopher J. ENDRES ◽  
Dhruba J. SENGUPTA ◽  
Jashvant D. UNADKAT
Keyword(s):  
Amino Acid ◽  
Yeast Cells ◽  
Nucleoside Transporter ◽  
Wild Type ◽  
Equilibrative Nucleoside Transporter ◽  
Ic50 Values ◽  
Km Value ◽  
Adenosine Transport ◽  
The Media ◽  
First Time

We developed a yeast-based assay for selection of hENT1 (human equilibrative nucleoside transporter 1) mutants that have altered affinity for hENT1 inhibitors and substrates. In this assay, expression of hENT1 in a yeast strain deficient in adenine biosynthesis (ade2) permits yeast growth on a plate lacking adenine but containing adenosine, a hENT1 substrate. This growth was prevented when inhibitors of hENT1 {e.g. NBMPR [S6-(4-nitrobenzyl)-mercaptopurine riboside], dilazep or dipyridamole} were included in the media. To identify hENT1 mutants resistant to inhibition by these compounds, hENT1 was randomly mutagenized and introduced into this strain. Mutation(s) that allowed growth of yeast cells in the presence of these inhibitors were then identified and characterized. Mutants harbouring amino acid changes at Leu92 exhibited resistance to NBMPR and dilazep but not dipyridamole. The IC50 values of NBMPR and dilazep for [3H]adenosine transport by one of these mutants L92Q (Leu92→Gln) were approx. 200- and 4-fold greater when compared with the value for the wild-type hENT1, whereas that for dipyridamole remained unchanged. Additionally, when compared with the wild-type transporter, [3H]adenosine transport by L92Q transporter was significantly resistant to inhibition by inosine and guanosine but not by adenosine or pyrimidines. The Km value for inosine transport was approx. 4-fold greater for the L92Q mutant (260±16 µM) when compared with the wild-type transporter (65±7.8 µM). We have identified for the first time an amino acid residue (Leu92) of hENT1 that, when mutated, selectively alters the affinity of hENT1 to transport the nucleosides inosine and guanosine and its sensitivity to the inhibitors NBMPR and dilazep.

scholarly journals A comprehensive model of purine uptake by the malaria parasite Plasmodium falciparum: identification of four purine transport activities in intraerythrocytic parasites

2008 ◽  
Vol 411 (2) ◽  
pp. 287-295 ◽  
Author(s):  
Neils B. Quashie ◽  
Dominique Dorin-Semblat ◽  
Patrick G. Bray ◽  
Giancarlo A. Biagini ◽  
Christian Doerig ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
De Novo ◽  
High Capacity ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Transport Activity ◽  
High Affinity ◽  
Parasite Plasmodium Falciparum ◽  
Adenosine Transport ◽  
Efficient Uptake

Plasmodium falciparum is incapable of de novo purine biosynthesis, and is absolutely dependent on transporters to salvage purines from the environment. Only one low-affinity adenosine transporter has been characterized to date. In the present study we report a comprehensive study of purine nucleobase and nucleoside transport by intraerythrocytic P. falciparum parasites. Isolated trophozoites expressed (i) a high-affinity hypoxanthine transporter with a secondary capacity for purine nucleosides, (ii) a separate high-affinity transporter for adenine, (iii) a low-affinity adenosine transporter, and (iv) a low-affinity/high-capacity adenine carrier. Hypoxanthine was taken up with 12-fold higher efficiency than adenosine. Using a parasite clone with a disrupted PfNT1 (P. falciparum nucleoside transporter 1) gene we found that the high-affinity hypoxanthine/nucleoside transport activity was completely abolished, whereas the low-affinity adenosine transport activity was unchanged. Adenine transport was increased, presumably to partly compensate for the loss of the high-affinity hypoxanthine transporter. We thus propose a model for purine salvage in P. falciparum, based on the highly efficient uptake of hypoxanthine by PfNT1 and a high capacity for purine nucleoside uptake by a lower affinity carrier.

scholarly journals Hypoxanthine enters human vascular endothelial cells (ECV 304) via the nitrobenzylthioinosine-insensitive equilibrative nucleoside transporter

1996 ◽  
Vol 317 (3) ◽  
pp. 843-848 ◽  
Author(s):  
Nelson OSSES ◽  
Jeremy D. PEARSON ◽  
David L. YUDILEVICH ◽  
Simon M. JARVIS
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Nucleoside Transport ◽  
Vascular Endothelial ◽  
Nucleoside Transporter ◽  
Equilibrative Nucleoside Transporter ◽  
Nucleoside Transport Inhibitors ◽  
Adenosine Transport ◽  
Transport Inhibitors

The transport properties of the nucleobase hypoxanthine were examined in the human umbilical vein endothelial cell line ECV 304. Initial rates of hypoxanthine influx were independent of extracellular cations: replacement of Na+ with Li+, Rb+, N-methyl-d-glucamine or choline had no significant effect on hypoxanthine uptake by ECV 304 cells. Kinetic analysis demonstrated the presence of a single saturable system for the transport of hypoxanthine in ECV 304 cells with an apparent Km of 320±10 μM and a Vmax of 5.6±0.9 pmol/106 cells per s. Hypoxanthine uptake was inhibited by the nucleosides adenosine, uridine and thymidine (apparent Ki 41±6, 240±27 and 59±8 μM respectively) and the nucleoside transport inhibitors nitrobenzylthioinosine (NBMPR), dilazep and dipyridamole (apparent Ki 2.5±0.3, 11±3 and 0.16±0.006 μM respectively), whereas the nucleobases adenine, guanine and thymine had little effect (50% inhibition at > 1 mM). ECV 304 cells were also shown to transport adenosine via both the NBMPR-sensitive and -insensitive nucleoside carriers. Hypoxanthine specifically inhibited adenosine transport via the NBMPR-insensitive system in a competitive manner (apparent Ki 290±14 μM). These results indicate that hypoxanthine entry into ECV 304 endothelial cells is mediated by the NBMPR-insensitive nucleoside carrier present in these cells.

scholarly journals Na+-dependent nucleoside transport in liver: two different isoforms from the same gene family are expressed in liver cells

1998 ◽  
Vol 330 (2) ◽  
pp. 997-1001 ◽  
Author(s):  
Antonio FELIPE ◽  
Raquel VALDES ◽  
Belén del SANTO ◽  
Jorge LLOBERAS ◽  
Javier CASADO ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Polyclonal Antibodies ◽  
High Specificity ◽  
Liver Cells ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Nucleoside Transporter ◽  
Transport Activity ◽  
Plasma Membrane Vesicles

Hepatocytes show a Na+-dependent nucleoside transport activity that is kinetically heterogeneous and consistent with the expression of at least two independent concentrative Na+-coupled nucleoside transport systems (Mercader et al. Biochem. J. 317, 835-842, 1996). So far, only a single nucleoside carrier-related cDNA (SPNT) has been isolated from liver cells (Che et al. J. Biol. Chem. 270, 13596-13599, 1995). This cDNA presumably encodes a plasma membrane protein responsible for Na+-dependent purine nucleoside transport activity. Thus, the liver must express, at least, a second nucleoside transporter which should be pyrimidine-preferring. Homology cloning using RT-PCR revealed that a second isoform is indeed present in liver. This second isoform turned out to be identical to the ‘epithelial-specific isoform’ called cNT1, which shows in fact high specificity for pyrimidine nucleosides. Although cNT1 mRNA is present at lower amounts than SPNT mRNA, the amounts of cNT1 protein, when measured using isoform-specific polyclonal antibodies, were even higher than the SPNT protein levels. Moreover, partially purified basolateral plasma membrane vesicles from liver were enriched in the SPNT but not in the cNT1 protein, which suggests that the subcellular localization of these carrier proteins is different. SPNT and cNT1 protein amounts in crude membrane extracts from 6 h-regenerating rat livers are higher than in the preparations from sham-operated controls (3.5- and 2-fold, respectively). These results suggest that liver parenchymal cells express at least two different isoforms of concentrative nucleoside carriers, the cNT1 and SPNT proteins, which show differential regulation and subcellular localization.

scholarly journals Nucleoside transport and proliferative rate in human thymocytes and lymphocytes

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 2038-2042 ◽  
Author(s):  
CL Smith ◽  
LM Pilarski ◽  
ML Egerton ◽  
JS Wiley
Keyword(s):  
Cytosine Arabinoside ◽  
Binding Sites ◽  
Flow Cytometric Analysis ◽  
Fold Increase ◽  
S Phase ◽  
Nucleoside Transporters ◽  
Labeling Index ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Equilibrative Nucleoside Transporter

The thymus is a site of active T-lymphoid cell proliferation and DNA synthesis. In this study, the capacity of human thymocytes for nucleoside transport was assessed both by cytosine arabinoside influx and by equilibrium binding of nitrobenzylmercaptopurine riboside (NBMPR), a specific ligand for the equilibrative nucleoside transporter of leukocytes. The proportion of freshly isolated thymocytes synthesizing DNA was 8.6% +/- 2.1% (n = 12) by 3H-thymidine labeling index and 7.8% +/- 2.9% (n = 4) S-phase cells by flow cytometric analysis of DNA content. In comparison, both methods gave proliferation S-phase values less than 1% for peripheral blood lymphocytes (PBLs). Thymocytes expressed a high density of specific NBMPR binding sites (26,068 +/- 8,776 sites per cell, n = 12) as compared with PBLs (1,123 +/- 553 sites per cell, n = 8). The initial influx of cytosine arabinoside into thymocytes was 14-fold greater than into PBLs, and in both cell types the influx of nucleoside was totally inhibited by 0.5 mumol/L NBMPR, which is known to inhibit the major equilibrative nucleoside transporter in white blood cells. Depletion of mature CD3+ cells from the thymocyte preparation by anti-CD3 antibody left a residual population with both increased labeling index and up to twofold greater density of NBMPR binding sites. When PBLs were cultured for 48 hours with the T-cell mitogen phytohemagglutinin, a 40-fold increase in labeling index was observed, together with a 30-fold increase in the density of specific NBMPR binding sites. Thus, fresh thymocytes from human thymus are actively proliferating and express high densities of a functional nucleoside transporter. The more immature cells in the thymocyte population which are proliferating more actively have a greater density of nucleoside transporters than the whole population. In contrast, mitotically inactive PBLs-have few nucleoside transporters, but after mitogenic stimulation PBLs express large numbers of this transmembrane molecule.

scholarly journals Nucleoside transport in rat cerebral-cortical synaptosomes. Evidence for two types of nucleoside transporters

1988 ◽  
Vol 249 (2) ◽  
pp. 557-564 ◽  
Author(s):  
C W Lee ◽  
S M Jarvis
Keyword(s):  
Dose Response ◽  
Binding Activity ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Transport Activity ◽  
Response Curves ◽  
Maximal Inhibition ◽  
Filtration Method ◽  
Ic50 Values ◽  
Ic50 Concentration

The transport of [U-14C]uridine was investigated in rat cerebral-cortical synaptosomes using an inhibitor-stop filtration method. Under these conditions the rapid efflux of uridine from the synaptosomes is prevented and uridine is not significantly metabolized in the synaptosome during the first 1 min of uptake. The dose-response curve for the inhibition of uridine transport by nitrobenzylthioinosine (NBMPR) was biphasic: approx. 40% of the transport activity was inhibited with an IC50 (concentration causing half-maximal inhibition) value of 0.5 nM, but the remaining activity was insensitive to concentrations as high as 1 microM. Similar biphasic dose-response curves were observed for dilazep inhibition, but both transport components were equally sensitive to dipyridamole inhibition. Uridine influx by both components was saturable (Km 300 +/- 51 and 214 +/- 23 microM, and Vmax. 12 +/- 3 and 16 +/- 3 pmol/s per mg of protein, for NBMPR-sensitive and NBMPR-insensitive components respectively), and inhibited by other nucleosides such as 2-chloroadenosine, adenosine, inosine, thymidine and guanosine with similar IC50 values for the two components. Inhibition of uridine transport by NBMPR was associated with high-affinity binding of NBMPR to the synaptosome membrane (Kd 58 +/- 15 pM). Binding of NBMPR to these sites was competitively blocked by uridine and adenosine and inhibited by dilazep and dipyridamole, with Ki values similar to those measured for inhibiting NBMPR-sensitive uridine influx. These results demonstrate that there are two components of nucleoside transport in our rat synaptosomal preparation that differ in their sensitivity to inhibition by NBMPR. Thus conclusions regarding nucleoside transport in rat brain based only on NBMPR-binding activity must be viewed with caution.

Quantitative analysis of nucleoside transporter and metabolism gene expression in chronic lymphocytic leukemia (CLL): identification of fludarabine-sensitive and -insensitive populations

Blood ◽  
2005 ◽  
Vol 105 (2) ◽  
pp. 767-774 ◽  
Author(s):  
John R. Mackey ◽  
Carlos M. Galmarini ◽  
Kathryn A. Graham ◽  
Anil A. Joy ◽  
Alain Delmer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
Disease Progression ◽  
Hierarchical Cluster ◽  
Complete Response ◽  
Lymphocytic Leukemia ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Equilibrative Nucleoside Transporter ◽  
Risk Of Disease

AbstractResistance to fludarabine is observed in the clinic, and molecular predictive assays for benefit from chemotherapy are required. Our objective was to determine if expression of nucleoside transport and metabolism genes was associated with response to fludarabine therapy in patients with chronic lymphocytic leukemia (CLL). CLL cells from 56 patients were collected prior to treatment with fludarabine. Quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) was performed on sample RNA to determine the relative levels of mRNA of 3 nucleoside transporters that mediate fludarabine uptake (human equilibrative nucleoside transporter 1 [hENT1], human equilibrative nucleoside transporter 2 [hENT2], and human concentrative nucleoside transporter 3 [hCNT3]), deoxycytidine kinase (dCK), and 3 5′-nucleotidases (ecto-5′nucleotidase [CD73], deoxynucleotidase-1 [dNT-1], and cytoplasmic high-Km 5-nucleotidase [CN-II]). Two-dimensional hierarchical cluster analysis of gene expression identified 2 distinct populations of CLL. Cluster 2 patients experienced a 3.4-fold higher risk of disease progression than cluster 1 patients (P = .0058, log-rank analysis). Furthermore, independent analysis of the individual genes of interest revealed statistically significant differences for risk of disease progression (adjusted hazard ratios [HRs]) with underexpression of dNT-1 (HR = 0.45; P = .042), CD73 (HR = 0.40; P = .022), and dCK (HR = 0.0.48; P = .035), and overexpression of hCNT3 (HR = 4.7; P = .0007) genes. Subjects with elevated hCNT3 expression experienced a lower complete response rate to fludarabine therapy (11% vs 69%; P = .002). No hCNT3-mediated plasma membrane nucleoside transport was detected in CLL samples expressing hCNT3 message, and hCNT3 protein was localized to the cytoplasm with immunohistochemical and confocal microscopy.

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