Publications

• Facilitated mitochondrial import of antiviral and anticancer nucleoside drugs by human equilibrative nucleoside transporter-3.

  Govindarajan R., Leung G.P., Zhou M., Tse C.M., Wang J., Unadkat J.D.

  Human equilibrative nucleoside transporter-3 (hENT3) was recently reported as a pH-dependent, intracellular (lysosomal) transporter capable of transporting anti-human immunodeficiency virus (HIV) dideoxynucleosides (ddNs). Because most anti-HIV ddNs (e.g., zidovudine, AZT) exhibit clinical mitocho ... >> More

  Human equilibrative nucleoside transporter-3 (hENT3) was recently reported as a pH-dependent, intracellular (lysosomal) transporter capable of transporting anti-human immunodeficiency virus (HIV) dideoxynucleosides (ddNs). Because most anti-HIV ddNs (e.g., zidovudine, AZT) exhibit clinical mitochondrial toxicity, we investigated whether hENT3 facilitates transport of anti-HIV ddNs into the mitochondria. Cellular fractionation and immunofluorescence microscopy studies in several human cell lines identified a substantial presence of hENT3 in the mitochondria, with additional presence at the cell surface of two placental cell lines (JAR, JEG3). Mitochondrial or cell surface hENT3 expression was confirmed in human hepatocytes and placental tissues, respectively. Unlike endogenous hENT3, yellow fluorescent protein (YFP)-tagged hENT3 was partially directed to the lysosomes. Xenopus oocytes expressing NH2-terminal-deleted hENT3 (expressed at the cell surface) showed pH-dependent interaction with several classes of nucleosides (anti-HIV ddNs, gemcitabine, fialuridine, ribavirin) that produce mitochondrial toxicity. Transport studies in hENT3 gene-silenced JAR cells showed significant reduction in mitochondrial transport of nucleosides and nucleoside drugs. Our data suggest that cellular localization of hENT3 is cell type dependent and the native transporter is substantially expressed in mitochondria and/or cell surface. hENT3-mediated mitochondrial transport may play an important role in mediating clinically observed mitochondrial toxicity of nucleoside drugs. In addition, our finding that hENT3 is a mitochondrial transporter is consistent with the recent finding that mutations in the hENT3 gene cause an autosomal recessive disorder in humans called the H syndrome. << Less

  Am. J. Physiol. 296:G910-G922(2009) [PubMed] [EuropePMC]

  This publication is cited by 12 other entries.

• Kinetic and pharmacological properties of cloned human equilibrative nucleoside transporters, ENT1 and ENT2, stably expressed in nucleoside transporter-deficient PK15 cells. Ent2 exhibits a low affinity for guanosine and cytidine but a high affinity for inosine.

  Ward J.L., Sherali A., Mo Z.P., Tse C.M.

  We stably transfected the cloned human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) into nucleoside transporter-deficient PK15NTD cells. Although hENT1 and hENT2 are predicted to be 50-kDa proteins, hENT1 runs as 40 kDa and hENT2 migrates as 50 and 47 kDa on SDS-polyacrylamide g ... >> More

  We stably transfected the cloned human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) into nucleoside transporter-deficient PK15NTD cells. Although hENT1 and hENT2 are predicted to be 50-kDa proteins, hENT1 runs as 40 kDa and hENT2 migrates as 50 and 47 kDa on SDS-polyacrylamide gel electrophoresis. Peptide N-glycosidase F and endoglycosidase H deglycosylate hENT1 to 37 kDa and hENT2 to 45 kDa. With hENT1 being more sensitive, there is a 7000-fold and 71-fold difference in sensitivity to nitrobenzylthioinosine (NBMPR) (IC(50), 0.4 +/- 0.1 nM versus 2.8 +/-0.3 microM) and dipyridamole (IC(50), 5.0 +/- 0.9 nM versus 356 +/-13 nM), respectively. [(3)H]NBMPR binds to ENT1 cells with a high affinity K(d) of 0.377 +/-0.098 nM, and each ENT1 cell has 34,000 transporters with a turnover number of 46 molecules/s for uridine. Although both transporters are broadly selective, hENT2 is a generally low affinity nucleoside transporter with 2.6-, 2.8-, 7. 7-, and 19.3-fold lower affinity than hENT1 for thymidine, adenosine, cytidine, and guanosine, respectively. In contrast, the affinity of hENT2 for inosine is 4-fold higher than hENT1. The nucleobase hypoxanthine inhibits [(3)H]uridine uptake by hENT2 but has minimal effect on hENT1. Taken together, these results suggest that hENT2 might be important in transporting adenosine and its metabolites (inosine and hypoxanthine) in tissues such as skeletal muscle where ENT2 is predominantly expressed. << Less

  J. Biol. Chem. 275:8375-8381(2000) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Distribution and functional characterization of equilibrative nucleoside transporter-4, a novel cardiac adenosine transporter activated at acidic pH.

  Barnes K., Dobrzynski H., Foppolo S., Beal P.R., Ismat F., Scullion E.R., Sun L., Tellez J., Ritzel M.W., Claycomb W.C., Cass C.E., Young J.D., Billeter-Clark R., Boyett M.R., Baldwin S.A.

  Adenosine plays multiple roles in the efficient functioning of the heart by regulating coronary blood flow, cardiac pacemaking, and contractility. Previous studies have implicated the equilibrative nucleoside transporter family member equilibrative nucleoside transporter-1 (ENT1) in the regulation ... >> More

  Adenosine plays multiple roles in the efficient functioning of the heart by regulating coronary blood flow, cardiac pacemaking, and contractility. Previous studies have implicated the equilibrative nucleoside transporter family member equilibrative nucleoside transporter-1 (ENT1) in the regulation of cardiac adenosine levels. We report here that a second member of this family, ENT4, is also abundant in the heart, in particular in the plasma membranes of ventricular myocytes and vascular endothelial cells but, unlike ENT1, is virtually absent from the sinoatrial and atrioventricular nodes. Originally described as a monoamine/organic cation transporter, we found that both human and mouse ENT4 exhibited a novel, pH-dependent adenosine transport activity optimal at acidic pH (apparent K(m) values 0.78 and 0.13 mmol/L, respectively, at pH 5.5) and absent at pH 7.4. In contrast, serotonin transport by ENT4 was relatively insensitive to pH. ENT4-mediated nucleoside transport was adenosine selective, sodium independent and only weakly inhibited by the classical inhibitors of equilibrative nucleoside transport, dipyridamole, dilazep, and nitrobenzylthioinosine. We hypothesize that ENT4, in addition to playing roles in cardiac serotonin transport, contributes to the regulation of extracellular adenosine concentrations, in particular under the acidotic conditions associated with ischemia. << Less

  Circ. Res. 99:510-519(2006) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Identification of a nucleoside/nucleobase transporter from Plasmodium falciparum, a novel target for anti-malarial chemotherapy.

  Parker M.D., Hyde R.J., Yao S.Y., McRobert L., Cass C.E., Young J.D., McConkey G.A., Baldwin S.A.

  Plasmodium, the aetiologic agent of malaria, cannot synthesize purines de novo, and hence depends upon salvage from the host. Here we describe the molecular cloning and functional expression in Xenopus oocytes of the first purine transporter to be identified in this parasite. This 422-residue prot ... >> More

  Plasmodium, the aetiologic agent of malaria, cannot synthesize purines de novo, and hence depends upon salvage from the host. Here we describe the molecular cloning and functional expression in Xenopus oocytes of the first purine transporter to be identified in this parasite. This 422-residue protein, which we designate PfENT1, is predicted to contain 11 membrane-spanning segments and is a distantly related member of the widely distributed eukaryotic protein family the equilibrative nucleoside transporters (ENTs). However, it differs profoundly at the sequence and functional levels from its homologous counterparts in the human host. The parasite protein exhibits a broad substrate specificity for natural nucleosides, but transports the purine nucleoside adenosine with a considerably higher apparent affinity (K(m) 0.32+/-0.05 mM) than the pyrimidine nucleoside uridine (K(m) 3.5+/-1.1 mM). It also efficiently transports nucleobases such as adenine (K(m) 0.32+/-0.10 mM) and hypoxanthine (K(m) 0.41+/-0.1 mM), and anti-viral 3'-deoxynucleoside analogues. Moreover, it is not sensitive to classical inhibitors of mammalian ENTs, including NBMPR [6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, or nitrobenzylthioinosine] and the coronary vasoactive drugs, dipyridamole, dilazep and draflazine. These unique properties suggest that PfENT1 might be a viable target for the development of novel anti-malarial drugs. << Less

  Biochem. J. 349:67-75(2000) [PubMed] [EuropePMC]

  This publication is cited by 10 other entries.

• Genetic evidence for the essential role of PfNT1 in the transport and utilization of xanthine, guanine, guanosine and adenine by Plasmodium falciparum.

  El Bissati K., Downie M.J., Kim S.K., Horowitz M., Carter N., Ullman B., Ben Mamoun C.

  The malaria parasite, Plasmodium falciparum, is unable to synthesize the purine ring de novo and is therefore wholly dependent upon purine salvage from the host for survival. Previous studies have indicated that a P. falciparum strain in which the purine transporter PfNT1 had been disrupted was un ... >> More

  The malaria parasite, Plasmodium falciparum, is unable to synthesize the purine ring de novo and is therefore wholly dependent upon purine salvage from the host for survival. Previous studies have indicated that a P. falciparum strain in which the purine transporter PfNT1 had been disrupted was unable to grow on physiological concentrations of adenosine, inosine and hypoxanthine. We have now used an episomally complemented pfnt1Delta knockout parasite strain to confirm genetically the functional role of PfNT1 in P. falciparum purine uptake and utilization. Episomal complementation by PfNT1 restored the ability of pfnt1Delta parasites to transport and utilize adenosine, inosine and hypoxanthine as purine sources. The ability of wild-type and pfnt1Delta knockout parasites to transport and utilize the other physiologically relevant purines adenine, guanine, guanosine and xanthine was also examined. Unlike wild-type and complemented P. falciparum parasites, pfnt1Delta parasites could not proliferate on guanine, guanosine or xanthine as purine sources, and no significant transport of these substrates could be detected in isolated parasites. Interestingly, whereas isolated pfnt1Delta parasites were still capable of adenine transport, these parasites grew only when adenine was provided at high, non-physiological concentrations. Taken together these results demonstrate that, in addition to hypoxanthine, inosine and adenosine, PfNT1 is essential for the transport and utilization of xanthine, guanine and guanosine. << Less

  Mol. Biochem. Parasitol. 161:130-139(2008) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Bidirectional transport of 2-chloroadenosine by equilibrative nucleoside transporter 4 (hENT4): Evidence for allosteric kinetics at acidic pH.

  Tandio D., Vilas G., Hammond J.R.

  Adenosine has been reported to be transported by equilibrative nucleoside transporter 4 (ENT4), encoded by the SLC29A4 gene, in an acidic pH-dependent manner. This makes hENT4 of interest as a therapeutic target in acidic pathologies where adenosine is protective (e.g. vascular ischaemia). We exam ... >> More

  Adenosine has been reported to be transported by equilibrative nucleoside transporter 4 (ENT4), encoded by the SLC29A4 gene, in an acidic pH-dependent manner. This makes hENT4 of interest as a therapeutic target in acidic pathologies where adenosine is protective (e.g. vascular ischaemia). We examined the pH-sensitivity of nucleoside influx and efflux by hENT4 using a recombinant transfection model that lacks the confounding influences of other nucleoside transporters (PK15-NTD). We established that [³H]2-chloroadenosine, which is resistant to metabolism by adenosine deaminase, is a substrate for hENT4. Transport of [³H]2-chloroadenosine at a pH of 6.0 in PK15-NTD cells stably transfected with SLC29A4 was biphasic, with a low capacity (V_max ~ 30 pmol/mg/min) high-affinity component (K_m ~ 50 µM) apparent at low substrate concentrations, which shifted to a high capacity (V_max ~ 500 pmol/mg/min) low affinity system (K_m > 600 µM) displaying positive cooperativity at concentrations above 200 µM. Only the low affinity component was observed at a neutral pH of 7.5 (K_m ~ 2 mM). Efflux of [³H]2-chloroadenosine from these cells was also enhanced by more than 4-fold at an acidic pH. Enhanced influx and efflux of nucleosides by hENT4 under acidic conditions supports its potential as a therapeutic target in pathologies such as ischaemia-reperfusion injury. << Less

  Sci. Rep. 9:13555-13555(2019) [PubMed] [EuropePMC]