Nucleoside Transporter Family Research Articles

Analysis of human equilibrative nucleoside transporter 1 (hENT1) protein in non-Hodgkin's lymphoma by immunohistochemistry

The human equilibrative nucleoside transporter 1 (hENT1) is a member of the equilibrative nucleoside transporter family that mediates cellular entry of gemcitabine, cytarabine, and fludarabine. Deficiency in hENT1 confers resistance to toxicity of these drugs in a variety of model systems. Since some nucleoside analogs have a role in treating patients with non-Hodgkin’s lymphoma (NHL), this study was undertaken to assess hENT1 abundance in NHL. A total of 115 cases of NHL of various subtypes and 15 reactive lymph nodes were evaluated for the presence of hENT1 protein using immunohistochemistry applied to frozen tissues. Samples were considered positive when ≥50% of neoplastic cells showed immunostaining. In reactive lymph nodes, hENT1 was confined to the germinal centers, whereas mantle zone B-cells and interfollicular T-cells were negative. In NHL, a relatively high frequency of hENT1 positivity was found in Burkitt lymphoma/leukemia (63%), diffuse large B-cell lymphoma (DLCL; 45%), and follicular lymphoma (40%). In DLCL, 26% of cases were positive for CD10, and CD10-positive DLCL cases were more likely to be hENT1 positive than CD10-negative cases (P=0.025). A lower frequency of hENT1 positivity was found in mantle cell lymphoma (13%) and peripheral T-cell lymphomas (37%). All marginal zone lymphomas (n=5), chronic lymphocytic leukemia small lymphocytic lymphomas (n=10), plasmacytoma (n=3), acute lymphoblastic lymphoma/leukemia, and anaplastic large-cell lymphomas (n=5) were negative. In conclusion, hENT1 was most frequently found in benign and malignant follicular center cells. Prospective studies to assess the value of hENT1 immunostaining in predicting resistance to nucleoside chemotherapy for NHL are warranted.

Functional Characterization of Novel Human and Mouse Equilibrative Nucleoside Transporters (hENT3 and mENT3) Located in Intracellular Membranes

The first mammalian examples of the equilibrative nucleoside transporter family to be characterized, hENT1 and hENT2, were passive transporters located predominantly in the plasma membranes of human cells. We now report the functional characterization of members of a third subgroup of the family, from human and mouse, which differ profoundly in their properties from previously characterized mammalian nucleoside transporters. The 475-residue human and mouse proteins, designated hENT3 and mENT3, respectively, are 73% identical in amino acid sequence and possess long N-terminal hydrophilic domains that bear typical (DE)XXXL(LI) endosomal/lysosomal targeting motifs. ENT3 transcripts and proteins are widely distributed in human and rodent tissues, with a particular abundance in placenta. However, in contrast to ENT1 and ENT2, the endogenous and green fluorescent protein-tagged forms of the full-length hENT3 protein were found to be predominantly intracellular proteins that co-localized, in part, with lysosomal markers in cultured human cells. Truncation of the hydrophilic N-terminal region or mutation of its dileucine motif to alanine caused the protein to be relocated to the cell surface both in human cells and in Xenopus oocytes, allowing characterization of its transport activity in the latter. The protein proved to be a broad selectivity, low affinity nucleoside transporter that could also transport adenine. Transport activity was relatively insensitive to the classical nucleoside transport inhibitors nitrobenzylthioinosine, dipyridamole, and dilazep and was sodium ion-independent. However, it was strongly dependent upon pH, and the optimum pH value of 5.5 probably reflected the location of the transporter in acidic, intracellular compartments.

Functional analysis of genetic variants in the human concentrative nucleoside transporter 3 (CNT3; SLC28A3)

The human concentrative nucleoside transporter, CNT3 (SLC28A3), plays an important role in mediating the cellular entry of a broad array of physiological nucleosides and synthetic anticancer nucleoside analog drugs. As a first step toward understanding the genetic basis for interindividual differences in the disposition and response to antileukemic nucleoside analogs, we examined the genetic and functional diversity of CNT3. In all, 56 variable sites in the exons and flanking intronic region of SLC28A3 were identified in a collection of 270 DNA samples from US populations (80 African-Americans, 80 European-Americans, 60 Asian-Americans, and 50 Mexican-Americans). Of the 16 coding region variants, 12 had not been previously reported. Also, 10 resulted in amino-acid changes and three of these had total allele frequencies of >/=1%. Nucleotide diversity (pi) at nonsynonymous and synonymous sites was estimated to be 1.81 x 10(4) and 18.13 x 10(4), respectively, suggesting that SLC28A3 is under negative selection. All nonsynonymous variants, constructed by site-directed mutagenesis and expressed in Xenopus laevis oocytes, transported purine and pyrimidine model substrates, except for c. 1099G>A (p. Gly367Arg). This rare variant alters an evolutionarily conserved site in the putative substrate recognition domain of CNT3. The presence of three additional evolutionarily conserved glycine residues in the vicinity of p. Gly367Arg that are also conserved in human paralogs suggest that these glycine residues are critical in the function of the concentrative nucleoside transporter family. The genetic analysis and functional characterization of CNT3 variants suggest that this transporter does not tolerate nonsynonymous changes and is important for human fitness.

The concentrative nucleoside transporter family (SLC28): new roles beyond salvage?

The concentrative nucleoside transporter (CNT) family (SLC28) has three members: SLC28A1 (CNT1), SLC28A2 (CNT2) and SLC28A3 (CNT3). The CNT1 and CNT2 transporters are co-expressed in liver parenchymal cells and macrophages, two suitable models in which to study cell cycle progression. Despite initial observations suggesting that these transporter proteins might contribute to nucleoside salvage during proliferation, their subcellular localization and regulatory properties suggest alternative roles in cell physiology. In particular, CNT2 is a suitable candidate for modulation of purinergic responses, since it is under the control of the adenosine 1 receptor. Increasing evidence also suggests a role for CNT2 in energy metabolism, since its activation relies on the opening of ATP-sensitive K(+) channels. Animal and cell models genetically modified to alter nucleoside transporter expression levels may help to elucidate the particular roles of CNT proteins in cell physiology.

The nucleoside transport proteins, NupC and NupG, from Escherichia coli: specific structural motifs necessary for the binding of ligands

A series of 46 natural nucleosides and analogues (mainly adenosine-based) were tested as inhibitors of [U-(14)C]uridine uptake by the concentrative, H(+)-linked nucleoside transport proteins NupC and NupG from Escherichia coli. The two evolutionarily unrelated transporters showed similar but distinct patterns of inhibition, revealing differing selectivities for the different nucleosides and their analogues. Binding of nucleosides to NupG required the presence of hydroxyl groups at each of the C-3' and C-5' positions of ribose, while binding to NupC required only the C-3' hydroxyl substituent. The greater importance of the ribose moiety for binding to NupG is consistent with the evolutionary relationship between this protein and the oligosaccharide: H(+) symporter (OHS) subfamily of the major facilitator superfamily (MFS) of transporters. For both proteins the natural alpha-configuration at C-3' and the natural beta-configuration at C-1' was mandatory for ligand binding. N-7 in the imidazole ring of adenosine and the amino group at C-6 were found not to be important for binding and both transporters showed flexibility for substitution at C-6/N(6); one or both of N-1 and N-3 were important for adenosine analogue binding to NupC but significantly less so for binding to NupG. From the different effects of 8-bromoadenosine on the two transporters it appears that adenosine selectively binds to NupC in an anti- rather than a syn-conformation, whereas NupG is less prescriptive. The pattern of inhibition of NupC by differing nucleoside analogues confirmed the functional relationship of the bacterial transporter to members of the human concentrative nucleoside transporter (CNT) family and reaffirmed the use of the bacterial protein as an experimental model for these physiologically and clinically important mammalian proteins. The specificity data for NupG have been used to develop a homology model of the protein's binding site, based on the X-ray crystallographic structure of the disaccharide transporter LacY from E. coli. We have also developed an efficient general protocol for the synthesis of adenosine and three of its analogues, which is illustrated by the synthesis of [1'-(13)C]adenosine.

Second-site Suppression of a Nonfunctional Mutation within the Leishmania donovani Inosine-Guanosine Transporter

LdNT2 is a member of the equilibrative nucleoside transporter family, which possesses several conserved residues located mainly within transmembrane domains. One of these residues, Asp(389) within LdNT2, was shown previously to be critical for transporter function without affecting ligand affinity or plasma membrane targeting. To further delineate the role of Asp(389) in LdNT2 function, second-site suppressors of the ldnt2-D389N null mutation were selected in yeast deficient in purine nucleoside transport and incapable of purine biosynthesis. A library of random mutants within the ldnt2-D389N background was screened in yeast for restoration of growth on inosine. Twelve different clones were obtained, each containing secondary mutations enabling inosine transport. One mutation, N175I, occurred in four clones and conferred augmented inosine transport capability compared with LdNT2 in yeast. N175I was subsequently introduced into an ldnt2-D389N construct tagged with green fluorescent protein and transfected into a Deltaldnt1/Deltaldnt2 Leishmania donovani knockout. GFP-N175I/D389N significantly suppressed the D389N phenotype and targeted properly to the plasma membrane and flagellum. Most interestingly, N175I increased the inosine K(m) by 10-fold within the D389N background relative to wild type GFP-LdNT2. Additional substitutions introduced at Asn(175) established that only large, nonpolar amino acids suppressed the D389N phenotype, indicating that suppression by Asn(175) has a specific size and charge requirement. Because multiple suppressor mutations alleviate the constraint imparted by the D389N mutation, these data suggest that Asp(389) is a conformationally sensitive residue. To impart spatial information to the clustering of second-site mutations, a three-dimensional model was constructed based upon members of the major facilitator superfamily using threading analysis. The model indicates that Asn(175) and Asp(389) lie in close proximity and that the second-site suppressor mutations cluster to one region of the transporter.

Identification and characterization of purine nucleoside transporters from Crithidia fasciculata

To initiate a molecular dissection into the mechanism by which purine transport is up-regulated in Crithidia, genes encoding nucleoside transporters from Crithidia fasciculata were cloned and functionally characterized. Sequence analysis revealed CfNT1 and CfNT2 to be members of the equilibrative nucleoside transporter family, and the genes isolated encompassed polypeptides of 497 and 502 amino acids, respectively, each with 11 predicted membrane-spanning domains. Heterologous expression of CfNT1 cRNA in Xenopus laevis oocytes or CfNT2 in nucleoside transport-deficient Leishmania donovani demonstrated that CfNT1 is a novel high affinity adenosine transporter that also recognizes inosine, hypoxanthine, and pyrimidine nucleosides, while CfNT2 is a high affinity permease specific for inosine and guanosine. Southern blot analysis revealed that CfNT2 is present as a single copy within the C. fasciculata genome. Starvation of parasites for purines increased CfNT2 transport activity by an order of magnitude, although Northern blot analysis indicated CfNT2 transcript levels increased by <2-fold. These data imply that this metabolic adaptation can mainly be ascribed to post-transcriptional events. Conversely, Southern analysis of CfNT1 suggests that it is a member of a highly homologous multi-copy gene family, indicating that adenosine transport by C. fasciculata is more complex than previously thought.

Identification and Characterization of a Novel Monoamine Transporter in the Human Brain

Precise control of monoamine neurotransmitter levels in the extracellular fluids of the brain is critical in maintaining efficient and robust neurotransmission. High affinity transporters in the solute carrier SLC6A family function in removing monoamines from the neurosynaptic cleft. Emerging evidence suggests that these transporters are only one part of a system of transporters that work in concert to maintain brain homeostasis of monoamines. Here we report the cloning and characterization of a new human plasma membrane monoamine transporter, PMAT. The PMAT cDNA encodes a protein of 530 amino acid residues with 10-12 transmembrane segments. PMAT is not homologous to known neurotransmitter transporters but exhibits low homology to members of the equilibrative nucleoside transporter family. When expressed in Madin-Darby canine kidney cells and Xenopus laevis oocytes, PMAT efficiently transports serotonin (K(m) = 114 mum), dopamine (K(m) = 329 mum), and the neurotoxin 1-methyl-4-phenylpyridinium (K(m) = 33 mum). In contrast, there is no significant interaction of PMAT with nucleosides or nucleobases. PMAT-mediated monoamine transport does not require Na(+) or Cl(-) but appears to be sensitive to changes in membrane potential. Northern blot analysis showed that PMAT is predominantly expressed in the human brain and widely distributed in the central nervous system. These studies demonstrate that PMAT may be a novel low affinity transporter for biogenic amines, which, under certain conditions, might supplement the role of the high affinity transporters in the brain.

Characterization of three novel members of the Arabidopsis thaliana equilibrative nucleoside transporter (ENT) family.

Research on metabolism of nucleotides and their derivatives has gained increasing interest in the recent past. This includes de novo synthesis, analysis of salvage pathways, breakdown and transport of nucleotides, nucleosides and nucleobases. To perform a further step towards the analysis of nucleoside transport in Arabidopsis, we incubated leaf discs with various radioactively labelled nucleosides. Leaf cells imported labelled nucleosides and incorporated these compounds into RNA, but not into DNA. Furthermore, we report on the biochemical properties of three so far uncharacterized members of the Arabidopsis ENT (equilibrative nucleoside transporter) family (AtENT4, AtENT6 and AtENT7). After heterologous expression in yeast, all three proteins exhibited broad substrate specificity and transported the purine nucleosides adenosine and guanosine, as well as the pyrimidine nucleosides cytidine and uridine. The apparent K(m) values were in the range 3-94 microM, and transport was inhibited most strongly by deoxynucleosides, and to a smaller extent by nucleobases. Typical inhibitors of mammalian ENT proteins, such as dilazep and NBMPR (nitrobenzylmercaptopurine ribonucleoside, also known as nitrobenzylthioinosine) surprisingly exerted almost no effect on Arabidopsis ENT proteins. Transport mediated by the AtENT isoforms differed in pH-dependency, e.g. AtENT7 was not affected by changes in pH, AtENT3, 4 and 6 exhibited a less pronounced pH-dependency, and AtENT1 activity was clearly pH-dependent. Using a GFP (green fluorescent protein)-fusion protein transiently expressed in tobacco leaf protoplasts, a localization of AtENT6 in the plant plasma membrane has been revealed.

Distribution of CNT2 and ENT1 transcripts in rat brain: selective decrease of CNT2 mRNA in the cerebral cortex of sleep-deprived rats.

Nucleoside transport processes regulate the levels of adenosine available to modulate neurotransmission, vascular tone and other physiological events. However, although equilibrative transporter transcripts or proteins have been mapped in the central nervous system of rats and humans, little is known about the presence and distribution of the complete family of nucleoside transporters in brain. In this study, we analysed the distribution of the transcript encoding the high affinity adenosine-preferring concentrative transporter CNT2 in the rat central nervous system and compared it with that of the equilibrative transporter ENT1. Furthermore, we evaluated the changes in expression of these two transporters in a situation of increased extracellular levels of adenosine, such as sleep deprivation. CNT2 mRNA was widespread in rat brain, although most prevalent in the amygdala, the hippocampus, specific neocortical regions and the cerebellum. The distribution of CNT2 mRNA only partially overlapped that of ENT1. Most of the cells labelled were neurones. Total sleep deprivation dramatically diminished the amounts of CNT2 mRNA, whereas ENT1 mRNA remained unchanged. This specific decrease in CNT2 transcript suggests a new physiological role for the transporter in the modulation of extracellular adenosine levels and the sleep/wakefulness cycle.

Functional redundancy of two nucleoside transporters of the ENT family (CeENT1, CeENT2) required for development of Caenorhabditis elegans.

The genome of Caenorhabditis elegans encodes multiple homologues of the two major families of mammalian equilibrative and concentrative nucleoside transporters. As part of a programme aimed at understanding the biological rationale underlying the multiplicity of eukaryote nucleoside transporters, we have now demonstrated that the nematode genes ZK809.4 (ent-1) and K09A9.3 (ent-2) encode equilibrative transporters, which we designate CeENT1 and CeENT2 respectively. These transporters resemble their human counterparts hENT1 and hENT2 in exhibiting similar broad permeant specificities for nucleosides, while differing in their permeant selectivities for nucleobases. They are insensitive to the classic inhibitors of mammalian nucleoside transport, nitrobenzylthioinosine, dilazep and draflazine, but are inhibited by the vasoactive drug dipyridamole. Use of green fluorescent protein reporter constructs indicated that the transporters are present in a limited number of locations in the adult, including intestine and pharynx. Their potential roles in these tissues were explored by using RNA interference to disrupt gene expression. Although disruption of ent-1 or ent-2 expression alone had no effect, simultaneous disruption of both genes yielded pronounced developmental defects involving the intestine and vulva.

Transmembrane domain 5 of the LdNT1.1 nucleoside transporter is an amphipathic helix that forms part of the nucleoside translocation pathway.

Transporters of the equilibrative nucleoside transporter (ENT) family promote the uptake of nucleosides, nucleobases, and a variety of therapeutic drugs in eukaryotes from protozoa to mammals. Despite its importance, the translocation pathway that mediates the internalization of these substrates has not been identified yet in any of the ENT carriers. Previous genetic studies on the LdNT1.1 nucleoside transporter from Leishmania donovani defined two amino acid residues in predicted transmembrane domains (TMD) 5 and 7 that may line this translocation pathway. The role of TMD5 in forming a portion of the aqueous channel was investigated using the substituted-cysteine accessibility method. A series of 22 cysteine substitution mutants spanning predicted TMD5 were created from a fully functional, cysteine-less, parental LdNT1.1. Cysteine replacement at six positions (M(176)C, T(186)C, S(187)C, Q(190)C, V(193)C, and K(194)C) produced permeases that were inhibited by incubation with sulfhydryl-specific methanethiosulfonate reagents, denoting their solvent accessibility to the translocation pathway. Adenosine was able to block this thiol modification, implying that access to the domain becomes restricted as a consequence of the substrate binding. Strikingly, the Q(190)C substitution interacted differentially with the substrates adenosine and uridine, suggesting that binding of adenosine but not uridine might directly occlude this position. When superimposed on a helical model, all six mutants clustered along one face of the amphipathic alpha-helix predicted for TMD5, strongly suggesting its involvement in the translocation pathway through LdNT1.1.

Glycine 154 of the equilibrative nucleoside transporter, hENT1, is important for nucleoside transport and for conferring sensitivity to the inhibitors nitrobenzylthioinosine, dipyridamole, and dilazep

hENT1 and hENT2 are members of the human equilibrative nucleoside transporter family. hENT1 is ubiquitously expressed and plays an important role in the disposition and pharmacological activity of nucleoside drugs and nucleosides, such as adenosine. hENT2 is expressed in only a few tissues (e.g. muscle). hENT1 and hENT2 differ in their affinity for nucleoside substrates and in their sensitivity to inhibitors, such as nitrobenzylthioinosine (NBMPR). hENT1 has higher (or equal) affinity to hENT2 for all natural nucleosides except inosine. hENT1 is also more sensitive to NBMPR inhibition ( ic 50∼0.4 –8 nM) when compared with hENT2 ( ic 50∼2.8 μM). This difference in inhibition potency is substantially dependent on the difference in amino acid at position 154 in hENT1 (glycine) and hENT2 (serine). Since NBMPR competitively inhibits nucleoside transporter activity, we hypothesized that G154 may also play a role in the transport of natural nucleosides and in the inhibition by other hENT1 inhibitors, dipyridamole (DP), and dilazep (DZ). Our results, using a yeast expression system, demonstrate that substituting glycine 154 of hENT1 with serine of hENT2 converts hENT1 to a transporter that exhibits partial characteristics of hENT2. For example, this conversion reduces sensitivity of hENT1 to the inhibitors NBMPR, DP, and DZ and reduces its transport affinity for the natural nucleosides cytidine and adenosine. However, this conversion renders hENT1 less sensitive to inhibition by anti-HIV drugs azidothymidine, dideoxyinosine, and the nucleobase, hypoxanthine. Collectively, these results suggest that glycine 154 plays an important role in the transport of nucleosides and in sensitivity to the inhibitors NBMPR, DP, and DZ.

Functional expression and characterization of a purine nucleobase transporter gene from Leishmania major.

Leishmania major, like all the other kinetoplastid protozoa, are unable to synthesize purines and rely on purine nucleobase and nucleoside acquisition across the parasite plasma membrane by specific permeases. Although, several genes have been cloned that encode nucleoside transporters in Leishmania and Trypanosoma brucei, much less progress has been made on nucleobase transporters, especially at the molecular level. The studies reported here have cloned and expressed the first gene for a L. major nucleobase transporter, designated LmaNT3. The LmaNT3 permease shows 33% identity to L. donovani nucleoside transporter 1.1 (LdNT1.1) and is, thus, a member of the equilibrative nucleoside transporter (ENT) family. ENT family members identified to date are nucleoside transporters, some of which also transport one or several nucleobases. Functional expression studies in Xenopus laevis oocytes revealed that LmaNT3 mediates high levels of uptake of hypoxanthine, xanthine, adenine and guanine. Moreover, LmaNT3 is an high affinity transporter with K(m) values for hypoxanthine, xanthine, adenine and guanine of 16.5 +/- 1.5, 8.5 +/- 0.6, 8.5 +/- 1.1, and 8.8 +/- 4.0 microM, respectively. LmaNT3 is, thus, the first member of the ENT family identified in any organism that functions as a nucleobase rather than nucleoside or nucleoside/nucleobase transporter.

Purification and properties of theEscherichia colinucleoside transporter NupG, a paradigm for a major facilitator transporter sub-family

NupG from Escherichia coli is the archetype of a family of nucleoside transporters found in several eubacterial groups and has distant homologues in eukaryotes, including man. To facilitate investigation of its molecular mechanism, we developed methods for expressing an oligohistidine-tagged form of NupG both at high levels (>20% of the inner membrane protein) in E. coli and in Xenopus laevis oocytes. In E. coli recombinant NupG transported purine (adenosine) and pyrimidine (uridine) nucleosides with apparent K(m) values of approximately 20-30 microM and transport was energized primarily by the membrane potential component of the proton motive force. Competition experiments in E. coli and measurements of uptake in oocytes confirmed that NupG was a broad-specificity transporter of purine and pyrimidine nucleosides. Importantly, using high-level expression in E. coli and magic-angle spinning cross-polarization solid-state nuclear magnetic resonance, we have for the first time been able directly to measure the binding of the permeant ([1'-(13)C]uridine) to the protein and to assess its relative mobility within the binding site, under non-energized conditions. Purification of over-expressed NupG to near homogeneity by metal chelate affinity chromatography, with retention of transport function in reconstitution assays, was also achieved. Fourier transform infrared and circular dichroism spectroscopy provided further evidence that the purified protein retained its 3D conformation and was predominantly alpha-helical in nature, consistent with a proposed structure containing 12 transmembrane helices. These findings open the way to elucidating the molecular mechanism of transport in this key family of membrane transporters.

Interaction of the novel adenosine uptake inhibitor 3-[1-(6,7-diethoxy-2-morpholinoquinazolin-4-yl)piperidin-4-yl]-1,6-dimethyl-2,4(1H,3H)-quinazolinedione hydrochloride (KF24345) with the es and ei subtypes of equilibrative nucleoside transporters.

Nucleosides such as adenosine, as well as many nucleoside-based drugs, permeate cell membranes via a family of equilibrative nucleoside transporters (ENTs). We assessed the effects of (3-[1-(6,7-diethoxy-2-morpholino-quinazolin-4-yl)piperidin-4-yl]-1,6-dimethyl-2,4(1H,3H)-quinazolinedione hydrochloride (KF24345), a novel anti-inflammatory agent that potentiates the actions of adenosine, on the es (inhibitor-sensitive) and ei (inhibitor-resistant) subtypes of ENTs in human, mouse, and rat cells. KF24345 was similar to the prototypical high-affinity inhibitor nitrobenzylthioinosine (NBMPR) for blocking the human es transporter (K(I) of approximately 0.4 nM), but was 50-fold more effective than NBMPR at blocking the human ei transporter (K(I) of approximately 100 nM). KF24345 displayed significantly less species heterogeneity in its affinity for the es transporter than did dipyridamole, a widely used inhibitor of nucleoside transport; KF24345 may thus prove useful as an inhibitor for studies of nucleoside metabolism in a range of animal models. Furthermore, KF24345 seemed to act as a noncompetitive inhibitor of both [(3)H]NBMPR binding and [(3)H]nucleoside uptake by human es transporters, and these kinetics were consistent with an observed slow dissociation of KF24345 from the inhibitor binding site. KF24345 also exhibited unusual biphasic profiles for inhibition of [(3)H]NBMPR binding to membranes prepared from a recombinant human es transporter model (PK15-hENT1), suggesting the presence of multiple populations of NBMPR binding proteins in these membranes. The atypical tight binding interaction of KF24345 with the es transporter may prove useful for the molecular delineation of inhibitor binding domains and will facilitate its use as an in vivo inhibitor of nucleoside transport in studies focused on the biological effects of adenosine.

Nucleoside anticancer drugs: the role of nucleoside transporters in resistance to cancer chemotherapy.

The clinical efficacy of anticancer nucleoside drugs depends on a complex interplay of transporters mediating entry of nucleoside drugs into cells, efflux mechanisms that remove drugs from intracellular compartments and cellular metabolism to active metabolites. Nucleoside transporters (NTs) are important determinants for salvage of preformed nucleosides and mediated uptake of antimetabolite nucleoside drugs into target cells. The focus of this review is the two families of human nucleoside transporters (hENTs, hCNTs) and their role in transport of cytotoxic chemotherapeutic nucleoside drugs. Resistance to anticancer nucleoside drugs is a major clinical problem in which NTs have been implicated. Single nucleotide polymorphisms (SNPs) in drug transporters may contribute to interindividual variation in response to nucleoside drugs. In this review, we give an overview of the functional and molecular characteristics of human NTs and their potential role in resistance to nucleoside drugs and discuss the potential use of genetic polymorphism analyses for NTs to address drug resistance.

Equilibrative Nucleoside Transporter Family Members from Leishmania donovani Are Electrogenic Proton Symporters

Leishmania donovani express two members of the equilibrative nucleoside transporter family; LdNT1 encoded by two closely related and linked genes, LdNT1.1 and LdNT1.2, that transport adenosine and pyrimidine nucleosides and LdNT2 that transports inosine and guanosine exclusively. LdNT1.1, LdNT1.2, and LdNT2 have been expressed in Xenopus laevis oocytes and found to be electrogenic in the presence of nucleoside ligands for which they mediate transport. Further analysis revealed that ligand uptake and transport currents through LdNT1-type transporters are proton-dependent. In addition to the flux of protons that is coupled to the transport reaction, LdNT1 transporters mediate a variable constitutive proton conductance that is blocked by substrates and dipyridamole. Surprisingly, LdNT1.1 and LdNT1.2 exhibit different electrogenic properties, despite their close sequence homology. This electrophysiological study provides the first demonstration that members of the equilibrative nucleoside transporter family can be electrogenic and establishes that these three permeases, unlike their mammalian counterparts, are probably concentrative rather than facilitative transporters.

Molecular and functional characterization of the first nucleobase transporter gene from African trypanosomes.

African trypanosomes are unable to synthesize purines and depend upon purine nucleoside and nucleobase transporters to salvage these compounds from their hosts. To understand the crucial role of purine salvage in the survival of these parasites, a central objective is to identify and characterize all of the purine permeases that mediate uptake of these essential nutrients. We have cloned and functionally expressed in a purine nucleobase transport deficient strain of Saccharomyces cerevisiae a novel nucleobase transporter gene, TbNT8.1, from Trypanosoma brucei. The permease encoded by this gene mediates the uptake of hypoxanthine, adenine, guanine, and xanthine with Kms in the low micromolar range. The TbNT8.1 protein is a member of the equilibrative nucleoside transporter (ENT) family of permeases that occur in organisms as diverse as protozoa and mammals. TbNT8.1 is distinct from other ENT permeases that have been identified in trypanosomes in utilizing multiple purine nucleobases, rather than purine nucleosides, as substrates and is hence the first bona fide nucleobase permease identified in these parasites. Furthermore, unlike the mRNAs for other purine transporters, TbNT8.1 mRNA is significantly more abundant in insect stage procyclic forms than in mammalian stage bloodstream forms, and the TbNT8.1 permease thus may represent a major route for purine nucleobase uptake in procyclic trypanosomes.

The concentrative nucleoside transporter family, SLC28.

The SLC28 family consists of three subtypes of sodium-dependent, concentrative nucleoside transporters, CNT1, CNT2, and CNT3 (SLC28A1, SLC28A2, and SLC28A3, respectively), that transport both naturally occurring nucleosides and synthetic nucleoside analogs used in the treatment of various diseases. These subtypes differ in their substrate specificities: CNT1 is pyrimidine-nucleoside preferring, CNT2 is purine-nucleoside preferring, and CNT3 transports both pyrimidine and purine nucleosides. Recent studies have identified key amino acid residues that are determinants of pyrimidine and purine specificity of CNT1 and CNT2. The tissue distributions of the CNTs vary: CNT1 is localized primarily in epithelia, whereas CNT2 and CNT3 have more generalized distributions. Nucleoside transporters in the SLC28 and SLC29 families play critical roles in nucleoside salvage pathways where they mediate the first step of nucleotide biosynthesis. In addition, these transporters work in concert to terminate adenosine signaling. SLC28 family members are crucial determinants of response to a variety of anticancer and antiviral nucleoside analogs, as they modulate the entry of these analogs into target tissues. Further, this family is involved in the absorption and disposition of many nucleoside analogs. Several CNT single nucleoside polymorphisms (SNPs) have been identified, but have yet to be characterized.