The equilibrative nucleoside transporter family,SLC29

The human SLC29 family of proteins contains four members, designated equilibrative nucleoside transporters (ENTs) because of the properties of the first-characterised family member, hENT1. They belong to the widely-distributed eukaryotic ENT family of equilibrative and concentrative nucleoside/nucleobase transporters and are distantly related to a lysosomal membrane protein, CLN3, mutations in which cause neuronal ceroid lipofuscinosis. A predicted topology of 11 transmembrane helices with a cytoplasmic N-terminus and an extracellular C-terminus has been experimentally confirmed for hENT1. The best-characterised members of the family, hENT1 and hENT2, possess similar broad substrate specificities for purine and pyrimidine nucleosides, but hENT2 in addition efficiently transports nucleobases. The ENT3 and ENT4 isoforms have more recently also been shown to be genuine nucleoside transporters. All four isoforms are widely distributed in mammalian tissues, although their relative abundance varies: ENT2 is particularly abundant in skeletal muscle. In polarised cells ENT1 and ENT2 are found in the basolateral membrane and, in tandem with concentrative transporters of the SLC28 family, may play a role in transepithelial nucleoside transport. The transporters play key roles in nucleoside and nucleobase uptake for salvage pathways of nucleotide synthesis, and are also responsible for the cellular uptake of nucleoside analogues used in the treatment of cancers and viral diseases. In addition, by regulating the concentration of adenosine available to cell surface receptors, they influence many physiological processes ranging from cardiovascular activity to neurotransmission.     

Two high affinity nucleoside transporters in Leishmania donovani

A rapid sampling kinetic technique has been used to evaluate the nucleoside transport functions of Leishmania donovani. The results indicated that L. donovani promastigotes possessed two independent purine nucleoside transporters with nonoverlapping substrate specificity. The first transported inosine, guanosine, and their analogs, while the second carried adenosine, analogs of adenosine, and the pyrimidine nucleosides, uridine, cytidine, and thymidine. The apparent Km values of the two nucleoside permeases for their purine nucleoside substrates were extraordinarily low, in the micromolar range. The organisms were capable of concentrating purine nucleosides from the medium and converting them to the nucleotide level with great efficiency and rapidity. Inosine and adenosine transport could be distinguished by different sensitivities to sulfhydryl reagents, suggesting structural differences between the two transporters. Finally, the two nucleoside transport systems of L. donovani were virtually refractory to inhibition by 4-nitrobenzylthioinosine and dipyridamole, two potent inhibitors of nucleoside entry into mammalian cells.     

Genomic organization and functional characterization of the human concentrative nucleoside transporter-3 isoform (hCNT3) expressed in mammalian cells

Human CNT3 encodes the concentrative nucleoside transport N3 system. Previous expression studies in oocytes showed that the K_m values for nucleosides of the cloned hCNT3 were 7- to 25-fold lower than the endogenous N3 transporter in HL60 cells. Therefore, in the present study we re-examined the kinetic properties of the cloned hCNT3 using mammalian cell expression systems by transient expression in Cos7L cells and stably expression in nucleoside transporter deficient PK15NTD cells. We demonstrated that hCNT3 is a Na-dependent, broadly-selective nucleoside transporter with affinities (<11 M) for nucleosides closely resembling the endogenous N3 transporter. Pharmacological studies showed that phloridzin is a mixed-type inhibitor of hCNT3 (K_i=15 M), and the dideoxyuridine analogs are poor substrates. By epitope-tagging, we further demonstrated that hCNT3 is N-glycosylated as PNGase F and Endo H deglycosylated hCNT3 from 67 kDa to 58 kDa. Searching the human genome database, we identified the genomic organization of hCNT3. This gene contains 19 exons and its exon-intron boundaries within the coding sequence exactly match with those of hCNT1 and hCNT2 with one additional exon in the N-terminus. Our data suggest that hCNT3 gene is evolutionarily conserved with hCNT1 and hCNT2. Physiologically, hCNT3 is a glycoprotein, which transports purine and pyrimidine nucleosides in a Na-dependent manner with high affinities.     

Role of CNT3 in the transepithelial flux of nucleosides and nucleoside-derived drugs

We examined the role of the concentrative nucleoside transporter CNT3 in the establishment of a transepithelial flux of natural nucleosides and their pharmacologically active derivatives in renal epithelial cell lines. Murine PCT cells grown on a transwell dish showed endogenous CNT3 activity at their apical membrane that was responsible for the sodium-dependent transepithelial flux of both purine and pyrimidine nucleosides. hCNT3 was also identified in human kidney and its role in the transport of nucleosides was tested. To this end, MDCK cells, lacking endogenous CNT3 activity, were genetically engineered to express the human orthologue of CNT3 (hCNT3-MDCK cells). In these cells, hCNT3 was inserted into the apical membrane, thus generating, as for PCT cells, a transepithelial flux of both nucleosides and nucleoside-derived drugs. Apical-to-basolateral transepithelial flux was present in all cells expressing a functional CNT3 transporter and was significantly higher than that found either in PCT cells in absence of sodium or in mock-transfected MDCK cells. Nevertheless in all cases a significant amount of the transported nucleoside was retained and transformed inside cells. However release to the opposite compartment was CNT3 dependent, not only in terms of absolute flux (much higher when an apical CNT3 transporter was active) but also regarding metabolic transformations of the apically absorbed nucleosides. These results underline a critical role of CNT3 in the renal reabsorption of nucleosides and their derivatives as well as in their intracellular metabolism.     

Stage-specific development of a novel adenosine transporter in Leishmania donovani amastigotes

Leishmania donovani, like all other kinetoplastida, is a purine auxotroph. Comparative studies of adenosine transport in L. donovani amastigotes and promastigotes revealed that, unlike the promastigote stage, the amastigote possesses two distinct adenosine transporters (T(1) and T(2)) both with high affinities (K(m), 1.14+/-0.05 and 2. 09+/-0.13 microM, respectively). One of these transporters (T(1)) appears to be identical with the adenosine/pyrimidine nucleoside transporter of the promastigote reported earlier. The other transporter (T(2)) is specific for the amastigote stage and transports only purine nucleosides. The biological significance of this stage-specific development of the second adenosine transporter has been briefly discussed.     

Two novel nucleobase/pentamidine transporters from Trypanosoma brucei

African trypanosomes are unable to synthesize purines de novo and must salvage preformed purine nucleosides and nucleobases from their hosts. The Trypanosoma brucei genome project has identified 12 members of the equilibrative nucleoside transporter family, most of which have been characterized previously as nucleoside and/or nucleobase transporters. Here the 11th member of this family, TbNT11.1, has been functionally expressed in null mutants of Leishmania that are deficient in purine nucleoside or nucleobase uptake and identified as a high-affinity purine nucleobase transporter. Expression of TbNT11.1 in Xenopus oocytes revealed that it is also a transporter for the diamidine drug pentamidine that is the principal drug employed to treat early stage human African trypanosomiasis and may thus contribute to the uptake of this therapeutically important compound. In addition, characterization of the 12th member of the family, TbNT12.1, reveals that it is an adenine/pentamidine transporter.     

Differential regulation of nucleoside and nucleobase transporters in Crithidia fasciculata and Trypanosoma brucei brucei

The regulation of the activity of purine transporters in two protozoan species, Crithidia fasciculata and Trypanosoma brucei brucei, was investigated in relation to purine availability and growth cycle. In C. fasciculata, two high-affinity purine nucleoside transporters were identified. The first, designated CfNT1, displayed a K(m) of 9.4 +/- 2.8 microM for adenosine and was inhibited by pyrimidine nucleosides as well as adenosine analogues; a second C. fasciculata nucleoside transporter (CfNT2) recognized inosine (K(m) = 0.38 +/- 0.06 microM) and guanosine but not adenosine. The activity of both transporters increased in cells at mid-logarithmic growth, as compared to cells in the stationary phase, and was also stimulated 5-15-fold following growth in purine-depleted medium. These increased rates were due to increased Vmax values (K(m) remained unchanged) and inhibited by cycloheximide (10 microM). In the procyclic forms of T. b. brucei, adenosine transport by the P1 transporter was upregulated by purine starvation but only after 48 h, whereas hypoxanthine transport was maximally increased after 24 h. The latter effect was due to the expression of an additional hypoxanthine transporter, H2, that is normally absent from procyclic forms of T. b. brucei and was characterised by its high affinity for hypoxanthine (K(m) approximately 0.2 microM) and its sensitivity to inhibition by guanosine. The activity of the H1 hypoxanthine transporter (K(m) approximately 10 microM) was unchanged. These results show that regulation of the capacity of the purine transporters is common in different protozoa, and that, in T. b. brucei, various purine transporters are under differential control.     

Sodium-coupled neutral amino acid (System N/A) transporters of the SLC38 gene family

The sodium-coupled neutral amino acid transporters (SNAT) of the SLC38 gene family resemble the classically-described System A and System N transport activities in terms of their functional properties and patterns of regulation. Transport of small, aliphatic amino acids by System A subtypes (SNAT1, SNAT2, and SNAT4) is rheogenic and pH sensitive. The System N subtypes SNAT3 and SNAT5 also countertransport H(+), which may be key to their operation in reverse, and have narrower substrate profiles than do the System A subtypes. Glutamine emerges as a favored substrate throughout the family, except for SNAT4. The SLC38 transporters undoubtedly play many physiological roles including the transfer of glutamine from astrocyte to neuron in the CNS, ammonia detoxification and gluconeogenesis in the liver, and the renal response to acidosis. Probing their regulation has revealed additional roles, and recent work has considered SLC38 transporters as therapeutic targets in neoplasia.     

Reduced purine accumulation is encoded on an amplified DNA in Leishmania mexicana amazonensis resistant to toxic nucleosides

Nucleoside analogs are potential anti-Leishmania agents. To better understand how these compounds might lose their effectiveness, Leishmania were independently selected for resistance to inosine dialdehyde or tubercidin. Each of the resistant cells exhibited resistance to inosine dialdehyde and tubercidin as well as to formycin B and allopurinol ribonucleoside. Resistant cells had a greatly reduced capability of accumulating exogenous adenosine, guanosine, thymidine and guanine. This decreased ability to accumulate nucleosides and at least one nucleobase appeared to be due to reduced activity of a number of distinct purine transporters, as the differences between purine metabolizing enzymes were not sufficiently different to account for the decreased accumulation capability. The resistance to toxic nucleosides and the decreased ability to accumulate purines were due to the presence in the resistant cells of an extrachromosomal DNA approximately 55 kb in size. The extrachromosomal DNA was not detected in wild-type cells or revertants which have lost resistance to toxic nucleosides. Except for a 1.2-kb difference, the extrachromosomal DNA from both independently selected resistant cells appeared to be identical. The resistant cells contained 2–4 times as much DNA homologous to the extrachromosomal DNA as compared to wild type cells. When cloned into an E. coli/Leishmania shuttle vector, a portion of the amplified DNA had the ability to confer upon wild-type cells resistance to the toxic purine nucleoside analogs tubercidin and inosine dialdehyde. These transformed cells also exhibited a decreased ability to accumulate non-toxic purine nucleosides.     

Functional characterization of nucleoside transporter gene replacements in Leishmania donovani

Leishmania donovani express two nucleoside transporters of non-overlapping ligand selectivity. To evaluate the physiological role of nucleoside transporters in L. donovani, homozygous null mutants of the genes encoding the LdNT1 adenosine–pyrimidine nucleoside transporter and the LdNT2 inosine–guanosine transporter were created singly and in combination by single targeted gene replacement followed by selection for loss-of-heterozygosity. The mutant alleles were verified by Southern blotting, and the effects of gene replacement on transport phenotype were evaluated by rapid sampling transport measurements and by drug resistance profiles. The Δldnt1, Δldnt2, and Δldnt1/Δldnt2 mutants were all capable of proliferation in defined culture medium supplemented with any of a spectrum of purine nucleobases or nucleosides, except that a Δldnt2 lesion conferred an inability to efficiently salvage exogenous xanthosine, a newly discovered ligand of LdNT2. Each of the three knockout strains was viable as promastigotes and axenic amastigotes and capable of maintaining an infection in J774 and bone marrow-derived murine macrophages. These genetic studies demonstrate: (1) that L. donovani promastigotes, axenic amastigotes, and tissue amastigotes are viable in the absence of nucleoside transport; (2) that nucleoside transporters are not essential for sustaining an infection in mammalian host cells; (3) that the phagolysosome of macrophages is likely to contain purines that are not LdNT1 or LdNT2 ligands, i.e., nucleobases. Furthermore, the Δldnt1, Δldnt2, and Δldnt1/Δldnt2 knockouts offer a unique genetically defined null background for the biochemical and genetic characterization of nucleoside transporter genes and cDNAs from phylogenetically diverse species and of genetically manipulated LdNT1 and LdNT2 constructs.     

Purine metabolism in the intact sporozoites and merozoites of Eimeria tenella

Intact Eimeria tenella sporozoites and merozoites did not incorporate radiolabeled formate or glycine into their purine nucleotides suggesting a lack of de novo purine synthesis. However, [U-14C]glucose was incorporated into the cellular purine and pyrimidine nucleotide pools of both forms probably via conversion to radiolabeled ribose-1-phosphate and/or 5'-phosphoribosyl-1-alpha-pyrophosphate and the resulting action of various purine and pyrimidine salvage enzymes. Both forms of the parasite salvaged radiolabeled purine bases and nucleosides in a similar fashion. These purines were incorporated into ribonucleotides and into RNA and DNA. Adenine and inosine were transformed to hypoxanthine. Adenosine was converted to both inosine and hypoxanthine. Hypoxanthine and xanthine were not oxidized to uric acid but were metabolized to nucleotides. Guanosine was cleaved to guanine; guanine was deaminated to xanthine. The results demonstrate the presence of several purine salvage pathways. Purine phosphoribosylating and nucleoside phosphorylating activities as well as purine nucleoside cleaving and adenosine, adenine and guanine deaminating activities were evident. The metabolic evidence suggests the enzymes required to convert the newly formed nucleoside monophosphates to ATP and GTP were present also.     

Screening of genetic variations of SLC15A2, SLC22A1, SLC22A2 and SLC22A6 genes

A growing list of membrane-spanning proteins involved in the transport of a large variety of drugs has been recognized and characterized to include peptide and organic anion/cation transporters. Given such an important role of transporter genes in drug disposition process, the role of single-nucleotide polymorphisms (SNPs) in such transporters as potential determinants of interindividual variability in drug disposition and pharmacological response has been investigated. To define the distribution of transporter gene SNPs across ethnic groups, we screened 450 DNAs in cohorts of 250 Korean, 50 Han Chinese, 50 Japanese, 50 African-American and 50 European-American ancestries for 64 SNPs in four transporter genes encoding proteins of the solute carrier family (SLC15A2, SLC22A1, SLC22A2 and SLC22A6). Of the 64 SNPs, 19 were core pharmacogenetic variants and 45 were HapMap tagging SNPs. Polymorphisms were genotyped using the golden gate genotyping assay. After genetic variability, haplotype structures and ethnic diversity were analyzed, we observed that the distributions of SNPs in a Korean population were similar to other Asian groups (Chinese and Japanese), and significantly different from African-American and European-American cohorts. Findings from this study would be valuable for further researches, including pharmacogenetic studies for drug responses.     

Characterization of nucleoside uptake and transport in Entamoeba histolytica

The uptake and transportation of purine and pyrimidine based nucleosides by trophozoites of axenically grown Entamoeba histolytica (HMI-IMSS) were studied. The trophozoites transported adenosine and its analog tubercidin (1 μM) at a significant rate but poor transportation was observed in case of uridine (about 10% relative rate), inosine (3%?), thymidine (2%?) and formycin B (1%?). The Km for adenosine was 160?±?42 μM. Unlabeled nucleosides (100 μM) inhibited adenosine and tubercidin transport. Adenosine related compounds 5′-deoxyadenosine and nebularin inhibited adenosine and tubercidine transport by 50%? or more. However, inosine related compounds guanosine, 3′-deoxyinosine and formycin B were less inhibitory. The pyrimidine nucleosides uridine, thymidine and cytidine were poorly inhibitory. 6-[(4 nitrobenzyl)-mercapto] purine ribonucleoside, an inhibitor of mammalian nucleoside transporter, inhibited adenosine or tubercidin transport in E. histolytica variably between 0–30%?at 10?μM, but dilazep, a known inhibitor, was inactive upto 10 μM. Increase in temperature from 22°C to 33°C enhanced the rate of transport of adenosine 4.5 fold, tubercidin 7.3 fold and of inosine 4 fold. These findings along with the structure activity figures suggested that transport was mediated and not passive.     

Sodium-coupled dicarboxylate and citrate transporters from the SLC13 family

The SLC13 family in humans and other mammals consists of sodium-coupled transporters for anionic substrates: three transporters for dicarboxylates/citrate and two transporters for sulfate. This review will focus on the di- and tricarboxylate transporters: NaDC1 (SLC13A2), NaDC3 (SLC13A3), and NaCT (SLC13A5). The substrates of these transporters are metabolic intermediates of the citric acid cycle, including citrate, succinate, and α-ketoglutarate, which can exert signaling effects through specific receptors or can affect metabolic enzymes directly. The SLC13 transporters are important for regulating plasma, urinary and tissue levels of these metabolites. NaDC1, primarily found on the apical membranes of renal proximal tubule and small intestinal cells, is involved in regulating urinary levels of citrate and plays a role in kidney stone development. NaDC3 has a wider tissue distribution and high substrate affinity compared with NaDC1. NaDC3 participates in drug and xenobiotic excretion through interactions with organic anion transporters. NaCT is primarily a citrate transporter located in the liver and brain, and its activity may regulate metabolic processes. The recent crystal structure of the Vibrio cholerae homolog, VcINDY, provides a new framework for understanding the mechanism of transport in this family. This review summarizes current knowledge of the structure, function, and regulation of the di- and tricarboxylate transporters of the SLC13 family.     

Localization of equilibrative nucleoside transporters in the rat brain

Adenosine is a recognized inhibitory neuromodulator and neuroprotective agent in the central nervous system. It is produced both intra- and extracellularly and transported across the cell membrane. Nucleoside transporters thus have a major impact on the extracellular adenosine levels, and consequently adenosine signalling. We have raised and characterized polyclonal antibodies against both the equilibrative nucleoside transporters 1 and 2, and report for the first time their distribution in rat brain at the cellular level. Double staining studies were performed to assess the localization of the transporters in neural and glial cells. Both transporters were present in practically all neurons. Some astrocytes showed equilibrative nucleoside transporter 1 staining, while equilibrative nucleoside transporter 2 staining on astrocytes was observed only sporadically.     

The glutamate/neutral amino acid transporter family SLC1: molecular, physiological and pharmacological aspects

The solute carrier family 1 (SLC1) includes five high-affinity glutamate transporters, EAAC1, GLT-1, GLAST, EAAT4 and EAAT5 (SLC1A1, SLC1A2, SLC1A3, SLC1A6, and SLC1A7, respectively) as well as the two neutral amino acid transporters, ASCT1 and ASCT2 (SLC1A4 and ALC1A5, respectively). Although each of these transporters have similar predicted structures, they exhibit distinct functional properties which are variations of a common transport mechanism. The high-affinity glutamate transporters mediate transport of l-Glu, l-Asp and d-Asp, accompanied by the cotransport of 3 Na(+) and 1 H(+), and the countertransport of 1 K(+), whereas ASC transporters mediate Na(+)-dependent exchange of small neutral amino acids such as Ala, Ser, Cys and Thr. The unique coupling of the glutamate transporters allows uphill transport of glutamate into cells against a concentration gradient. This feature plays a crucial role in protecting neurons against glutamate excitotoxicity in the central nervous system. During pathological conditions, such as brain ischemia (e.g. after a stroke), however, glutamate exit can occur due to "reversed glutamate transport", which is caused by a reversal of the electrochemical gradients of the coupling ions. Selective inhibition of the neuronal glutamate transporter EAAC1 (SLC1A1) may be of therapeutic interest to block glutamate release from neurons during ischemia. On the other hand, upregulation of the glial glutamate transporter GLT1 (SLC1A2) may help protect motor neurons in patients with amyotrophic lateral sclerosis (ALS), since loss of function of GLT1 has been associated with the pathogenesis of certain forms of ALS.     

The proton oligopeptide cotransporter family SLC15 in physiology and pharmacology

Mammalian members of the SLC15 family are electrogenic transporters that utilize the proton-motive force for uphill transport of short chain peptides and peptido-mimetics into a variety of cells. The prototype transporters of this family are PEPT1 (SLC15A1) and PEPT2 (SLC15A2), which mediate the uptake of peptide substrates into intestinal and renal epithelial cells. More recently, other sites of functional expression of the two proteins have been identified such as bile duct epithelium (PEPT1), glia cells and epithelia of the choroid plexus, lung and mammary gland (PEPT2). Both proteins can transport essentially every possible di- and tripeptide regardless of the substrate's net charge, but operate stereoselectively. Based on peptide-like structures, various drugs and prodrugs are transported as well, allowing efficient intestinal absorption of the compounds via PEPT1. In kidney tubules both peptide transporters can mediate the renal reabsorption of the filtered compounds thus affecting their pharmacokinetics. Recently, two new peptide transporters, PHT1 (SLC15A4) and PHT2 (SLC15A3), were identified in mammals. They possess an overall amino acid identity with the PEPT-series of 20% to 25%. PHT1 and PHT2 were shown to transport free histidine and certain di- and tripeptides, but it is not yet clear whether they are located on the plasma membrane or represent lysosomal transporters for the proton-dependent export of histidine and dipeptides from lysosomal protein degradation into the cytosol.     

Identification and characterization of trypanocides by functional expression of an adenosine transporter from Trypanosoma brucei in yeast

The causative agents of sleeping sickness, Trypanosoma brucei rhodesiense and T. brucei gambiense, do not synthesize purines de novo but salvage purine bases and nucleosides from their hosts. We used yeast as an expression system for functional characterization of the trypanosomal adenosine transporter TbAT1. A selection of purine analogs and flavonoids were tested for their ability to interfere with adenosine transport, with the aims of identifying (a) trypanocidal TbAT1 substrates, and (b) inhibitors of trypanosomal purine transport. Cordycepin (3'-deoxyadenosine) was a TbAT1 substrate of high activity against T. brucei rhodesiense (IC50 0.2 nM). Inhibitors of mammalian nucleoside transport were not active, while the flavonol silibinin was a potent, noncompetitive inhibitor of TbAT1-mediated adenosine transport in yeast. Silibinin also inhibited melarsen-induced lysis of bloodstream form trypanosomes. IC50 values to T. brucei rhodesiense and to human carcinoma cells were 0.6 and 140 microM, respectively, indicating a good selectivity towards the parasites. Further studies are necessary to elucidate the effects of flavonoids on trypanosomal purine transport and their potential as trypanocides.