Pflugers Arch - Eur J Physiol (2004) 447:735–743 DOI 10.1007/s00424-003-1103-2
The ABC of Solute Carriers
Guest Editor: Matthias A. Hediger
Stephen A. Baldwin · Paul R. Beal · Sylvia Y. M. Yao · Anne E. King · Carol E. Cass · James D. Young
The equilibrative nucleoside transporter family, SLC29
Received: 17 March 2003 / Accepted: 28 April 2003 / Published online: 28 June 2003 Springer-Verlag 2003
Abstract The human SLC29 family of proteins contains four members, designated equilibrative nucleoside transporters (ENTs) because of the properties of the firstcharacterised 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 S. A. Baldwin ()) · P. R. Beal School of Biochemistry and Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK e-mail:
[email protected] Fax: +44-113-3433173 A. E. King School of Biomedical Sciences, University of Leeds, Leeds, LS2 9JT, UK S. Y. M. Yao · J. D. Young Membrane Transport Research Group, Department of Physiology, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada C. E. Cass Membrane Transport Research Group, Department of Oncology (Cross Cancer Institute), University of Alberta, Edmonton, Alberta, T6G 2H7, Canada
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. Keywords Adenosine · Cancer · Cardiovascular · ENT · Equilibrative · Nitrobenzylthioinosine · Nucleoside · Transporter
History Studies performed over the past 30 years have revealed that most mammalian cells exhibit low-affinity, equilibrative nucleoside transport processes, now known to be mediated by members of the SLC29 family (reviewed in [13]). Some tissues, typified by liver, small intestine and kidney, have also been found to exhibit concentrative, sodium-dependent nucleoside transport, mediated by members of the SLC28 family (reviewed elsewhere in this volume). Prior to identification of their genes, the equilibrative transporters were classified, on the basis of their sensitivity to inhibition by nitrobenzylthioinosine (nitrobenzylmercaptopurine riboside; NBMPR), as es (equilibrative sensitive) or ei (equilibrative insensitive) [4]. Purification and N-terminal sequencing of the archetypal es transporter from human erythrocytes enabled us to clone a human placental cDNA encoding the corresponding transporter, designated hENT1, in 1996 [14]. cDNA clones encoding the ei-type transporter, hENT2, were subsequently isolated in our laboratory by virtue of its homology with hENT1 [15], and in parallel by a functional complementation approach in Belt’s laboratory [9]. cDNAs encoding the remaining two members of the family, hENT3 and hENT4, have more
BK000627
Widely distributed, possibly intracellular
Widely distributed
F
Not determined
Not determined
Purine and pyrimidine nucleosides and nucleobases
Purine and pyrimidine nucleosides and some nucleobases Adenosine
Ubiquitous, plasma membrane (basolateral in polarised renal epithelial cells) and perinuclear membranes Ubiquitous, plasma membrane (basolateral in polarised renal epithelial cells). Particularly abundant in skeletal muscle F Purine and pyrimidine nucleosides
ENT4
ENT2 SLC29A2
ENT3
ENT1 SLC29A1
Fig. 1 Phylogenetic tree of the human SLC29 amino acid sequences, showing distant relationship to the human CLN3 protein (GenBank accession NP_000077) A acquired defect, C cotransporter, E exchanger, F facilitated transporter, G genetic defect, O orphan transporter, P pseudogene
7 (precise locus not yet known)
NM_001532 11q13
NM_018344
NM_004955 6p21.1– p21.2
10q22.1
Sequence Accession ID Aliases Protein name Human gene name
Table 1 SLC29—the nucleoside transporter family
Tissue distribution and cellular/subcellular expression Transport type /coupling ions Predominant substrates
Link to disease
Human gene locus
HNP36: exon 4 splice variant leading to N- terminal truncation and lack of transport function hENT2A: exon 9 splice variant leading to C-terminal truncation and lack of transport function
Splice variants and their specific features
736
Fig. 2 Topographical model of hENT1. This model is based on the results of glycosylation scanning mutagenesis studies and other approaches detailed in [40]. Predicted membrane-spanning ahelices are numbered and the site of N-glycosylation indicated. Where residues are identical in 50% of the Pfam 01733 members listed [19], the most common residue is indicated using the single letter code. Met33 and Gly154 of hENT1, implicated in coronary vasodilator and NBMPR sensitivity respectively as described in the text, are in red. Coloured boxes indicate the regions implicated from chimaera studies in the recognition of nucleobase substrates (yellow) and coronary vasodilator drugs and NBMPR (yellow and green)
recently been identified and isolated as a result of the completion of the human genome project ([1, 19]; S.A. Baldwin et al., unpublished work). The SLC29 family (Table 1) is part of a larger group of equilibrative and concentrative nucleoside and nucleobase transporters (the ENT family; Pfam 01733) found in many eukaryotes [19]. PSI-BLAST [3] searching of the GenBank protein sequence database has also revealed a distant but significant (expect value 3e-73 after four iterations) sequence similarity between hENT1 and the human protein CLN3 (battenin; Pfam 02487) (Fig. 1). This 438-residue lysosomal protein is predicted to exhibit
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a similar transmembrane topology to members of the SLC29 family (see below). Although its function remains unclear, it has been proposed to play a role in lysosomal pH homeostasis and mutations in the corresponding gene are associated with the neurodegenerative disease neuronal ceroid lipofuscinosis [43].
by NBMPR (Fig. 3a) with approximately equal potency (Ki~5 nM) in the human and rodent proteins [14, 17, 22, 51]. However, rENT1 can be distinguished from hENT1 and mENT1 in being essentially insensitive to inhibition by the coronary vasodilators dipyridamole (Fig. 3a) and dilazep [14, 22, 51].
Functional characteristics
ENT2 (SLC29A2)
The best characterised members of the SLC29 family, ENT1 and ENT2, are known to be facilitated diffusion systems although they are homologous to active, protonlinked transporters in kinetoplastid protozoa [19]. The cation-dependence of ENT3 and ENT4 has not yet been fully characterised. All four members of the family share an ability to transport adenosine, but differ in their abilities to transport other nucleosides and nucleobases (Fig. 3a).
Human ENT2 (hENT2) is a 456-residue protein 46% identical in amino acid sequence to hENT1 and 88% identical to the 456-residue mouse (mENT2) and rat (rENT2) homologues [9, 15, 22, 51]. An mRNA splice variant lacking part of exon 4 has been described as the product of a delayed early-response gene [49]. Because of a resultant frameshift, this mRNA encodes a 326-residue truncated protein, designated hHNP36, that lacks the first three transmembrane (TM) helices of hENT2 and is nonfunctional as a transporter [9]. A second, widely distributed splice variant, bearing a 40-bp deletion in exon 9, yields a 301-residue C-terminally truncated protein (hENT2A) that similarly lacks activity [25]. ENT2 mRNA is expressed in a wide range of tissues including brain, heart, placenta, thymus, pancreas, prostate and kidney, but is particularly abundant in skeletal muscle [9, 15]. Antibodies raised against the central cytoplasmic loop of HNP36/hENT2 were reported to stain nucleolar structures in cultured cells [49]. However, we have used immunocytochemistry to show cell surface location of ENT2 in rat cardiomyocytes [6], and GFP fusions of hENT2 are targeted to the basolateral membrane when expressed in polarised MDCK cells [25]. h/rENT2 transport a broad range of purine and pyrimidine nucleosides, although with a lower apparent affinity than h/rENT1 except in the case of inosine [9, 15, 46]. However, they differ from h/rENT1 in also being able efficiently to transport a wide range of purine and pyrimidine nucleobases, except for cytosine, which is not transported by rENT2 (Fig. 3a) [9, 54]. Although the apparent affinities for nucleobases are lower than those for the corresponding nucleosides, the turnover number for transport is higher, such that at physiological concentrations the efficiencies of nucleoside and nucleobase transport are similar [54]. A second key difference from hENT1 is that hENT2 can transport AZT and also exhibits a much greater capacity to transport ddC and ddI [52]. hENT2 therefore probably represents an important route for cellular uptake of these clinically important drugs, used in human immunodeficiency virus (HIV) therapy. Thirdly, in contrast to h/rENT1, h/rENT2-mediated transport is only very weakly, if at all, inhibited by NBMPR, dipyridamole or dilazep [9, 15, 22, 51].
Family members ENT1 (SLC29A1) Human ENT1 (hENT1) is a 456-residue protein 78% identical in sequence to its 457-residue rat homologue (rENT1) and 79% identical to the 460-residue mouse protein (mENT1.1) [14, 17, 51]. Splice variants of hENT1 have not been reported, but a 458-residue variant of the mouse homologue (mENT1.2), generated by alternative splicing at the end of exon 7, is widely distributed [17]. It lacks a potential casein kinase II phosphorylation site but appears functionally identical to mENT1.1 [17]. Studies at both the mRNA and protein levels have revealed that ENT1 is almost ubiquitously distributed in human and rodent tissues, although its abundance varies between tissues ([14, 17]; S.A. Baldwin et al., unpublished work). For example, in human brain the transporter is most abundant in the frontal and parietal lobes of the cortex [20]. Immunocytochemical studies on rat kidney cortex sections have shown that the transporter is located on the basolateral surface of tubular epithelial cells, in contrast to the concentrative nucleoside transporter (CNT) protein rCNT1, which is located on the apical surface [6, 16]. hENT1 tagged with GFP or YFP is similarly targeted to the basolateral membrane when expressed in MadinDarby canine kidney (MDCK) cells [23, 25]. In kidney ENT1 may therefore operate in tandem with CNT proteins to bring about transepithelial nucleoside flux. h/rENT1 transport a wide range of purine and pyrimidine nucleosides, with Km values ranging from 50 mM (adenosine) to 680 mM (cytidine), but are unable to transport the pyrimidine base uracil [14, 46]. Similarly, they only poorly transport the antiviral nucleosides 20 ,30 dideoxycytidine (ddC) and 20 ,30 -dideoxyinosine (ddI), and do not transport 30 -azido-30 -deoxythymidine (AZT), indicating the importance of the 30 -hydroxyl group for substrate recognition (Fig. 3b) [52]. Transport is inhibited
ENT3 Human ENT3 (hENT3) is a 475-residue protein 29% identical in sequence to hENT1 and 74% identical to its
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739
475-residue mouse homologue mENT3 [19]. h/mENT3 differ from ENT1 and ENT2 in possessing a very long (51 residues), hydrophilic N-terminal region preceding TM1. This region contains two dileucine motifs, similar to those that mediate the sorting of other membrane proteins at the trans-Golgi network, endosomes and plasma membrane [34]. It is therefore possible that these isoforms reside predominantly in an intracellular compartment rather than at the cell surface. Analysis of multiple tissue RNA arrays indicates that hENT3 is widely expressed in human tissues but is particular abundant in placenta, from which tissue the cDNA was originally cloned ([19], S.A. Baldwin et al., unpublished work). Kinetic characterisation of hENT3 is currently in progress, but our preliminary studies have revealed that the substrate specificity and inhibitor sensitivity of hENT3 are akin to those of hENT2, although the apparent affinity for transported nucleosides is lower and the sensitivity to dipyridamole higher (S.A. Baldwin et al., unpublished work).
ENT4 Human ENT4 (hENT4) is a 530-residue protein 86% identical in sequence to its 528-residue mouse homologue (mENT4). Originally identified by genome database analysis [1], hENT4 is more closely related to the products of the Drosophila melanogaster gene CG11010 (28% identity) and the Anopheles gambiae gene agCG56160 (30% identity) than to hENT1 (18% identity), indicating an ancient divergence from the other members of the SLC29 family. However, recent characterisation of the cDNAs encoding h/mENT4 in our laboratory has confirmed that these proteins are indeed nucleoside transporters, capable of low-affinity adenosine transport (unpublished observations). Analysis of multiple tissue RNA arrays indicates that hENT4 is likely to be ubiquitously expressed in human tissues (S.A. Baldwin et al., unpublished work).
Physiological implications
Fig. 3a–d Physiological roles and therapeutic applications of SLC29 family transporters. a Substrate and inhibitor specificity of hENT1 and hENT2. As described in the text, the transporters have broad substrate specificities: the structures of representative purine and pyrimidine nucleoside and nucleobase substrates are shown. Kinetic evidence indicates that nitrobenzylthioinosine and dipyridamole bind competitively with substrate to the exofacial conformation of the substrate-binding site. b Transport and action of chemotherapeutic nucleoside analogues. Structures of representative analogues are shown, with substituents that differ from natural nucleosides highlighted in red. Cytarabine is used primarily for treatment of acute myelogenous and lymphoblastic leukaemias. Unlike cytarabine, gemcitabine has activity on solid tumours and is used to treat pancreatic, lung and breast cancers. Zalcitabine and zidovudine are 3´-deoxynucleoside analogues used for treatment of HIV infections. Following uptake and activation by metabolism to triphosphates, the analogues inhibit viral replication or inhibit cellular DNA synthesis/repair, leading to apoptotic cell death. c Role of nucleoside transporters in the heart. Imbalance between O2 supply and demand during ischaemia or hypoxia leads to increased generation of adenosine in cardiomyocytes from ATP breakdown, mediated by myokinase (4) and intracellular 5´-nucleotidase (1) and potentiated by inhibition of the salvage enzyme adenosine kinase (3) under these conditions. Interstitial adenosine arises from cellular release via equilibrative nucleoside transporters (ENT) and from breakdown of extracellular AMP by ecto-5´-nucleotidase (2). Adenosine then increases coronary blood flow by acting on endothelial cell A2 receptors and decreases heart rate, in particular via A1-mediated activation of potassium channels in supraventricular tissues. A1 and A3 receptors also appear to mediate the cardioprotective effects of adenosine. ENT inhibitors such as dipyridamole potentiate the cardioprotective and other effects of adenosine, probably by selectively inhibiting uptake by ENT1 into endothelial cells where the nucleoside is broken down by the action of adenosine deaminase (5) and nucleoside phosphorylase (6). Continued release of adenosine from cardiomyocytes may involve dipyridamole-insensitive transporters such as ENT2. d ENT1 inhibitors such as NMBPR suppress nociceptive neurotransmission in the substantia gelatinosa (SG) of the spinal dorsal horn. Inhibition of uptake by ENT1 leads to accumulation of adenosine, which acts presynaptically via A1 receptors to diminish release of the neurotransmitter glutamate [2]. Inhibition of ENTs is therefore potentially of therapeutic value for the control of pain
Equilibrative nucleoside transporters play an important role in the provision of nucleosides, derived from the diet or produced by tissues such as the liver, for salvage pathways of nucleotide synthesis in those cells deficient in de novo biosynthetic pathways. The latter include erythrocytes, leukocytes, bone marrow cells and some cells in the brain [13]. The co-existence in many cell types of both hENT1 and hENT2, which exhibit similar nucleoside specificities, may reflect the importance of the hENT2 substrate hypoxanthine as a source of purines for salvage: in the bone marrow the concentration of this nucleobase can reach 30 mM [42]. Similarly, this ability to transport hypoxanthine and the higher apparent affinity of hENT2 for inosine [46] have been suggested to reflect a role in the efflux or uptake of these adenosine metabolites during muscle exercise and recovery respectively [9]. The nucleoside adenosine also regulates many physiological processes including coronary blood flow, myocardial O2 supply-demand balance, inflammation and neurotransmission via binding to at least four different subtypes of G-protein coupled receptors (A1, A2A, A2B and A3) [37]. By influencing the concentration of adenosine available to these receptors, ENTs play important regulatory roles in such processes (Fig. 3c). For example, they are likely to modulate the effect of adenosine in the sinoatrial (SA) node of the heart, which we have recently shown contains an abundance of ENT1 in the rat [30]. Pharmacological evidence for this role has been provided in the guinea pig SA node, where dipyridamole was found to potentiate the A1 receptormediated chronotropic effects of adenosine via inhibition of uptake [28]. Similarly, rENT1 is abundant in the superficial dorsal horn of the spinal cord, and we have shown that its inhibition by NBMPR and consequent elevation of adenosine concentrations modulates glutamatergic synaptic transmission via presynaptic A1 receptors (Fig. 3d) [2]. hENT1 also modulates A1 receptor-
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mediated adenosine control of K+ channel function in human airway A549 epithelial cells [41].
Regulation of expression The hENT1 content of cultured human cancer cells appears to be coordinated with the cell cycle, levels of the transporter approximately doubling between the G1 and G2-M phases [32]. Experiments with inhibitors of nucleotide synthesis suggest that transporter synthesis and/or cell surface expression is regulated in response to cellular deoxynucleotide levels, but the mechanism of regulation is unknown [32]. In addition to these changes in transporter levels, a recent report has described the rapid activation of cell surface hENT1 in cultured cells in response to phorbol ester treatment [8]. This response appears to involve PKC d and/or e, but it is not yet clear whether changes in the phosphorylation state of the transporter itself are involved [8]. Less is known about the of regulation of hENT2 levels. Although expression of the mouse HNP36 splice variant is greatly enhanced by treatment of quiescent fibroblasts with serum or growth factors as part of the delayed-early response [49], similar regulation of hENT2 has not been detected [9]. Moreover, while colony stimulating factor has been reported to stimulate es-type transport activity, presumably mediated by mENT1, in a mouse macrophage cell line no changes in ei-type activity were observed [27], and so a role for ENT2 in the proliferative response remains unclear.
Structure/function relationships Glycosylation scanning mutagenesis and use of antibodies as topological probes have confirmed the originallyproposed 11 TM topology of hENT1, in which the Nterminus is cytoplasmic while the C-terminus is extracellular (Fig. 2) [40]. hENT1 is N-glycosylated at a single site and hENT2 at two sites in the large extracellular loop linking TM1 and 2, but glycosylation is not essential for activity in either case [44, 47]. h/mENT3 similarly bear a glycosylation site in the TM1–2 loop, and h/mENT4 bear a site in the C-terminal tail, but their glycosylation status remains unknown. Investigations of the properties of chimaeras between different human and rat ENTs have thrown considerable light on the regions of the proteins involved in substrate and inhibitor interaction (Fig. 2). Such studies have revealed that the region encompassing TM3–6 contains residues responsible for sensitivity or resistance both to coronary vasodilators [38] and to NBMPR [39]. The TM1–6 region appears to be responsible for the ability of ENT2 efficiently to transport 3´-deoxynucleosides [52], while the TM5–6 region has been implicated in the ability of this transporter to recognise nucleobases [54]. The properties of point mutants have also been revealing. For example, the effects of mutating hENT1 residue Met33 in
TM1 (Fig. 2) to that found at the corresponding location in hENT2 (Ile), and of the reciprocal mutation in hENT2, have provided evidence for a role of this location in sensitivity or resistance respectively to coronary vasodilators [45]. Similarly, position 154 in hENT1 TM4 (Fig. 2) has been implicated in sensitivity to NBMPR: mutation of Gly154 to Ser, the residue present in hENT2, renders the protein resistant to NBMPR [19]. The proximity of this residue to the inhibitor/substrate-binding site is supported by the finding that chemical modification of a cysteine residue in the corresponding location in rENT2 inhibits transport, and that this modification is prevented by the presence of substrate [53]. Mutation of Gly179 in TM5 of hENT1 to Ala profoundly inhibits transport activity and this residue has been suggested to play a direct role in NBMPR binding [36]. However, the drastic effect of mutating this absolutely conserved residue would also be consistent with a role in helix-helix packing.
Pathological implications There are currently no reports implicating SLC29 transporters in the pathogenesis of human disease. However the transporters, in particular hENT1, play a critical role in the cellular uptake of currently used anticancer nucleoside drugs including cladribine, cytarabine, fludarabine, gemcitabine and capecitabine (reviewed in [7]). These drugs act via incorporation into nucleic acids, by interference with nucleic acid synthesis or by interference with the metabolism of physiological nucleosides (Fig. 3b) [11]. Increased hENT1 abundance may contribute to the relative selectivity of nucleoside chemotherapy for malignant cells: high cellular proliferation rates have been associated with elevated es-type transporter levels and the in vitro sensitivity of acute lymphoblastic leukaemia cells to cladribine has been correlated with the abundance of these proteins (reviewed in [7]). A corollary is that down-regulation of transporter expression or selection of transporter-deficient cells may contribute to clinical resistance to cytotoxic nucleoside analogues: this mechanism has been documented for cytarabine chemotherapy [12, 48]. Measurement of transporter abundance may therefore provide a predictive tool for guiding the appropriate use of such drugs in individual patients: in recent immunocytochemical studies, we found that the abundance of hENT1 in biopsy samples varied markedly between different breast cancer and Hodgkin’s disease patients [24, 33].
Pharmacology and therapeutic applications Because ENTs are bi-directional, they can mediate not only the uptake but also the efflux of therapeutic nucleoside analogues. The cytotoxic effect of exposure of cultured acute lymphocytic leukaemia cells to cladribine has been reported to be enhanced by subsequent treatment with NBMPR to prevent drug efflux [50].
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Hence, selective inhibition of ENTs may represent a means of improving the antitumour efficacy of nucleoside drugs, in particular if these are taken up into cells by concentrative nucleoside transporters. In addition to NBMPR and the coronary vasodilator drugs such as dipyridamole, dilazep and lidoflazine analogues, many inhibitors of serine/threonine and of tyrosine kinases, such as the Bcr-Abl tyrosine kinase inhibitor STI-571 (Gleevec) used for treatment of chronic myelogenous leukaemia, have recently also been shown to be moderately potent inhibitors of ENT1 and/or ENT2 (IC50 values for inhibition of 5 mM uridine uptake in the range 60– 1000 nM) [18]. Given the potent pharmacological effects of adenosine, it is possible that some of the effects of these compounds reported in the literature reflect not the involvement of the kinases, but alteration in extracellular adenosine concentration resulting from inhibition of uptake. More importantly, potential antagonism between kinase inhibitors and nucleoside analogue drugs may influence the outcome of combination therapies with these agents. ENT inhibitors, by virtue of their effect on extracellular adenosine concentrations, can also modulate a variety of physiological processes, potentially leading to therapeutic benefits. For example, by inhibiting nucleoside uptake into endothelial and other cells the coronary vasodilator draflazine (Fig. 3a) substantially increases and prolongs the cardiovascular effects of adenosine [10]. The latter exerts beneficial, cardioprotective effects in the ischaemic/reperfused myocardium, mediated at least in part via activation of A1 and possibly also A3 receptors, and probably involving protein kinase C and mitochondrial KATP channels [5, 29]. Such processes are likely to underlie the finding that infusion of the ENT inhibitor R75231 before coronary artery occlusion enhanced postischaemic recovery of function and reduced infarct size in the pig [26]. The beneficial effects of administering the ENT inhibitor dipyridamole during percutaneous transluminal coronary angioplasty in humans are likely to have a similar origin [29]. Transport inhibitors are also potentially of value in the context of ischaemic neuronal injury: pre-ischaemic administration of the pro-drug NBMPRphosphate has been shown to increase brain adenosine levels and reduce ischaemia-induced loss of hippocampal CA1 neurons in the rat [55]. A second area of potential therapeutic application concerns the treatment of chronic pain. Adenosine A1 receptor activation in the spinal cord has antinociceptive effects [35], and intrathecal administration of inhibitors of adenosine metabolism produces antinociception in the rat [31]. Inhibitors of cellular adenosine uptake might potentially have a similar beneficial action, and nucleoside transport inhibitors have been reported to significantly enhance opioid-mediated antinociception in the mouse [21]. Support for the potential utility of this approach has come from our recent demonstration that ENT1 is abundant in the dorsal horn of the rat spinal cord, and that its inhibition by NBMPR leads to presynaptic A1
receptor-mediated inhibition of glutamatergic synaptic transmission in the substantia gelatinosa (Fig. 3d) [2].
Perspective The recent identification and characterisation of four equilibrative (SLC29) and three concentrative (SLC28) types of nucleoside transporter in humans represent important advances. However, we are still at an early stage in our understanding of the individual roles and significance of these proteins for whole organism physiology. Gaining greater understanding, via transgenic and other approaches, is an important task for the future, as is developing a better picture of the molecular mechanisms of these proteins. Progress in both areas will be essential if the therapeutic potential of these transport proteins is to be fully exploited. Acknowledgements This work was supported by the Wellcome Trust, the Medical Research Council of the UK, the Canadian Institutes of Health Research, the National Cancer Institute of Canada, with funds from the Canadian Cancer Society, the Alberta Cancer Board and the Natural Sciences and Engineering Research Council of Canada. J.D.Y. is a Heritage Scientist of the Alberta Heritage Foundation for Medical Research and C.E.C. holds a Canada Research Chair in Oncology.
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