The human concentrative (Na ؉ -linked) plasma membrane transport proteins hCNT1 and hCNT2 are selective for pyrimidine nucleosides (system cit) and purine nucleosides (system cif), respectively. Both have homologs in other mammalian species and belong to a gene family (CNT) that also includes hfCNT, a newly identified broad specificity pyrimidine and purine Na ؉ -nucleoside symporter (system cib) from the ancient marine vertebrate, the Pacific hagfish (Eptatretus stouti). We now report the cDNA cloning and characterization of cib homologs of hfCNT from human mammary gland, differentiated human myeloid HL-60 cells, and mouse liver. The 691-and 703-residue human and mouse proteins, designated hCNT3 and mCNT3, respectively, were 79% identical in amino acid sequence and contained 13 putative transmembrane helices. hCNT3 was 48, 47, and 57% identical to hCNT1, hCNT2, and hfCNT, respectively. When produced in Xenopus oocytes, both proteins exhibited Na ؉ -dependent cib-type functional activities. hCNT3 was electrogenic, and a sigmoidal dependence of uridine influx on Na ؉ concentration indicated a Na ؉ : uridine coupling ratio of at least 2:1 for both hCNT3 and mCNT3 (cf 1:1 for hCNT1/2). Phorbol myristate acetateinduced differentiation of HL-60 cells led to the parallel appearance of cib-type activity and hCNT3 mRNA. Tissues containing hCNT3 transcripts included pancreas, bone marrow, trachea, mammary gland, liver, prostrate, and regions of intestine, brain, and heart. The hCNT3 gene mapped to chromosome 9q22.2 and included an upstream phorbol myristate acetate response element.Most nucleosides, including those with antineoplastic and/or antiviral activities (1, 2), are hydrophilic, and specialized plasma membrane nucleoside transporter (NT) 1 proteins are required for uptake into or release from cells (3, 4). NT-mediated transport is therefore a critical determinant of metabolism and, for nucleoside drugs, their pharmacologic actions (5). NTs also regulate adenosine concentrations in the vicinity of cell surface receptors and have profound effects on neurotransmission, vascular tone, and other processes (6, 7).Seven nucleoside transport processes 2 that differ in their cation dependence, permeant selectivities and inhibitor sensitivities have been observed in human and other mammalian cells and tissues. The major concentrative systems (cit, cif, and cib) are inwardly directed Na ϩ -dependent processes and have been primarily described in specialized epithelia such as intestine, kidney, liver, and choroid plexus, in other regions of the brain, and in splenocytes, macrophages, and leukemic cells (3, 4). Concentrative NT transcripts have also been found in heart, skeletal muscle, placenta, and pancreas. The equilibrative (bidirectional) transport processes (es and ei) have generally lower substrate affinities and occur in most, possibly all, cell types (3, 4). Epithelia (e.g. intestine and kidney) and some nonpolarized cells (e.g. leukemic cells) coexpress both concentrative and equilibrative NTs, whereas other nonpola...

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 proteintagged 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.

Equilibrative nucleoside transport processes in mammalian cells are either nitrobenzylthioinosine (NBMPR)-sensitive (es) or NBMPR-insensitive (ei). Previously, we isolated a cDNA from human placenta encoding the 456-residue glycoprotein hENT1. When expressed in Xenopus oocytes, hENT1 mediated es-type transport activity and was inhibited by coronary vasoactive drugs (dipyridamole and dilazep) that may compete with nucleosides and NBMPR for binding to the substrate binding site. We now report the molecular cloning and functional expression of es and ei homologs of hENT1 from rat tissues; rENT1 (457 residues) was 78% identical to hENT1 in amino acid sequence, and rENT2 (456 residues) was 49 -50% identical to rENT1/hENT1 and corresponded to a full-length form of the delayed-early proliferative response gene product HNP36, a protein of unknown function previously cloned in truncated form. rENT1 was inhibited by NBMPR (IC 50 ‫؍‬ 4.6 nM at 10 M uridine), whereas rENT2 was NBMPR-insensitive (IC 50 > 1 M). Both proteins mediated saturable uridine influx (K m ‫؍‬ 0.15 and 0.30 mM, respectively), were broadly selective for purine and pyrimidine nucleosides, including adenosine, and were relatively insensitive to inhibition by dipyridamole and dilazep (IC 50 > 1 M). These observations demonstrate that es and ei nucleoside transport activities are mediated by separate, but homologous, proteins and establish a function for the HNP36 gene product.

The human (h) and rat (r) equilibrative (Na ؉ -independent) nucleoside transporters (ENTs) hENT1, rENT1, hENT2, and rENT2 belong to a family of integral membrane proteins with 11 transmembrane domains (TMs) and are distinguished functionally by differences in sensitivity to inhibition by nitrobenzylthioinosine and coronary vasoactive drugs. Structurally, the proteins have a large glycosylated loop between TMs 1 and 2 and a large cytoplasmic loop between TMs 6 and 7. In the present study, hENT1, rENT1, hENT2, and rENT2 were produced in Xenopus laevis oocytes and investigated for their ability to transport pyrimidine and purine nucleobases. hENT2 and rENT2 efficiently transported radiolabeled hypoxanthine, adenine, guanine, uracil, and thymine (apparent K m values 0.7-2.6 mM), and hENT2, but not rENT2, also transported cytosine. These findings were independently confirmed by hypoxanthine transport experiments with recombinant hENT2 produced in purine-cytosine permease (FCY2)-deficient Saccharomyces cerevisiae and provide the first direct demonstration that the ENT2 isoform is a dual mechanism for the cellular uptake of nucleosides and nucleobases, both of which are physiologically important salvage metabolites. In contrast, recombinant hENT1 and rENT1 mediated negligible oocyte fluxes of hypoxanthine relative to hENT2 and rENT2. Chimeric experiments between rENT1 and rENT2 using splice sites at rENT1 residues 99 (end of TM 2), 171 (between TMs 4 and 5), and 231 (end of TM 6) identified TMs 5-6 of rENT2 (amino acid residues 172-231) as a determinant of nucleobase transport activity, suggesting that this domain forms part(s) of the ENT2 substrate translocation channel.Plasma membrane transport processes for nucleosides and nucleobases play key roles in many aspects of mammalian physiology and pharmacology (1-5). In particular, uptake of exogenous nucleosides and nucleobases is the first step of nucleotide synthesis in tissues such as bone marrow and intestinal epithelium (and certain parasitic organisms) that lack de novo pathways for purine biosynthesis (5-7). The same transport processes also mediate cellular uptake of many synthetic nucleoside and nucleobase analogs used in cancer, viral, and parasite chemotherapy (3)(4)(5)8). Independent transport processes specific for nucleosides or nucleobases as well as shared mechanisms of nucleoside and nucleobase transport have been described (2, 4).In human and other mammalian cells and tissues, uptake of nucleosides is brought about by members of the concentrative (Na ϩ -dependent) nucleoside transporter (CNT) 1 and equilibrative (Na ϩ -independent) nucleoside transporter (ENT) families (3, 5). CNTs have been described primarily in specialized epithelia, whereas ENTs occur in most, possibly all, cell types and tissues. Three CNT and two ENT isoforms have been identified. Human (h) and rat (r) CNT1 and CNT2 both transport uridine, but are otherwise selective for pyrimidine (hCNT1 and rCNT1) and purine (hCNT2 and rCNT2) nucleosides (9 -14). In contrast, hCNT3 and its mous...

The concentrative nucleoside transporter (CNT) protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. hCNT3, a Na ؉ /nucleoside symporter, transports a broad range of physiological purine and pyrimidine nucleosides as well as anticancer and antiviral nucleoside drugs, and belongs to a different CNT subfamily than hCNT1/2. H ؉ -dependent Escherichia coli NupC and Candida albicans CaCNT are also CNT family members. The present study utilized heterologous expression in Xenopus oocytes to investigate the specificity, mechanism, energetics, and structural basis of hCNT3 cation coupling. hCNT3 exhibited uniquely broad cation interactions with Na Physiological nucleosides and synthetic nucleoside analogs have important biochemical, physiological, and pharmacological activities in humans. Adenosine, for example, has purinoreceptor-mediated functions in processes such as modulation of immune responses, platelet aggregation, renal function, and coronary vasodilation (1, 2). Nucleosides also provide salvage precursors for nucleic acid biosynthesis, and nucleoside drugs are commonly used in the therapy of cancer and viral infections (3, 4). Most nucleosides, including those with antineoplastic and/or antiviral activities, are hydrophilic and require specialized plasma membrane nucleoside transporter (NT) 1 proteins for their uptake into or release from cells (5-7).Multiple transport systems for nucleosides have been observed in human and other mammalian cells and tissues (7-9). The concentrative systems (cit, cif, and cib) 2 are inwardly directed Na ϩ -dependent processes present primarily in intestinal and renal epithelia and other specialized cells (7-9). The equilibrative systems (es and ei) mediate bidirectional downhill transport of nucleosides, have generally lower substrate affinities than the concentrative systems, and occur in most, possibly all, cell types (7-9). Systems cit and cif transport adenosine and uridine, but are otherwise pyrimidine and purine nucleoside-selective, respectively, whereas systems cib, es, and ei transport both pyrimidine and purine nucleosides. System es is inhibited by nanomolar concentrations of nitrobenzylthioinosine (NBMPR), whereas system ei also transports nucleobases (7-10).Molecular cloning studies have identified the human and rodent integral membrane proteins responsible for each of these nucleoside transport activities (11-18). They belong to two previously unrecognized and structurally unrelated protein families (CNT and ENT), and their relationship to the processes defined by functional studies is: CNT1 (cit), CNT2 (cif), CNT3 (cib), ENT1 (es), and ENT2 (ei) (11)(12)(13)(14)(15)(16)(17)(18). In addition to ENT1 and ENT2, the ENT protein family also contains three further human and rodent members (ENT3, ENT4, and CLN3) (19 -23). Human and other eukaryote CNTs have 13 predicted transmembrane helices (TMs), with an intracellular N terminus and an extracellular C terminus (24). NupC, an H ϩ -coupled CNT from Escherichia coli, has a similar predicted to...