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 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...
Background: Gemcitabine, a pyrimidine analogue of deoxycytidine, is an anticancer nucleoside drug that requires functional plasma membrane nucleoside transporter proteins to reach its intracellular targets and cause cytotoxicity. Because of technical difficulties inherent in studying nucleoside transport in human cells, we rigorously defined gemcitabine membrane transportability by producing each of the available human (h) and rat (r) recombinant nucleoside transporters (NTs) individually in Xenopus laevis oocytes. Methods: Oocytes were microinjected with in vitro-transcribed RNAs derived from complementary DNAs encoding (C = concentrative) rCNT1, rCNT2, hCNT1, hCNT2, (E = equilibrative) rENT1, rENT2, hENT1, and hENT2. Uptake of [ 3 H]gemcitabine and [ 14 C] uridine was measured 3 days after microinjection to determine kinetic constants. We also used the two-electrode, voltage-clamp technique to investigate the electrophysiology of hCNT1-mediated gemcitabine transport. Results: Gemcitabine was transported by most of the tested proteins (the exceptions being the purine-selective rCNT2 and hCNT2), with the greatest uptake occurring in oocytes producing recombinant rCNT1 and hCNT1. Influxes of gemcitabine mediated by hCNT1, hENT1, and hENT2 were saturable and conformed to Michaelis-Menten kinetics with apparent K m values of 24, 160, and 740 µM, respectively. Gemcitabine had a limited ability to cross the lipid bilayer of oocyte membranes by simple diffusion. External application of gemcitabine to oocytes producing recombinant hCNT1 induced an inward current, which demonstrated that hCNT1 functions as a Na
Human concentrative nucleoside transporter 1 (hCNT1) mediates active transport of nucleosides and anticancer and antiviral nucleoside drugs across cell membranes by coupling influx to the movement of Na + down its electrochemical gradient. The two-microelectrode voltage-clamp technique was used to measure steady-state and presteady-state currents of recombinant hCNT1 produced in Xenopus oocytes. Transport was electrogenic, phloridzin sensitive and specific for pyrimidine nucleosides and adenosine. Nucleoside analogues that induced inwardly directed Na + currents included the anticancer drugs 5-fluorouridine, 5-fluoro-2 -deoxyuridine, cladribine and cytarabine, the antiviral drugs zidovudine and zalcitabine, and the novel thymidine mimics 1-(2-deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-methylbenzene and 1-(2-deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-iodobenzene. Apparent K m values for 5-fluorouridine, 5-fluoro-2 -deoxyuridine and zidovudine were 18, 15 and 450 µM, respectively. hCNT1 was Na + specific, and the kinetics of steady-state uridineevoked Na + currents were consistent with an ordered simultaneous transport model in which Na + binds first followed by uridine. Membrane potential influenced both ion binding and carrier translocation. The Na + -nucleoside coupling stoichiometry, determined directly by comparing the uridine-induced inward charge movement to [ 14 C]uridine uptake was 1 : 1. hCNT1 presteady-state currents were used to determine the fraction of the membrane field sensed by Na + (61%), the valency of the movable charge (−0.81) and the average number of transporters present in the oocyte plasma membrane (6.8 × 10 10 per cell). The hCNT1 turnover rate at −50 mV was 9.6 molecules of uridine transported per second.
The human concentrative (Na+-linked) plasma membrane transport proteins hCNT1 and hCNT2, found primarily in specialized epithelia, are selective for pyrimidine nucleosides (system cit) and purine nucleosides (system cif), respectively. Both have orthologs in other mammalian species and belong to a gene family (CNT) that also includes members in lower vertebrates, insects, nematodes, pathogenic yeast and bacteria. The CNT transporter family also includes a newly identified human and mouse CNT3 transporter isoform. This paper reviews the studies of CNT transport proteins that led to the identification of hCNT3 and mCNT3, and gives an overview of the structural and functional properties of these latest CNT family members. hCNT3 and mCNT3 have primary structures that place them in a CNT subfamily separate from CNT1/2, transport a wide range of physiological pyrimidine and purine nucleosides and antineoplastic and antiviral nucleoside drugs (system cib), and exhibit a Na+:uridine coupling ratio of at least 2:1 (cf 1:1 for hCNT1/2). Cells and tissues containing hCNT3 transcripts include mammary gland, differentiated HL-60 cells, pancreas, bone marrow, trachea, liver, prostrate and regions of intestine, brain and heart. In HL-60 cells, hCNT3 is transcriptionally regulated by phorbol myristate (PMA). The hCNT3 gene, which contains an upstream PMA response element, mapped to 9q22.2 (cf chromosome 15 for hCNT1 and hCNT2).
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