Mark F. Vickers

Nucleoside Transporters of Mammalian Cells

In this review, we have summarized recent advances in our understanding of the biology of nucleoside transport arising from new insights provided by the isolation and functional expression of cDNAs encoding the major nucleoside transporters of mammalian cells. Nucleoside transporters are required for permeation of nucleosides across biological membranes and are present in the plasma membranes of most cell types. There is growing evidence that functional nucleoside transporters are required for translocation of nucleosides between intracellular compartments and thus are also present in organellar membranes. Functional studies during the 1980s established that nucleoside transport in mammalian cells occurs by two mechanistically distinct processes, facilitated diffusion and Na(+)-nucleoside cotransport. The determination of the primary amino acid sequences of the equilibrative and concentrative transporters of human and rat cells has provided a structural basis for the functional differences among the different transporter subtypes. Although nucleoside transporter proteins were first purified from human erythrocytes a decade ago, the low abundance of nucleoside transporter proteins in membranes of mammalian cells has hindered analysis of relationships between transporter structure and function. The molecular cloning of cDNAs encoding nucleoside transporters and the development of heterologous expression systems for production of recombinant nucleoside transporters, when combined with recombinant DNA technologies, provide powerful tools for characterization of functional domains within transporter proteins that are involved in nucleoside recognition and translocation. As relationships between molecular structure and function are determined, it should be possible to develop new approaches for optimizing the transportability of nucleoside drugs into diseased tissues, for development of new transport inhibitors, including reagents that are targeted to the concentrative transporters, and, eventually, for manipulation of transporter function through an understanding of the regulation of transport activity.

Distinct regional distribution of human equilibrative nucleoside transporter proteins 1 and 2 (hENT1 and hENT2) in the central nervous system

Nucleoside transport processes play an important role in human cells in salvage of nucleosides used in the biosynthesis of nucleic acids and in regulating endogenous adenosine concentrations in the human central nervous system (CNS). By altering the levels of adenosine available to interact with cell-surface receptors, nucleoside transporters have profound effects on the ability of adenosine to modulate neurotransmission, vascular tone and other physiological events. Although the human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) are believed to play a crucial role in modulating brain function, their distribution within the major divisions of the human CNS is not known. In this work, antibodies specific for hENT1 and hENT2 were produced against fragments of the transporter proteins and used for immunoblot analysis of enriched membrane fractions prepared from several regions of the human brain. While hENT1 was most prevalent in the frontal and parietal lobes of the cerebral cortex, thalamus, midbrain and basal ganglia, hENT2 was concentrated in the cerebellum and brainstem regions, particularly the pons. The apparent reciprocal distribution of hENT1 and hENT2 in human brain suggests that these nucleoside transporter proteins are produced in distinct regions of the CNS where they function in nucleoside salvage and/or regulation of exogenous adenosine. Within the brain regions that were investigated, the pattern of hENT1 distribution correlated well with adenosine A(1) receptor abundance. The regional co-localization of hENT1 and A(1) receptor protein suggests an important role of hENT1-mediated transport process in the control of neuromodulatory actions mediated by adenosine A(1) receptors in human brain.

Functional production and reconstitution of the human equilibrative nucleoside transporter (hENT1) in Saccharomyces cerevisiae Interaction of inhibitors of nucleoside transport with recombinant hENT1 and a glycosylation-defective derivative (hENT1/N48Q)

We have produced recombinant human equilibrative nucleoside transporter (hENT1) in the yeast Saccharomyces cerevisiae and have compared the binding of inhibitors of equilibrative nucleoside transport with the wild-type transporter and a N-glycosylation-defective mutant transporter. Equilibrium binding of 3H-labelled nitrobenzylmercaptopurine ribonucleoside {6-[(4-nitrobenzyl)thio]-9-beta-d-ribofuranosyl purine; NBMPR} to hENT1-producing yeast revealed a single class of high-affinity sites that were shown to be in membrane fractions by (1) equilibrium binding (means+/-S.D.) of [3H]NBMPR to intact yeast (Kd 1.2+/-0.2 nM; Bmax 5.0+/-0.5 pmol/mg of protein) and membranes (Kd 0.7+/-0.2 nM; Bmax 6.5+/-1 pmol/mg of protein), and (2) reconstitution of hENT1-mediated [3H]thymidine transport into proteoliposomes that was potently inhibited by NBMPR. Dilazep and dipyridamole inhibited NBMPR binding to hENT1 with IC50 values of 130+/-10 and 380+/-20 nM respectively. The role of N-linked glycosylation in the interaction of NBMPR with hENT1 was examined by the quantification of binding of [3H]NBMPR to yeast producing either wild-type hENT1 or a glycosylation-defective mutant (hENT1/N48Q) in which Asn-48 was converted into Gln. The Kd for binding of NBMPR to hENT1/N48Q was 10. 5+/-1.6 nM, indicating that the replacement of an Asn residue with Gln decreased the affinity of hENT1 for NBMPR. The decreased affinity of hENT1/N48Q for NBMPR was due to an increased rate of dissociation (koff) and a decreased rate of association (kon) of specifically bound [3H]NBMPR because the values for hENT1-producing and hENT1/N48Q-producing yeast were respectively 0.14+/-0.02 and 0. 36+/-0.05 min-1 for koff, and (1.2+/-0.1)x10(8) and (0.40+/-0. 04)x10(8) M-1.min-1 for kon. These results indicated that the conservative conversion of an Asn residue into Gln at position 48 of hENT1 and/or the loss of N-linked glycosylation capability altered the binding characteristics of the transporter for NBMPR, dilazep and dipyridamole.

A Comparison of the Transportability, and Its Role in Cytotoxicity, of Clofarabine, Cladribine, and Fludarabine by Recombinant Human Nucleoside Transporters Produced in Three Model Expression Systems

2-Chloro-9-(2′-deoxy-2′-fluoro-β-d-arabinofuranosyl)adenine (Cl-F-ara-A, clofarabine), a purine nucleoside analog with structural similarity to 2-chloro-2′-deoxyadenosine (Cl-dAdo, cladribine) and 9-β-d-arabinofuranosyl-2-fluoroadenine (F-ara-A, fludarabine), has activity in adult and pediatric leukemias. Mediated transport of the purine nucleoside analogs is believed to occur through the action of two structurally unrelated protein families, the equilibrative nucleoside transporters (ENTs) and the concentrative nucleoside transporters (CNTs). The current work assessed the transportability of Cl-F-ara-A, Cl-dAdo, and F-ara-A in cultured human leukemic CEM cells that were either nucleoside transport-defective or possessed individual human nucleoside transporter types and in Xenopus laevis oocytes and Saccharomyces cerevisiae yeast that produced individual recombinant human nucleoside transporter types. Cells producing hENT1 or hCNT3 exhibited the highest uptake of Cl-F-ara-A, whereas nucleoside transport-deficient cells and cells producing hCNT1 lacked uptake altogether. When Cl-F-ara-A transport rates by hENT1 were compared with those of Cl-dAdo and F-ara-A, Cl-dAdo had the highest efficiency of transport, although Cl-F-ara-A showed the greatest accumulation during 5-min exposures. In cytotoxicity studies with the CEM lines, Cl-F-ara-A was more cytotoxic to cells producing hENT1 than to the nucleoside transport-deficient cells. The efficiency of Cl-F-ara-A transport by oocytes with recombinant transporters was hCNT3 > hENT2 > hENT1 > hCNT2; no transport was observed with hCNT1. Affinity studies with recombinant transporters produced in yeast showed that hENT1, hENT2, and hCNT3 all had higher affinities for Cl-F-ara-A than for either Cl-dAdo or F-ara-A. These results suggest that the nature and activity of the plasma membrane proteins capable of inward transport of nucleosides are important determinants of Cl-F-ara-A activity in human cells.

Uridine recognition motifs of human equilibrative nucleoside transporters 1 and 2 produced in Saccharomyces cerevisiae.

The sugar moiety of nucleosides has been shown to play a major role in permeant‐transporter interaction with human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2). To better understand the structural requirements for interactions with hENT1 and hENT2, a series of uridine analogs with sugar modifications were subjected to an assay that tested their abilities to inhibit [3H]uridine transport mediated by recombinant hENT1 and hENT2 produced in Saccharomyces cerevisiae. hENT1 displayed higher affinity for uridine than hENT2. Both transporters barely tolerated modifications or inversion of configuration at C(3′). The C(2′)‐OH at uridine was a structural determinant for uridine‐hENT1, but not for uridine‐hENT2, interactions. Both transporters were sensitive to modifications at C(5′) and hENT2 displayed more tolerance to removal of C(5′)‐OH than hENT1; addition of an O‐methyl group at C(5′) greatly reduced interaction with either hENT1 or hENT2. The changes in binding energies between transporter proteins and the different uridine analogs suggested that hENT1 formed strong interactions with C(3′)‐OH and moderate interactions with C(2′)‐OH and C(5′)‐OH of uridine, whereas hENT2 formed strong interactions with C(3′)‐OH, weak interactions with C(5′)‐OH, and no interaction with C(2′)‐OH. †In honor and celebration of the 70th birthday of Professor Leroy B. Townsend. #These authors contributed equally to this work.

Functional production of mammalian concentrative nucleoside transporters in Saccharomyces cerevisiae.

The transport of nucleosides and nucleobases in the yeast Saccharomyces cerevisiae is reviewed and the use of this organism to study recombinant mammalian concentrative nucleoside transport (CNT) proteins is described. A selection strategy based on the ability of an expressed nucleoside transporter cDNA to mediate thymidine uptake by yeast under a selective condition that depletes endogenous thymidylate was used to assess the transport capacity of heterologous transporter proteins. The pyrimidine-nucleoside selective concentrative transporters from human (hCNT1) and rat (rCNT1) complemented the imposed thymidylate depletion in S. cerevisiae, as did N-terminally truncated versions of hCNT1 and rCNT1 lacking up to 31 amino acids. Transporter-mediated rescue of S. cerevisiae by both nucleoside transporters was inhibited by cytidine, uridine and adenosine, but not by guanosine or inosine. This work represents the development of a new model system for the functional production of recombinant nucleoside transporters of the CNT family of membrane proteins.