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Human equilibrative nucleoside transporter 1 (hENT1), the first identified member of the ENT family of integral membrane proteins, is the primary mechanism for cellular uptake of physiologic nucleosides and many antineoplastic and antiviral nucleoside drugs. hENT1, which is potently inhibited by nitrobenzylthioinosine (NBMPR), possesses 11 transmembrane helical domains with an intracellular N-terminus and an extracellular C-terminus. As a protein with 10 endogenous cysteine residues, it is sensitive to inhibition by the membrane permeable sulfhydryl-reactive reagent

The human and rat equilibrative nucleoside transporter proteins hENT1, rENT1, hENT2 and rENT2 belong to a family of integral membrane proteins with 11 potential transmembrane segments (TMs), and are distinguished functionally by differences in transport of nucleobases and sensitivity to inhibition by nitrobenzylthioinosine (NBMPR) and vasoactive drugs. In the present study, we have produced recombinant hENT1, rENT1, hENT2 and rENT2 in Xenopus oocytes and investigated uridine transport following exposure to the impermeant thiol-reactive reagent p-chloromercuriphenyl sulphonate (PCMBS). PCMBS caused reversible inhibition of uridine influx by rENT2, but had no effect on hENT1, hENT2 or rENT1. This difference correlated with the presence in rENT2 of a unique Cys residue (Cys140) in the outer half of TM4 that was absent from the other ENTs. Mutation of Cys140 to Ser produced a functional protein (rENT2/C140S) that was insensitive to inhibition by PCMBS, identifying Cys140 as the exofacial Cys residue in rENT2 responsible for PCMBS inhibition. Uridine protected wild-type rENT2 against PCMBS inhibition, suggesting that Cys140 in TM4 lies within or is closely adjacent to the substrate-translocation channel of the transporter. TM4has been shown previously to be within a structural domain (TMs 3Ő6) responsible for interactions with NBMPR, vasoactive drugs and nucleobases.

Plasmodium, the aetiologic agent of malaria, cannot synthesize purines de novo, and hence depends upon salvage from the host. Here we describe the molecular cloning and functional expression in Xenopus oocytes of the first purine transporter to be identified in this parasite. This 422-residue protein, which we designate PfENT1, is predicted to contain 11 membrane-spanning segments and is a distantly related member of the widely distributed eukaryotic protein family the equilibrative nucleoside transporters (ENTs). However, it differs profoundly at the sequence and functional levels from its homologous counterparts in the human host. The parasite protein exhibits a broad substrate specificity for natural nucleosides, but transports the purine nucleoside adenosine with a considerably higher apparent affinity (Km 0.32±0.05 mM) than the pyrimidine nucleoside uridine (Km 3.5±1.1 mM). It also efficiently transports nucleobases such as adenine (Km 0.32±0.10 mM) and hypoxanthine (Km 0.41±0.1 mM), and anti-viral 3ʹ-deoxynucleoside analogues. Moreover, it is not sensitive to classical inhibitors of mammalian ENTs, including NBMPR {6-[(4-nitrobenzyl)thio]-9-β-D-ribofuranosylpurine, or nitrobenzylthioinosine} and the coronary vasoactive drugs, dipyridamole, dilazep and draflazine. These unique properties suggest that PfENT1 might be a viable target for the development of novel anti-malarial drugs.

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.

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).