Topology of a Human Equilibrative, Nitrobenzylthioinosine (NBMPR)-sensitive Nucleoside Transporter (hENT1) Implicated in the Cellular Uptake of Adenosine and Anti-cancer Drugs

Abstract

The human equilibrative nucleoside transporter hENT1, the first identified member of the ENT family of integral membrane proteins, is the primary mechanism for the cellular uptake of physiologic nucleosides, including adenosine, and many anti-cancer nucleoside drugs. We have produced recombinant hENT1 in Xenopus oocytes and used native and engineered N-glycosylation sites in combination with immunological approaches to experimentally define the membrane architecture of this prototypic nucleoside transporter. hENT1 (456 amino acid residues) is shown to contain 11 transmembrane helical segments with an amino terminus that is intracellular and a carboxyl terminus that is extracellular. Transmembrane helices are linked by short hydrophilic regions, except for a large glycosylated extracellular loop between transmembrane helices 1 and 2 and a large central cytoplasmic loop between transmembrane helices 6 and 7. Sequence analyses suggest that this membrane topology is common to all mammalian, insect, nematode, protozoan, yeast, and plant members of the ENT protein family.Nucleoside transporters play key roles in physiology and pharmacology (1). Uptake of exogenous nucleosides, for example, is a critical 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 (2, 3). The same transport mechanisms function as drug transporters and mediate uptake of many synthetic nucleoside analogs used in cancer (and viral) chemotherapy (2). Nucleoside transporters also control the extracellular concentration of adenosine in the vicinity of its cell surface receptors and regulate processes such as neurotransmission and cardiovascular activity (1-3). Adenosine itself is used clinically to treat cardiac arrhythmias, and nucleoside transport inhibitors such as dipyridamole, dilazep, and draflazine function as coronary vasodilators. In mammals, plasma membrane transport of nucleosides is brought about by members of the concentrative, Na ϩ -dependent (CNT) 1 and equilibrative, Na ϩ -independent (ENT) nucleoside transporter families (1-3). CNTs are expressed in a tissue-specific fashion; ENTs are present in most, possibly all, cell types.Two ENT isoforms have been identified in human and rat tissues (4 -7). Human (h) and rat (r) ENT1 and ENT2 (456 -457 amino acid residues) transport both purine and pyrimidine nucleosides, including adenosine, and are distinguished functionally by a difference in sensitivity to inhibition by NBMPR: hENT1 and rENT1 are potently inhibited by NBMPR (K d 1-10 nM) and have the functional designation equilibrative-sensitive (es), while hENT2 and rENT2 are unaffected by micromolar concentrations of NBMPR and have the functional designation equilibrative-insensitive (ei) (4 -7). They also differ in sensitivity to inhibition by vasodilator drugs (hENT1 Ͼ hENT2 Ͼ rENT1 ϭ rENT2) and by the ability of hENT2 and rENT2 to transport nucleobases as well as nucleosides (1,3,7,8). ENTs are widely distributed i...

The human equilibrative nucleoside transporter 1 mediates in vitro cytarabine sensitivity in childhood acute myeloid leukaemia

“…In multivariate analysis, hENT1 mRNA expression was the most important factor determining sensitivity to ara-C. This might be explained by the fact that entry of ara-C into the cell is mainly dependent on hENT1-mediated transport (Wiley et al, 1982;Pastor-Anglada et al, 1998;Sundaram et al, 2001;Clarke et al, 2002). In contrast, 2-CdA, DAC and dFdC differ from ara-C with respect to their preferential nucleoside transporters and can be transported across the cell membrane by other members of the nucleoside transporter family as well (Damaraju et al, 2003).…”

“…Ara-C is a hydrophilic molecule and as such requires facilitated diffusion via nucleoside-specific membrane transport carriers to enter cells (Cass et al, 1998;Clarke et al, 2002). The human equilibrative nucleoside transporter (hENT1) is responsible for 80% of ara-C influx in human leukemic blast cells (Sundaram et al, 2001;Clarke et al, 2002). Inside the cell, conversion of ara-C into ara-CMP by deoxycytidine kinase (dCK) is believed to be the rate-limiting step in the metabolism of ara-C (Liliemark et al, 1985;Plunkett et al, 1987).…”

Second-site Suppression of a Nonfunctional Mutation within the Leishmania donovani Inosine-Guanosine Transporter

“…Crystal structures of MFS members encompass 12 TMs with cytosolic NH 2 and COOH termini and a large intracellular loop between TMs 6 -7. ENTs are similar to MFS members in their predicted membrane topology accommodating a large loop between TMs 6 and 7, although they are conjectured to have 11 TMs with an extracellular COOH terminus (34). Although the exclusion of TM12 in the ENT prediction from the MFS template is a concern, an extensive mutagenesis study of TM12 of the lactose permease, an MFS member, indicates that residues in this domain do not participate in ligand binding or translocation (35).…”

Functional Characterization of Novel Human and Mouse Equilibrative Nucleoside Transporters (hENT3 and mENT3) Located in Intracellular Membranes

“…Family members are predicted to share a common topology of 11 transmembrane (TM) ␣-helices, with a cytoplasmic N terminus and extracellular C terminus, and typically possess a large cytoplasmic loop linking TM6 and -7 (9). Direct experimental evidence for this topology has been obtained in the case of the archetypal family member hENT1 (10).…”