The most widely distributed nucleoside transport processes in mammals are equilibrative, and can be divided into two classes on the basis of their sensitivity to the nucleoside analogue nitrobenzylthioinosine (NBMPR). Transport processes of the es (equilibrative sensitive) class are sensitive to inhibition by nanomolar concentrations of NBMPR, whereas transport processes of the ei (equilibrative insensitive) class are relatively insensitive to this inhibitor. In humans, both types of process are also potently inhibited by the coronary vasodilator drugs dipyridamole, dilazep and draflazine, although the es-type process is considerably more sensitive. Recent cloning studies from our laboratories have shown that the proteins responsible for these activities belong to a novel family of transport proteins that we have designated the equilibrative nucleoside transporter or ENT family: the ENT1 transporter isoform possesses es-type activity, while the ENT2 isoform possesses ei-type activity. The two isoforms exhibit similar broad substrate selectivities for purine and pyrimidine nucleosides but only the ENT2 transporters also efficiently transport nucleobases. Glycosylation scanning mutagenesis and other approaches have shown that the 456-residue human protein hENT1 probably contains 11 transmembrane α-helices (TMs), with an extracellular site of glycosylation in the loop connecting TMs 1 and 2, an intracellular N-terminus and an extracellular C-terminus.

Transporters homologous to the mammalian ENTs have been identified in a wide range of other eukaryotes, including yeast, plants and protozoans. Interestingly, some protozoan ENTs catalyse active, proton-linked transport rather than equilibrative transport. In order to identify the regions of the transporters that are involved in solute recognition, we have examined the functional properties of chimaeras constructed from the human and rat ENT1 and ENT2 proteins, which differ in their substrate and inhibitor specificities. Such studies have identified regions within the N-terminal half of the proteins that are responsible for recognition of NBMPR, dipyridamole and dilazep. The varying abilities of these chimaeras to transport nucleobases have similarly confirmed the importance of the N-terminal half of the transporters in solute recognition. Site-directed mutagenesis is now being employed to identify the specific residues involved.

In contrast to the widely distributed equilibrative transporters, the concentrative transport processes of mammals have primarily been described in specialised tissues such as intestinal and renal epithelia, liver and choroid plexus. They are typically insensitive to NBMPR and can be divided into three major classes on the basis of their permeant selectivities. The cit (concentrative, insensitive to NBMPR and accepts thymidine as a permeant) processes accept pyrimidine nucleosides and adenosine, although the latter is a poor substrate. The cif (concentrative, insensitive to NBMPR and accepts formycin B as a permeant) processes accept purine nucleosides and uridine. The cib (concentrative, insensitive to NBMPR, accepts a broad range of permeants) processes accept both purine and pyrimidine nucleosides. Recent cloning studies in our laboratories and elsewhere have revealed that all three processes are catalysed by members of second transporter family, unrelated to the ENT family, which we have designated the concentrative nucleoside transporter or CNT family. Transporters with cit-type activity are designated CNT1, those with cif-type activity are designated CNT2 and those with cib-type activity are designated CNT3. Using glycosylation scanning mutagenesis and other techniques, we have recently shown that unlike the ENTs these proteins probably possess 13 TMs. The properties of chimaeras between the CNT1 and CNT2 isoforms have revealed that putative TMs 7 and 8 play a part in determining the substrate specificity of transport and mutagenesis studies are beginning to identify specific residues that may be involved in solute recognition.

The identification and molecular cloning of mammalian representatives of the ENT and CNT families of proteins has greatly increased our understanding of the molecular mechanisms of nucleoside and nucleobase transport. Knowledge of these mechanisms should be of value in the design of more specific and effective nucleoside analogue drugs for use in the treatment of neoplastic, viral and parasitic diseases. However, much remains to be discovered, including the biological rationale underlying the diversity of nucleoside transporters found in nature.

Research in our laboratories is supported in part by The Wellcome Trust and the Medical Research Council of the UK, the National Cancer Institute of Canada, with funds from the Canadian Cancer Society, and grants from the Canadian Institutes of Health Research. J.D.Y. is a Heritage Scientist of the Alberta Heritage Foundation for Medical Research and C.E.C. is Canada Research Chair in Oncology.