Abstract

Na⁺-dependent nucleoside transporters mediate the intracellular uptake of purine and pyrimidine nucleosides. The N1, N2, and N3 Na⁺-nucleoside transporters differ in substrate selectivity. N1 is purine-selective, N2 is pyrimidine-selective, and N3 is broadly selective. Recently, we created a chimeric transporter, T8, from the cloned rat N1 and N2 transporters. Whereas most chimeric proteins exhibit the characteristics of one of the two parent proteins, limited studies suggested that T8 possesses either a combined substrate selectivity of N1 and N2 or the selectivity of N3. The purpose of this study was to determine the substrate profile, transport mechanisms, and Na⁺-coupling stoichiometry of T8 and to compare these measurements with those of wild-type N1, N2, and N3. In Xenopus laevis oocytes expressing T8, Na⁺-dependent uptake of ³H-labeled purine (adenosine, inosine, and guanosine) and pyrimidine nucleosides (uridine, thymidine, and cytidine) was significantly enhanced (3.5–18.6-fold), which suggests that T8 accepts both purine and pyrimidine nucleosides as permeants. T8-mediated uptake of [³H]thymidine was competitively inhibited by inosine, and T8-mediated uptake of [³H]inosine was competitively inhibited by thymidine, which suggests that purine and pyrimidine nucleosides share a common binding site. Base-modified ribo- and 2′-deoxyribonucleosides were potent inhibitors of T8. In contrast, 2′,3′-dideoxyinosine, 2′,3′-dideoxycytidine, and 3′-azidothymidine, which are known inhibitors of N1 or N2, did not inhibit T8-mediated uptake. These data suggest that the substrate profile of T8 is not a combination of those of N1 and N2; rather, it is similar to that of N3. However, the Na⁺/nucleoside stoichiometric ratio of T8 was determined to be 1, consistent with both N1 and N2 but different from N3.