Molecular Determinants of Substrate Selectivity in Na+-dependent Nucleoside Transporters

Abstract In mammalian cells, the salvage of purine and pyrimidine nucleosides is mediated by both facilitated and Na+-dependent nucleoside transporters. These transporters also play important roles in the transmembrane flux of therapeutic nucleoside analogs, which are widely used in the treatment of cancer and viral infections. The N1, N2, and N3 Na+-dependent nucleoside transporters differ in terms of their transport selectivity for purine and pyrimidine nucleosides. N1 is purine-selective, N2 is pyrimidine-selective, and N3 is broadly selective. To identify structural domains involved in substrate binding and molecular determinants responsible for distinct transport selectivity, chimeric transporters were made from the cloned rat N1 and N2 transporters. Of the 14 transmembrane domains (TM) of N1 and N2, transplanting TM8–9 of N1 into N2 converted N2 from a pyrimidine- to a purine-selective transporter. Transplanting only TM8 generated a chimera with characteristics similar to the N3 transporter that has yet to be cloned. These data suggest that TM8–9 confer substrate selectivity and may form at least part of a substrate-binding site in Na+-dependent nucleoside transporters.