Concentrative nucleoside transporter (CNT) proteins mediate active nucleoside transport using the electrochemical gradient of the coupling cation. The molecular mechanisms underlying interactions with both nucleosides and cations were investigated by heterelogous expression of recombinant CNT family members in Xenopus oocytes. Substituted cysteine accessibility method (SCAM) analysis in combination with radioisotope flux assays and electrophysiological studies revealed novel topological features within the C-terminal half of human (h)CNTs and identified residues of functional importance. The hCNT (SLC28) protein family is represented by three members. hCNT1 and hCNT2 are pyrimidine nucleoside- and purine nucleoside-selective, respectively, while hCNT3 transports both pyrimidine and purine nucleosides. hCNT1 and hCNT2 function exclusively as Na+-coupled nucleoside transporters and share a 1:1 Na+:nucleoside stoichiometry. Belonging to a CNT subfamily phylogenetically distinct from hCNT1/2, hCNT3 utilizes electrochemical gradients of Na+, Li+ or H+ to drive nucleoside transport and exhibits 2:1 Na+:nucleoside and 1:1 H+:nucleoside stoichiometries. Non-mammalian H+-coupled CNT family members that have been functionally characterized include NupC from Escherichia coli. Both Na+ and H+ activate CNTs through mechanisms to increase nucleoside apparent binding affinity. Multiple alignments of CNT family members reveal strong sequence similarities within the C-terminal halves of the proteins, and hCNT1/3 and other chimeric studies have demonstrated that this region determines both nucleoside and cation interactions with the transporter. In hCNT3, access of pchloromercuribenzene sulfonate (PCMBS) to introduced cysteine residues within putative transmembrane segments (TMs) 7, 8, 9 and 11A revealed novel discontinuous regions within -helical structures, whereas putative TMs 10, 11, 12 and 13 exhibited conventional -helical characteristics. Putative TM 11A, which contains the highly conserved CNT family motif (G/A)XKX3NEFVA(Y/M/F), was shown to be membrane associated and, most likely, membrane spanning, TMs 7-11 having a reversed orientation in the membrane compared to previous models of CNT topology. Furthermore, putative TMs 7, 8, 9, 11A and 12 were shown to contribute functional and structural elements to a common nucleoside/cation translocation pore. These studies, which were extended to TMs 7 and 8 of hCNT1 and to corresponding TMs of E. coli NupC, provide important structural and functional insights into the nature of CNT nucleoside/cation cotransport.

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