Identification of Amino Acid Residues Responsible for the Pyrimidine and Purine Nucleoside Specificities of Human Concentrative Na+Nucleoside Cotransporters hCNT1 and hCNT2*

hCNT1 and hCNT2 mediate concentrative (Na+-linked) cellular uptake of nucleosides and nucleoside drugs by human cells and tissues. The two proteins (650 and 658 residues, 71 kDa) are 72% identical in sequence and contain 13 putative transmembrane helices (TMs). When produced in Xenopusoocytes, recombinant hCNT1 is selective for pyrimidine nucleosides (system cit), whereas hCNT2 is selective for purine nucleosides (system cif). Both transport uridine. We have used (i) chimeric constructs between hCNT1 and hCNT2, (ii) sequence comparisons with a newly identified broad specificity concentrative nucleoside transporter (system cib) from Eptatretus stouti, the Pacific hagfish (hfCNT), and (iii) site-directed mutagenesis of hCNT1 to identify two sets of adjacent residues in TMs 7 and 8 of hCNT1 (Ser319/Gln320and Ser353/Leu354) that, when converted to the corresponding residues in hCNT2 (Gly313/Met314and Thr347/Val348), changed the specificity of the transporter from citto cif. Mutation of Ser319in TM 7 of hCNT1 to Gly enabled transport of purine nucleosides, whereas concurrent mutation of Gln320to Met (which had no effect on its own) augmented this transport. The additional mutation of Ser353to Thr in TM 8 converted hCNT1/S319G/Q320M, from cibto cif, but with relatively low adenosine transport activity. Additional mutation of Leu354to Val (which had no effect on its own) increased the adenosine transport capability of hCNT1/S319G/Q320M/S353T, producing a full cif-type transporter phenotype. On its own, the S353T mutation converted hCNT1 into a transporter with novel uridine-selective transport properties. Helix modeling of hCNT1 placed Ser319(TM 7) and Ser353(TM 8) within the putative substrate translocation channel, whereas Gln320(TM 7) and Leu354(TM 8) may exert their effects through altered helix packing.