Serine 318 is essential for the pyrimidine selectivity of the N2 Na+-nucleoside transporter.
Molecular cloning has isolated two subtypes of Na+-nucleoside transporters; one is pyrimidine-selective (N2), and the other is purine-selective (N1). Using chimeric rat N2/N1 transporters, we previously demonstrated that transmembrane domains (TM) 8 and 9 are the major sites for substrate binding and discrimination. Interestingly, when TM8 of N2 was replaced by that of N1, the resulting chimera, T8, lost the pyrimidine selectivity of N2 and accepted both purine and pyrimidine nucleosides. Five residues differ between rat N2 and N1 in TM8. To identify the critical residues responsible for transport selectivity, the five residues in N2 were systematically changed to their equivalents in N1. Replacing the serine residue at position 318 to its equivalent N1 residue, glycine, caused N2 to lose its selectivity for pyrimidine nucleosides and accept purine nucleosides as substrates. In contrast, replacing the other four residues did not change the pyrimidine selectivity of N2. Furthermore, when glycine 318 in chimera T8 was changed back to serine, the chimeric transporter regained pyrimidine selectivity. These observations suggest that serine 318 is located in the nucleoside permeation pathway and is responsible for the substrate selectivity of N2. An adjacent residue, glutamine 319, was found to be important in modulating the apparent affinity for nucleosides.
Wang
J
J
Departments of Biopharmaceutical Sciences and Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143, USA.
Giacomini
K M
KM
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011743:Pyrimidines / Q000378:metabolism*; D051381:Rats; D012694:Serine / Q000378:metabolism*; D013379:Substrate Specificity; D027981:Symporters; D014982:Xenopus laevis
D002352:Carrier Proteins; D011743:Pyrimidines; D027981:Symporters; C073034:sodium-nucleoside cotransporter; D012694:Serine