title: Functional analysis of the human concentrative nucleoside transporter-1 variant hCNT1S546P provides insight into the sodium-binding pocket.
abstract: SLC28 genes, encoding concentrative nucleoside transporter proteins (CNT), show little genetic variability, although a few single nucleotide polymorphisms (SNPs) have been associated with marked functional disturbances. In particular, human CNT1S546P had been reported to result in negligible thymidine uptake. In this study we have characterized the molecular mechanisms responsible for this apparent loss of function. The hCNT1S546P variant showed an appropriate endoplasmic reticulum export and insertion into the plasma membrane, whereas loss of nucleoside translocation ability affected all tested nucleoside and nucleoside-derived drugs. Site-directed mutagenesis analysis revealed that it is the lack of the serine residue itself responsible for the loss of hCNT1 function. This serine residue is highly conserved, and mutation of the analogous serine in hCNT2 (Ser541) and hCNT3 (Ser568) resulted in total and partial loss of function, respectively. Moreover, hCNT3, the only member that shows a 2Na(+)/1 nucleoside stoichiometry, showed altered Na(+) binding properties associated with a shift in the Hill coefficient, consistent with one Na(+) binding site being affected by the mutation. Two-electrode voltage-clamp studies using the hCNT1S546P mutant revealed the occurrence of Na(+) leak, which was dependent on the concentration of extracellular Na(+) indicating that, although the variant is unable to transport nucleosides, there is an uncoupled sodium transport.
authors: Departament de Bioquímica i Biologia Molecular, Institut de Biomedicina, Universitat de Barcelona & CIBER EHD, Barcelona, Spain.
authors: Pedro
authors: P
authors: Cano-Soldado
authors: Edurne
authors: E
authors: Gorraitz
authors: Ekaitz
authors: E
authors: Errasti-Murugarren
authors: F Javier
authors: FJ
authors: Casado
authors: M Pilar
authors: MP
authors: Lostao
authors: Marçal
authors: M
authors: Pastor-Anglada
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D014644:Genetic Variation / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011392:Proline / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D012694:Serine / Q000235:genetics; D012964:Sodium / Q000172:deficiency / Q000378:metabolism*
chemical_list: D026901:Membrane Transport Proteins; C065171:cif nucleoside transporter; D012694:Serine; D011392:Proline; D012964:Sodium