title: The human concentrative nucleoside transporter-3 C602R variant shows impaired sorting to lipid rafts and altered specificity for nucleoside-derived drugs.
abstract: The human concentrative nucleoside transporter-3 C602R (hCNT3C602R), a recently identified human concentrative nucleoside transporter-3 (hCNT3) variant, has been shown to interact with natural nucleosides with apparent K(m) values similar to those of the wild-type transporter, although binding of one of the two sodium ions required for nucleoside translocation is impaired, resulting in decreased V(max) values (Mol Pharmacol 73:379-386, 2008). We have further analyzed the properties of this hCNT3 variant by determining its localization in plasma membrane lipid domains and its interaction with nucleoside-derived drugs used in anticancer and antiviral therapies. When expressed heterologously in HeLa cells, wild-type hCNT3 localized to both lipid raft and nonlipid raft domains. Treatment of cells with the cholesterol-depleting agent methyl-beta-cyclodextrin resulted in a marked decrease in hCNT3-related transport activity that was associated with the loss of wild-type hCNT3 from lipid rafts. It is noteworthy that although exogenously expressed hCNT3C602R was present in nonlipid raft domains at a level similar to that of the wild-type transporter, the mutant transporter was present at much lower amounts in lipid rafts. A substrate profile analysis showed that interactions with a variety of nucleoside-derived drugs were altered in the hCNT3C602R variant and revealed that sugar hydroxyl residues are key structural determinants for substrate recognition by the hCNT3C602R variant.
authors.affiliation: Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona and CIBER EHD, Avda Diagonal 645, Edifici annex, Planta-1, 08028 Barcelona, Spain.
authors.forename: Ekaitz
authors.initials: E
authors.lastname: Errasti-Murugarren
authors.forename: Miriam
authors.initials: M
authors.lastname: Molina-Arcas
authors.forename: F Javier
authors.initials: FJ
authors.lastname: Casado
authors.forename: Marçal
authors.initials: M
authors.lastname: Pastor-Anglada
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D004285:Dogs; D006367:HeLa Cells; D006801:Humans; D050356:Lipid Metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009705:Nucleosides / Q000494:pharmacology*; D011110:Polymorphism, Genetic
chemical_list: D017931:DNA Primers; D026901:Membrane Transport Proteins; D009705:Nucleosides; C065171:cif nucleoside transporter