TY - JOUR T1 - Selectivity of the Polyspecific Cation Transporter rOCT1 Is Changed by Mutation of Aspartate 475 to Glutamate JF - Molecular Pharmacology JO - Mol Pharmacol SP - 1254 LP - 1261 DO - 10.1124/mol.56.6.1254 VL - 56 IS - 6 AU - Valentin Gorboulev AU - Christopher Volk AU - Petra Arndt AU - Aida Akhoundova AU - Hermann Koepsell Y1 - 1999/12/01 UR - http://molpharm.aspetjournals.org/content/56/6/1254.abstract N2 - After site-directed mutagenesis, the organic cation transporter rOCT1 was expressed in Xenopus laevis oocytes or human embryonic kidney cells and functionally characterized. rOCT1 belongs to a new family of polyspecific transporters that includes transporters for organic cations and anions and the Na+-carnitine cotransporter. When glutamate was substituted for Asp475 (middle of the proposed 11th transmembrane α-helix), theV max values for choline, tetraethylammonium (TEA), N 1-methylnicotinamide, and 1-methyl-4-phenylpyridinium were reduced by 89 to 98%. The apparentK m values were also decreased (choline by 15-fold, TEA by 8-fold,N 1-methylnicotinamide by 4-fold) or remained constant (1-methyl-4-phenylpyridinium). After the mutation, the membrane potential dependence of the K mvalue for [3H]choline uptake was abolished. The affinity of n-tetraalkyl ammonium compounds to inhibit TEA uptake was increased. This affinity and its increase by the D475E mutation were increased with the length of the n-alkyl chains. After expression in X. laevis oocytes, the IC50 ratios of wild-type and D475E mutant were 1.7 (tetramethylammonium), 4.3 (TEA), 5.0 (tetrapropylammonium), 5.0 (tetrabutylammonium), and 65 (tetrapentylammonium). Cationic inhibitors with ring structures were differentially affected: the IC50value for TEA inhibition by cyanine 863 remained unchanged, whereas it was increased for quinine. The data suggest that rOCT1 contains a large cation-binding pocket with several interaction domains that may be responsible for high-affinity binding of structurally different cations and that Asp475 is located close to one of these interaction domains. ER -