Selectivity of the polyspecific cation transporter rOCT1 is changed by mutation of aspartate 475 to glutamate

After site-directed mutagenesis, the organic cation transporter rOCT1 was e xpressed in Xenopus laevis oocytes or human embryonic kidney cells and func tionally characterized. rOCT1 belongs to a new family of polyspecific trans porters that includes transporters for organic cations and anions and the N a+-carnitine cotransporter. When glutamate was substituted for Asp475 (midd le of the proposed 11th transmembrane alpha-helix), the Vmax values for cho line, tetraethylammonium (TEA), N-1-methylnicotinamide, and 1-methyl-4-phen ylpyridinium were reduced by 89 to 98%. The apparent K-m values were also d ecreased (choline by 15-fold, TEA by 8-fold, N-1-methylnicotinamide by 4-fo ld) or remained constant (1-methyl-4-phenylpyridinium). After the mutation, the membrane potential dependence of the K-m value for [H-3] choline uptak e was abolished. The affinity of n-tetraalkyl ammonium compounds to inhibit TEA uptake was increased. This affinity and its increase by the D475E muta tion 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 (tetrab utylammonium), and 65 (tetrapentylammonium). Cationic inhibitors with ring structures were differentially affected: the IC50 value for TEA inhibition by cyanine 863 remained unchanged, whereas it was increased for quinine. Th e data suggest that rOCT1 contains a large cation-binding pocket with sever al 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.