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

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Gorboulev V, Volk C, Arndt P, Akhoundova A, Koepsell H

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

Mol Pharmacol. 1999 Dec;56(6):1254-61.

PubMed ID

10570053 [ View in PubMed

]

Abstract

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 alpha-helix), the V(max) values for choline, tetraethylammonium (TEA), N(1)-methylnicotinamide, and 1-methyl-4-phenylpyridinium were reduced by 89 to 98%. The apparent K(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(m) value for [(3)H]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 IC(50) 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 IC(50) value 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.

DrugBank Data that Cites this Article

Drug Transporters

Drug	Transporter	Kind	Organism	Pharmacological Action	Actions
Choline	Solute carrier family 22 member 1	Protein	Humans	Unknown	Substrate Inhibitor	Details
Choline salicylate	Solute carrier family 22 member 1	Protein	Humans	Unknown	Substrate Inhibitor	Details