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 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.
Footnotes
- Received May 24, 1999.
- Accepted August 20, 1999.
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Send reprint requests to: Prof. Dr. Hermann Koepsell, Anatomisches Institut der Universität, Koellikerstrasse 6, D-97070, Würzburg, Germany, E-mail:anat010{at}mail.uni-wuerzburg.de
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↵1 V.G. and C.V. contributed equally to the work.
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This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 174/A22.
- The American Society for Pharmacology and Experimental Therapeutics
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