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 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.
Gorboulev
V
V
Anatomisches Institut, Bayerische Julius-Maximilians-Universität, Würzburg, Germany.
Volk
C
C
Arndt
P
P
Akhoundova
A
A
Koepsell
H
H
D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000235:genetics; D001224:Aspartic Acid / Q000378:metabolism*; D002352:Carrier Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D017136:Ion Transport; D008565:Membrane Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D027702:Organic Cation Transporter 1; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D019789:Tetraethylammonium / Q000494:pharmacology; D014982:Xenopus laevis
D002352:Carrier Proteins; D008565:Membrane Proteins; D027702:Organic Cation Transporter 1; D000644:Quaternary Ammonium Compounds; D001224:Aspartic Acid; D018698:Glutamic Acid; D019789:Tetraethylammonium; D001120:Arginine; C027917:tetramethylammonium