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Vol. 56, Issue 6, 1254-1261, December 1999
Anatomisches Institut, Bayerische
Julius-Maximilians-Universität, Würzburg, Germany
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 
-helix), the
Vmax values for choline, tetraethylammonium
(TEA), N1-methylnicotinamide, and
1-methyl-4-phenylpyridinium were reduced by 89 to 98%. The apparent
Km values were also decreased (choline by
15-fold, TEA by 8-fold,
N1-methylnicotinamide by 4-fold) or remained
constant (1-methyl-4-phenylpyridinium). After the mutation, the
membrane potential dependence of the Km value 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 IC50
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.
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