期刊
MOLECULAR BRAIN RESEARCH
卷 75, 期 2, 页码 208-215出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/S0169-328X(99)00288-0
关键词
uptake; substrate-induced efflux; rotating disk electrode voltammetry; mutagenesis; serine; amphetamine
资金
- NIDA NIH HHS [DA11176, DA10896, K02 DA00179] Funding Source: Medline
The effect of structural modification of the human dopamine transporter protein on hi-directional transport was explored using site-directed mutagenesis and rotating disk electrode voltammetry. The substrate-induced DA efflux, as inferred from the K-m or K-i, was dependent on common structural features for uptake of the substrate inducer: reduced by beta-hydroxylation, stereoselective to alpha-methylation, and relatively insensitive to a switch of a single phenolic hydroxyl group between m- and p-positions. The potencies for substrates to compete with external DA uptake and to induce DA efflux were similar and highly correlated. Despite these similarities, the efflux of internal DA was substantially slower than the uptake of its inducers. Mutation of serine-528 of the hDAT to alanine (S528A) did not change the structure-activity relationships, maximal uptake rates, and the cation dependence for the uptake of external substrates, although it modestly reduced K-m or K-i of most tested substrates. In contrast, it substantially enhanced substrate-induced DA efflux, with maximal efflux rates doubled for all tested inducers. Simultaneous monitoring of tyramine uptake and resulting DA efflux revealed that S528A accelerated the DA efflux relative to tyramine uptake. Saturation analysis suggested that the mutation significantly enhanced the efflux kinetics of internal DA but it exerted little effect on the uptake kinetics of external DA. These findings suggest that Ser-528 may play a role in stabilizing a hDAT conformation unfavorable for outward transport of internal DA, thereby contributing to the efficiency of the transporter. (C) 2000 Elsevier Science B.V. All rights reserved.
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