Differential effect of structural modification of human dopamine transporter on the inward and outward transport of dopamine

The effect of structural modification of the human dopamine transporter pro tein on hi-directional transport was explored using site-directed mutagenes is and rotating disk electrode voltammetry. The substrate-induced DA efflux , as inferred from the K-m or K-i, was dependent on common structural featu res for uptake of the substrate inducer: reduced by beta-hydroxylation, ste reoselective to alpha-methylation, and relatively insensitive to a switch o f a single phenolic hydroxyl group between m- and p-positions. The potencie s 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. M utation of serine-528 of the hDAT to alanine (S528A) did not change the str ucture-activity relationships, maximal uptake rates, and the cation depende nce for the uptake of external substrates, although it modestly reduced K-m or K-i of most tested substrates. In contrast, it substantially enhanced s ubstrate-induced DA efflux, with maximal efflux rates doubled for all teste d inducers. Simultaneous monitoring of tyramine uptake and resulting DA eff lux revealed that S528A accelerated the DA efflux relative to tyramine upta ke. Saturation analysis suggested that the mutation significantly enhanced the efflux kinetics of internal DA but it exerted little effect on the upta ke kinetics of external DA. These findings suggest that Ser-528 may play a role in stabilizing a hDAT conformation unfavorable for outward transport o f internal DA, thereby contributing to the efficiency of the transporter. ( C) 2000 Elsevier Science B.V. All rights reserved.