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J Biol Chem. 2017 Mar 10;292(10):4235-4243. doi: 10.1074/jbc.M116.760140. Epub 2017 Jan 17.

Occupancy of the Zinc-binding Site by Transition Metals Decreases the Substrate Affinity of the Human Dopamine Transporter by an Allosteric Mechanism.

Author information

1
From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University Vienna, Waehringerstrasse 13a, 1090 Vienna, Austria.
2
From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University Vienna, Waehringerstrasse 13a, 1090 Vienna, Austria walter.sandtner@meduniwien.ac.at.

Abstract

The human dopamine transporter (DAT) has a tetrahedral Zn2+-binding site. Zn2+-binding sites are also recognized by other first-row transition metals. Excessive accumulation of manganese or of copper can lead to parkinsonism because of dopamine deficiency. Accordingly, we examined the effect of Mn2+, Co2+, Ni2+, and Cu2+ on transport-associated currents through DAT and DAT-H193K, a mutant with a disrupted Zn2+-binding site. All transition metals except Mn2+ modulated the transport cycle of wild-type DAT with affinities in the low micromolar range. In this concentration range, they were devoid of any action on DAT-H193K. The active transition metals reduced the affinity of DAT for dopamine. The affinity shift was most pronounced for Cu2+, followed by Ni2+ and Zn2+ (= Co2+). The extent of the affinity shift and the reciprocal effect of substrate on metal affinity accounted for the different modes of action: Ni2+ and Cu2+ uniformly stimulated and inhibited, respectively, the substrate-induced steady-state currents through DAT. In contrast, Zn2+ elicited biphasic effects on transport, i.e. stimulation at 1 μm and inhibition at 10 μm A kinetic model that posited preferential binding of transition metal ions to the outward-facing apo state of DAT and a reciprocal interaction of dopamine and transition metals recapitulated all experimental findings. Allosteric activation of DAT via the Zn2+-binding site may be of interest to restore transport in loss-of-function mutants.

KEYWORDS:

dopamine; electrophysiology; metal; monoamine transporter; neurotransmitter transport

PMID:
28096460
PMCID:
PMC5354487
DOI:
10.1074/jbc.M116.760140
[Indexed for MEDLINE]
Free PMC Article

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