title: Occupancy of the Zinc-binding Site by Transition Metals Decreases the Substrate Affinity of the Human Dopamine Transporter by an Allosteric Mechanism.
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.
authors.affiliation: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University Vienna, Waehringerstrasse 13a, 1090 Vienna, Austria.
authors.forename: Yang
authors.initials: Y
authors.lastname: Li
authors.affiliation: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University Vienna, Waehringerstrasse 13a, 1090 Vienna, Austria.
authors.forename: Felix P
authors.initials: FP
authors.lastname: Mayer
authors.affiliation: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University Vienna, Waehringerstrasse 13a, 1090 Vienna, Austria.
authors.forename: Peter S
authors.initials: PS
authors.lastname: Hasenhuetl
authors.affiliation: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University Vienna, Waehringerstrasse 13a, 1090 Vienna, Austria.
authors.forename: Verena
authors.initials: V
authors.lastname: Burtscher
authors.affiliation: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University Vienna, Waehringerstrasse 13a, 1090 Vienna, Austria.
authors.forename: Klaus
authors.initials: K
authors.lastname: Schicker
authors.affiliation: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University Vienna, Waehringerstrasse 13a, 1090 Vienna, Austria.
authors.forename: Harald H
authors.initials: HH
authors.lastname: Sitte
authors.affiliation: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University Vienna, Waehringerstrasse 13a, 1090 Vienna, Austria.
authors.forename: Michael
authors.initials: M
authors.lastname: Freissmuth
authors.affiliation: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University Vienna, Waehringerstrasse 13a, 1090 Vienna, Austria walter.sandtner@meduniwien.ac.at.
authors.forename: Walter
authors.initials: W
authors.lastname: Sandtner
mesh_terms: D000494:Allosteric Regulation; D001665:Binding Sites; D050483:Dopamine Plasma Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008670:Metals / Q000378:metabolism*; D011485:Protein Binding; D013379:Substrate Specificity; D028561:Transition Elements / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D008670:Metals; D028561:Transition Elements; D015032:Zinc
keywords: dopamine; electrophysiology; metal; monoamine transporter; neurotransmitter transport