Dual Action of Zn2+ on the Transport Cycle of the Dopamine Transporter.
The dopamine transporter shapes dopaminergic neurotransmission by clearing extracellular dopamine and by replenishing vesicular stores. The dopamine transporter carries an endogenous binding site for Zn(2+), but the nature of the Zn(2+)-dependent modulation has remained elusive: both, inhibition and stimulation of DAT have been reported. Here, we exploited the high time resolution of patch-clamp recordings to examine the effects of Zn(2+) on the transport cycle of DAT: we recorded peak currents associated with substrate translocation and steady-state currents reflecting the forward transport mode of DAT. Zn(2+) depressed the peak current but enhanced the steady-state current through DAT. The parsimonious explanation is preferential binding of Zn(2+) to the outward facing conformation of DAT, which allows for an allosteric activation of DAT, in both, the forward transport mode and substrate exchange mode. We directly confirmed that Zn(2+) dissociated more rapidly from the inward- than from the outward-facing state of DAT. Finally, we formulated a kinetic model for the action of Zn(2+) on DAT that emulated all current experimental observations and accounted for all previous (in part contradictory) findings. Importantly, the model predicts that the intracellular Na(+) concentration determines whether substrate uptake by DAT is stimulated or inhibited by Zn(2+). This prediction was directly verified. The mechanistic framework provided by the current model is of relevance for the rational design of allosteric activators of DAT. These are of interest for treating de novo loss-of-function mutations of DAT associated with neuropsychiatric disorders such as attention deficit hyperactivity disorder (ADHD).
From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse 13a, A-1090 Vienna, Austria.
Yang
Y
Li
From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse 13a, A-1090 Vienna, Austria.
Peter S
PS
Hasenhuetl
From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse 13a, A-1090 Vienna, Austria.
Klaus
K
Schicker
From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse 13a, A-1090 Vienna, Austria.
Harald H
HH
Sitte
From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse 13a, A-1090 Vienna, Austria.
Michael
M
Freissmuth
From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse 13a, A-1090 Vienna, Austria walter.sandtner@meduniwien.ac.at.
Walter
W
Sandtner
D001289:Attention Deficit Disorder with Hyperactivity / Q000235:genetics / Q000378:metabolism; D001693:Biological Transport, Active / Q000235:genetics; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008564:Membrane Potentials; D009154:Mutation; D015032:Zinc / Q000378:metabolism*
D050483:Dopamine Plasma Membrane Transport Proteins; D015032:Zinc
dopamine; dopamine transporter; electrophysiology; kinetics; neurotransmitter release; neurotransmitter transport