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J. Biol. Chem., Vol. 279, Issue 48, 49671-49679, November 26, 2004

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Zinc Potentiates an Uncoupled Anion Conductance Associated with the Dopamine Transporter^*

Anne-Kristine Meinild, Harald H. Sitte¶, and Ulrik Gether||

From the Molecular Neuropharmacology Group, Department of Pharmacology, The Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark and the ^¶Institute of Pharmacology, Medical University Vienna, Währingerstrasse 13a, A-1090 Vienna, Austria

Binding of Zn²⁺ to an endogenous binding site in the dopamine transporter (DAT) leads to inhibition of dopamine (DA) uptake and enhancement of carrier-mediated substrate efflux. To elucidate the molecular mechanism for this dual effect, we expressed the DAT and selected mutants in Xenopus laevis oocytes and applied the two-electrode voltage clamp technique together with substrate flux studies employing radiolabeled tracers. Under voltage clamp conditions we found that Zn²⁺ (10 µM) enhanced the current induced by both DA and amphetamine. This was not accompanied by a change in the uptake rate but by a marked increase in the charge/DA flux coupling ratio as assessed from concomitant measurements of [³H]DA uptake and currents in voltage-clamped oocytes. These data suggest that Zn²⁺ facilitates an uncoupled ion conductance mediated by DAT. Whereas this required substrate in the wild type (WT), we observed that Zn²⁺ by itself activated such a conductance in a previously described mutant (Y335A). This signifies that the conductance is not strictly dependent on an active transport process. Ion substitution experiments in Y335A, as well as in WT, indicated that the uncoupled conductance activated by Zn²⁺ was mainly carried by Cl^–. Experiments in oocytes under non-voltage-clamped conditions revealed furthermore that Zn²⁺ could enhance the depolarizing effect of substrates in oocytes expressing WT. The data suggest that by potentiating an uncoupled Cl^– conductance, Zn²⁺ is capable of modulating the membrane potential of cells expressing DAT and as a result cause simultaneous inhibition of uptake and enhancement of efflux.

Received for publication, July 8, 2004 , and in revised form, September 8, 2004.

^* This work was supported by funds from the Danish Health Science Research Council (to A. K. M. and U. G.), National Institute of Health Grant P01 DA 12408, funds from the Lundbeck Foundation (to U. G.), funds from the Meyer Foundation (to U. G.), funds from the Novo Nordic Foundation (to A. K. M.), funds from the Alfred Benzon Foundation (to H. H. S.), Hygiene-Fonds of the University of Vienna and the Austrian Science Foundation/FWF Grant P14509 (to H. H. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this paper.

^|| To whom correspondence should be addressed: Molecular Neuropharmacology Group, Dept. of Pharmacology, Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark. Tel.: 45-3532-7548; Fax: 45-3532-7610, E-mail: gether{at}neuropharm.ku.dk.

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