Meinild, Anne-Kristine5; Sitte, Harald H3; Gether, Ulrik6
1 Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Københavns Universitet2 Molecular Integrative Physiology, Department of Biology, Faculty of Science, Københavns Universitet3 unknown4 Neuropharm and Genetics Lab, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Københavns Universitet5 Molecular Integrative Physiology, Department of Biology, Faculty of Science, Københavns Universitet6 Neuropharm and Genetics Lab, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Københavns Universitet
Binding of Zn(2+) 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 Zn(2+) (10 mum) 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 [(3)H]DA uptake and currents in voltage-clamped oocytes. These data suggest that Zn(2+) facilitates an uncoupled ion conductance mediated by DAT. Whereas this required substrate in the wild type (WT), we observed that Zn(2+) 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 Zn(2+) was mainly carried by Cl(-). Experiments in oocytes under non-voltage-clamped conditions revealed furthermore that Zn(2+) could enhance the depolarizing effect of substrates in oocytes expressing WT. The data suggest that by potentiating an uncoupled Cl(-) conductance, Zn(2+) is capable of modulating the membrane potential of cells expressing DAT and as a result cause simultaneous inhibition of uptake and enhancement of efflux.
Journal of Biological Chemistry, 2004, Vol 279, Issue 48, p. 49671-9