The Role of Zinc Ions in Reverse Transport Mediated by Monoamine Transporters*
Abstract
The human dopamine transporter (hDAT) contains an endogenous high affinity Zn2+ binding site with three coordinating residues on its extracellular face (His193, His375, and Glu396). Upon binding to this site, Zn2+ causes inhibition of [3H]1-methyl-4-phenylpyridinium ([3H]MPP+) uptake. We investigated the effect of Zn2+ on outward transport by superfusing hDAT-expressing HEK-293 cells preloaded with [3H]MPP+. Although Zn2+ inhibited uptake, Zn2+facilitated [3H]MPP+ release induced by amphetamine, MPP+, or K+-induced depolarization specifically at hDAT but not at the human serotonin and the norepinephrine transporter (hNET). Mutation of the Zn2+coordinating residue His193 to Lys (the corresponding residue in hNET) eliminated the effect of Zn2+ on efflux. Conversely, the reciprocal mutation (K189H) conferred Zn2+sensitivity to hNET. The intracellular [3H]MPP+ concentration was varied to generate saturation isotherms; these showed that Zn2+ increasedV max for efflux (rather than KM-Efflux-intracellular). Thus, blockage of inward transport by Zn2+ is not due to a simple inhibition of the transporter turnover rate. The observations provide evidence against the model of facilitated exchange-diffusion and support the concept that inward and outward transport represent discrete operational modes of the transporter. In addition, they indicate a physiological role of Zn2+, because Zn2+ also facilitated transport reversal of DAT in rat striatal slices.
- hDAT
- human dopamine transporter
- hNET
- human norepinephrine transporter
- hSERT
- human serotonin transporter
- HEK-293
- human embryonic kidney 293 cells
- MPP+
- 1-methyl-4-phenylpyridinium
- wt
- wild type
- TM
- transmembrane segment
- CMV
- cytomegalovirus
- Received December 21, 2001.
- Revision received April 5, 2002.
- The American Society for Biochemistry and Molecular Biology, Inc.











