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.

  • Abbreviations:
    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.
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