The role of zinc ions in reverse transport mediated by monoamine transporters

The human dopamine transporter (hDAT) contains an endogenous high affinity Zn2+ binding site with three coordinating residues on its extracellular face (His(193), His(375), and Glu(396)). Upon binding to this site, Zn2+ causes inhibition of [H-3]1-methyl-4-phenylpyridinium ([H-3]MPP+) uptake. We investigated the effect of Zn2+ on outward transport by superfusing hDAT-expressing HEK-293 cells preloaded with [H-3]MPP+. Although Zn2+ inhibited uptake, Zn2+ facilitated [H-3]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 His(193) 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 [H-3]MPP+ concentration was varied to generate saturation isotherms; these showed that Zn2+ increased V-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