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

Abstract

The human dopamine transporter (hDAT) contains an endogenous high affinity Zn 2+ binding site with three coordinating residues on its extracellular face (His 193, His 375, and Glu 396). Upon binding to this site, Zn 2+ causes inhibition of [ 3H]1-methyl-4-phenylpyridinium ([ 3H]MPP +) uptake. We investigated the effect of Zn 2+ on outward transport by superfusing hDAT-expressing HEK-293 cells preloaded with [ 3H]MPP +. Although Zn 2+ inhibited uptake, Zn 2+ 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 Zn 2+ coordinating residue His 193 to Lys (the corresponding residue in hNET) eliminated the effect of Zn 2+ on efflux. Conversely, the reciprocal mutation (K189H) conferred Zn 2+ sensitivity to hNET. The intracellular [ 3H]MPP + concentration was varied to generate saturation isotherms; these showed that Zn 2+ increased V max for efflux (rather than K M-Efflux-intracellular). Thus, blockage of inward transport by Zn 2+ 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 Zn 2+, because Zn 2+ also facilitated transport reversal of DAT in rat striatal slices.