Occupancy of the Zinc-binding Site by Transition Metals Decreases the Substrate Affinity of the Human Dopamine Transporter by an Allosteric Mechanism

The human dopamine transporter (DAT) has a tetrahedral Zn(2+)-binding site. Zn(2+)-binding sites are also recognized by other first-row transition metals. Excessive accumulation of manganese or of copper can lead to parkinsonism because of dopamine deficiency. Accordingly, we examined the effect of Mn(2+), Co(2+), Ni(2+), and Cu(2+) on transport-associated currents through DAT and DAT-H193K, a mutant with a disrupted Zn(2+)-binding site. All transition metals except Mn(2+) modulated the transport cycle of wild-type DAT with affinities in the low micromolar range. In this concentration range, they were devoid of any action on DAT-H193K. The active transition metals reduced the affinity of DAT for dopamine. The affinity shift was most pronounced for Cu(2+), followed by Ni(2+) and Zn(2+) (= Co(2+)). The extent of the affinity shift and the reciprocal effect of substrate on metal affinity accounted for the different modes of action: Ni(2+) and Cu(2+) uniformly stimulated and inhibited, respectively, the substrate-induced steady-state currents through DAT. In contrast, Zn(2+) elicited biphasic effects on transport, i.e. stimulation at 1 μm and inhibition at 10 μm. A kinetic model that posited preferential binding of transition metal ions to the outward-facing apo state of DAT and a reciprocal interaction of dopamine and transition metals recapitulated all experimental findings. Allosteric activation of DAT via the Zn(2+)-binding site may be of interest to restore transport in loss-of-function mutants.