Occupancy of the Zinc Binding-site by Transition Metals decreases the Substrate Affinity of the Human Dopamine Transporter by an Allosteric Mechanism. [Neurobiology]

The human dopamine transporter (DAT) has a tetrahedral Zn2+-binding site. Zn2+-binding sites are also recognized by other first row transition metals. Excessive accumulation of manganese or of copper can lead to Parkinsonism due to dopamine deficiency. Accordingly, we examined the effect of Mn2+, Co2+, Ni2+ and Cu2+on transport-associated currents through DAT and DAT-H193K, a mutant with a disrupted Zn2+-binding site. All transition metals - but Mn2+ - modulated the transport cycle of wild type DAT with affinities in the low µM 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 Cu2+, followed by Ni2+ and Zn2+ (= Co2+). The extent of the affinity shift and the reciprocal effect of substrate on metal affinity accounted for the different modes of action: Ni2+ and Cu2+ uniformly stimulated and inhibited, respectively, the substrate-induced steady-state currents through DAT. In contrast, Zn2+ elicited biphasic effects on transport, i.e. stimulation at 1 µM and inhibition at 10 µM. A kinetic model, which posited (i) preferential binding of transition metal ions to the outward facing apo-state of DAT and (ii) a reciprocal interaction of dopamine and transition metals, recapitulated all experimental findings. Allosteric activation of DAT via the Zn2+ binding site may be of interest to restore transport in loss-of-function mutants.

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