Abstract
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 because of 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 except Mn2+ 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 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 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 Zn2+-binding site may be of interest to restore transport in loss-of-function mutants.
Highlights
ResultsEffects of Transition Metals on the Steady-state Current through dopamine transporter (DAT)—When challenged with substrate, monoamine transporters of the SLC6 family produce two types of currents that can be recorded in the whole-cell patch clamp configuration: an initial capacitive peak current that reflects the binding of substrate and co-substrate ions to the transporter and their movement in the electric field of the membrane [18, 19] and a sustained, steady-state current that reflects movement of the transporter through the transport cycle [18]
Occupancy of the Zinc-binding Site by Transition Metals Decreases the Substrate Affinity of the Human Dopamine Transporter by an Allosteric Mechanism*□S
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 dopamine transporter (DAT)
Summary
Effects of Transition Metals on the Steady-state Current through DAT—When challenged with substrate, monoamine transporters of the SLC6 family produce two types of currents that can be recorded in the whole-cell patch clamp configuration: an initial capacitive peak current that reflects the binding of substrate and co-substrate ions to the transporter and their movement in the electric field of the membrane [18, 19] and a sustained, steady-state current that reflects movement of the transporter through the transport cycle [18]. Contrary to Co2ϩ and Ni2ϩ, Cu2ϩ reversibly inhibited the steady-state current through DAT (Fig. 1D) with an IC50 of 4.4 M (2.3– 8.2 M, 95% confidence interval) These observations suggest that all tested transition metals except Mn2ϩ either stimulate or inhibit the transport cycle of DAT. The mutation of His193 to Lys eliminates high-affinity Zn2ϩ binding to DAT [20, 21] It is evident from the current traces shown, B–E, and the summary shown in Fig. 1F that both the stimulation of the steady-state current by Co2ϩ, Ni2ϩ, and Zn2ϩ and its inhibition by Cu2ϩ were abrogated in DAT-H193K. We recorded currents through wild-type DAT in transiently transfected HEK293 cells These experiments recapitulated the data shown, i.e. stimulation of substrate-induced currents by Co2ϩ, Ni2ϩ, and Zn2ϩ, their inhibition by Cu2ϩ, and the absence of any effect in the presence of Mn2ϩ (data not shown).
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