Binding of Zn2+ to the endogenous Zn2+ binding site in the human dopamine transporter leads to potent inhibition of [3H]dopamine uptake. Here we show that mutation of an intracellular tyrosine to alanine (Y335A) converts this inhibitory Zn2+ switch into an activating Zn2+ switch, allowing Zn2+-dependent activation of the transporter. The tyrosine is part of a conserved YXXΦ trafficking motif (X is any residue and Φ is a residue with a bulky hydrophobic group), but Y335A did not show alterations in surface targeting or protein kinase C-mediated internalization. Despite wild-type levels of surface expression, Y335A displayed a dramatic decrease in [3H]dopamine uptake velocity (Vmax) to less than 1% of the wild type. In addition, Y335A showed up to 150-fold decreases in the apparent affinity for cocaine, mazindol, and related inhibitors whereas the apparent affinity for several substrates was increased. However, the presence of Zn2+ in micromolar concentrations increased the Vmax up to 24-fold and partially restored the apparent affinities. The capability of Zn2+ to restore transport is consistent with a reversible, constitutive shift in the distribution of conformational states in the transport cycle upon mutation of Tyr-335. We propose that this shift is caused by disruption of intramolecular interactions important for stabilizing the transporter in a conformation in which extracellular substrate can bind and initiate transport, and accordingly that Tyr-335 is critical for regulating isomerization between discrete states in the transport cycle
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