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Proc Natl Acad Sci U S A. 2002 Feb 5;99(3):1683-8. Epub 2002 Jan 29.

Generation of an activating Zn(2+) switch in the dopamine transporter: mutation of an intracellular tyrosine constitutively alters the conformational equilibrium of the transport cycle.

Author information

  • 1Division of Cellular and Molecular Physiology, Department of Medical Physiology 12.5, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark.

Abstract

Binding of Zn(2+) to the endogenous Zn(2+) binding site in the human dopamine transporter leads to potent inhibition of [(3)H]dopamine uptake. Here we show that mutation of an intracellular tyrosine to alanine (Y335A) converts this inhibitory Zn(2+) switch into an activating Zn(2+) switch, allowing Zn(2+)-dependent activation of the transporter. The tyrosine is part of a conserved YXX Phi trafficking motif (X is any residue and Phi 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 [(3)H]dopamine uptake velocity (V(max)) 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 Zn(2+) in micromolar concentrations increased the V(max) up to 24-fold and partially restored the apparent affinities. The capability of Zn(2+) 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.

PMID:
11818545
[PubMed - indexed for MEDLINE]
PMCID:
PMC122251
Free PMC Article

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