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

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...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2002-02, Vol.99 (3), p.1683
Main Authors: Loland, Claus Juul, Norregaard, Lene, Litman, Thomas, Gether, Ulrik
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amino Acid Substitution
Animals
Binding Sites
Biological Transport
Cercopithecus aethiops
Conserved Sequence
COS Cells
Dopamine Plasma Membrane Transport Proteins
Humans
Kidney
Kinetics
Membrane Glycoproteins
Membrane Transport Proteins - chemistry
Membrane Transport Proteins - genetics
Membrane Transport Proteins - metabolism
Molecular Sequence Data
Mutagenesis, Site-Directed
Nerve Tissue Proteins
Protein Conformation
Protein Kinase C - metabolism
Protein Structure, Secondary
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
Transfection
Tyrosine
Zinc - metabolism
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Summary: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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.032386299