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

Research output: Contribution to journal › Journal article › Research › peer-review

Standard

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. / Loland, Claus Juul; Norregaard, Lene; Litman, Thomas; Gether, Ulrik.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 99, No. 3, 05.02.2002, p. 1683-8.

Research output: Contribution to journal › Journal article › Research › peer-review

Harvard

Loland, CJ, Norregaard, L, Litman, T & Gether, U 2002, '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', Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 3, pp. 1683-8. https://doi.org/10.1073/pnas.032386299

APA

Loland, C. J., Norregaard, L., Litman, T., & Gether, U. (2002). 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. Proceedings of the National Academy of Sciences of the United States of America, 99(3), 1683-8. https://doi.org/10.1073/pnas.032386299

Vancouver

Loland CJ, Norregaard L, Litman T, Gether U. 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. Proceedings of the National Academy of Sciences of the United States of America. 2002 Feb 5;99(3):1683-8. https://doi.org/10.1073/pnas.032386299

Author

Loland, Claus Juul ; Norregaard, Lene ; Litman, Thomas ; Gether, Ulrik. / 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. In: Proceedings of the National Academy of Sciences of the United States of America. 2002 ; Vol. 99, No. 3. pp. 1683-8.

Bibtex

@article{0946daf7fee5453f8ad6bed30ddc1324,
title = "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",
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.",
keywords = "Amino Acid Sequence, Amino Acid Substitution, Animals, Binding Sites, Biological Transport, COS Cells, Cercopithecus aethiops, Conserved Sequence, Dopamine Plasma Membrane Transport Proteins, Humans, Kidney, Kinetics, Membrane Glycoproteins, Membrane Transport Proteins, Molecular Sequence Data, Mutagenesis, Site-Directed, Nerve Tissue Proteins, Protein Conformation, Protein Kinase C, Protein Structure, Secondary, Recombinant Proteins, Transfection, Tyrosine, Zinc",
author = "Loland, {Claus Juul} and Lene Norregaard and Thomas Litman and Ulrik Gether",
year = "2002",
month = feb,
day = "5",
doi = "10.1073/pnas.032386299",
language = "English",
volume = "99",
pages = "1683--8",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
publisher = "The National Academy of Sciences of the United States of America",
number = "3",

}

RIS

TY - JOUR

T1 - Generation of an activating Zn(2+) switch in the dopamine transporter

T2 - mutation of an intracellular tyrosine constitutively alters the conformational equilibrium of the transport cycle

AU - Loland, Claus Juul

AU - Norregaard, Lene

AU - Litman, Thomas

AU - Gether, Ulrik

PY - 2002/2/5

Y1 - 2002/2/5

N2 - 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.

AB - 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.

KW - Amino Acid Sequence

KW - Amino Acid Substitution

KW - Animals

KW - Binding Sites

KW - Biological Transport

KW - COS Cells

KW - Cercopithecus aethiops

KW - Conserved Sequence

KW - Dopamine Plasma Membrane Transport Proteins

KW - Humans

KW - Kidney

KW - Kinetics

KW - Membrane Glycoproteins

KW - Membrane Transport Proteins

KW - Molecular Sequence Data

KW - Mutagenesis, Site-Directed

KW - Nerve Tissue Proteins

KW - Protein Conformation

KW - Protein Kinase C

KW - Protein Structure, Secondary

KW - Recombinant Proteins

KW - Transfection

KW - Tyrosine

KW - Zinc

U2 - 10.1073/pnas.032386299

DO - 10.1073/pnas.032386299

M3 - Journal article

C2 - 11818545

VL - 99

SP - 1683

EP - 1688

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 3

ER -

ID: 47293009