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

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

SN - 0027-8424

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

IS - 3

ER -