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.
authors.affiliation: Division of Cellular and Molecular Physiology, Department of Medical Physiology 12.5, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: Claus Juul
authors.initials: CJ
authors.lastname: Loland
authors.forename: Lene
authors.initials: L
authors.lastname: Norregaard
authors.forename: Thomas
authors.initials: T
authors.lastname: Litman
authors.forename: Ulrik
authors.initials: U
authors.lastname: Gether
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D019556:COS Cells; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D050483:Dopamine Plasma Membrane Transport Proteins; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014443:Tyrosine; D015032:Zinc / Q000378:metabolism*
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D011493:Protein Kinase C; D015032:Zinc