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 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.
Loland
Claus Juul
CJ
Division of Cellular and Molecular Physiology, Department of Medical Physiology 12.5, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark.
Norregaard
Lene
L
Litman
Thomas
T
Gether
Ulrik
U
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*
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