Identification of intracellular residues in the dopamine transporter critical for regulation of transporter conformation and cocaine binding.
Recently we showed evidence that mutation of Tyr-335 to Ala (Y335A) in the human dopamine transporter (hDAT) alters the conformational equilibrium of the transport cycle. Here, by substituting, one at a time, 16 different bulky or charged intracellular residues, we identify three residues, Lys-264, Asp-345, and Asp-436, the mutation of which to alanine produces a phenotype similar to that of Y335A. Like Y335A, the mutants (K264A, D345A, and D436A) were characterized by low uptake capacity that was potentiated by Zn(2+). Moreover, the mutants displayed lower affinity for cocaine and other inhibitors, suggesting a role for these residues in maintaining the structural integrity of the inhibitor binding crevice. The conformational state of K264A, Y335A, and D345A was investigated by assessing the accessibility to MTSET ([2-(trimethylammonium)ethyl]-methanethiosulfonate) of a cysteine engineered into position 159 (I159C) in transmembrane segment 3 of the MTSET-insensitive "E2C" background (C90A/C306A). Unlike its effect at the corresponding position in the homologous norepinephrine transporter (NET I155C), MTSET did not inhibit uptake mediated by E2C I159C. Furthermore, no inhibition was observed upon treatment with MTSET in the presence of dopamine, cocaine, or Zn(2+). Without Zn(2+), E2C I159C/K264A, E2C I159C/Y335A, and E2C I159C/D345A were also not inactivated by MTSET. In the presence of Zn(2+) (10 microm), however, MTSET (0.5 mm) caused up to approximately 60% inactivation. As in NET I155C, this inactivation was protected by dopamine and enhanced by cocaine. These data are consistent with a Zn(2+)-dependent partial reversal of a constitutively altered conformational equilibrium in the mutant transporters. They also suggest that the conformational equilibrium produced by the mutations resembles that of the NET more than that of the DAT. Moreover, the data provide evidence that the cocaine-bound state of both DAT mutants and of the NET is structurally distinct from the cocaine-bound state of the DAT.
Molecular Neuropharmacology Group, Department of Pharmacology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark.
Claus Juul
CJ
Loland
Charlotta
C
Grånäs
Jonathan A
JA
Javitch
Ulrik
U
Gether
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D019921:Biotinylation; D015153:Blotting, Western; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D003042:Cocaine / Q000737:chemistry* / Q000494:pharmacology; D004298:Dopamine / Q000737:chemistry; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009419:Nerve Tissue Proteins; D009638:Norepinephrine / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C493214:SLC6A3 protein, human; D014443:Tyrosine; D003042:Cocaine; D015032:Zinc; D000409:Alanine; D004298:Dopamine; D009638:Norepinephrine