Identification of intracellular residues in the dopamine transporter critical for regulation of transporter conformation and cocaine binding
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Identification of intracellular residues in the dopamine transporter critical for regulation of transporter conformation and cocaine binding. / Loland, Claus Juul; Grånäs, Charlotta; Javitch, Jonathan A; Gether, Ulrik.
In: The Journal of Biological Chemistry, Vol. 279, No. 5, 30.01.2004, p. 3228-38.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Identification of intracellular residues in the dopamine transporter critical for regulation of transporter conformation and cocaine binding
AU - Loland, Claus Juul
AU - Grånäs, Charlotta
AU - Javitch, Jonathan A
AU - Gether, Ulrik
PY - 2004/1/30
Y1 - 2004/1/30
N2 - 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.
AB - 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.
KW - Alanine
KW - Amino Acid Sequence
KW - Animals
KW - Biotinylation
KW - Blotting, Western
KW - COS Cells
KW - Cell Membrane
KW - Cocaine
KW - Dopamine
KW - Dopamine Plasma Membrane Transport Proteins
KW - Dopamine Uptake Inhibitors
KW - Humans
KW - Inhibitory Concentration 50
KW - Kinetics
KW - Membrane Glycoproteins
KW - Membrane Transport Proteins
KW - Molecular Sequence Data
KW - Mutagenesis, Site-Directed
KW - Mutation
KW - Nerve Tissue Proteins
KW - Norepinephrine
KW - Protein Binding
KW - Protein Conformation
KW - Sequence Homology, Amino Acid
KW - Transfection
KW - Tyrosine
KW - Zinc
U2 - 10.1074/jbc.M304755200
DO - 10.1074/jbc.M304755200
M3 - Journal article
C2 - 14597628
VL - 279
SP - 3228
EP - 3238
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 5
ER -
ID: 47293920