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Dopamine transporter site-directed mutations differentially alter substrate transport and cocaine binding
1Laboratory of Molecular Neurobiology, Addiction Research Center/National Institute on Drug Abuse, Baltimore, MD.
Summary
Specific amino acids in the dopamine transporter (DAT) are vital for its function. Mutations in aspartate and serine residues significantly impact dopamine uptake and neurotoxin binding.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Polar amino acids in hydrophobic regions of the dopamine transporter (DAT) are structurally similar to those in catecholamine receptors.
- Understanding the role of these amino acids is crucial for deciphering DAT function and its interaction with ligands.
Purpose of the Study:
- To investigate the functional significance of specific polar amino acids within hydrophobic regions of the dopamine transporter (DAT).
- To identify residues critical for dopamine uptake, neurotoxin binding, and cocaine analog interaction.
Main Methods:
- Site-directed mutagenesis of the dopamine transporter (DAT) cDNA to alter polar amino acid residues.
- Expression of mutated DAT in cells and assessment of [3H]dopamine, [3H]MPP+, and [3H]CFT uptake and binding affinities.
Main Results:
- Replacing aspartate at position 79 significantly reduced [3H]dopamine and [3H]MPP+ uptake and decreased [3H]CFT binding affinity.
- Mutating serine residues at positions 356 and 359 in the seventh hydrophobic region decreased [3H]dopamine and [3H]MPP+ uptake but had less effect on [3H]CFT binding.
- Altering serines in the eighth hydrophobic region did not affect DAT function or ligand binding.
Conclusions:
- Aspartate (position 79) and serine (positions 356-359) residues in specific hydrophobic transmembrane regions are essential for dopamine transporter (DAT) function.
- These findings differentiate residues critical for cocaine binding versus dopamine uptake, offering insights into DAT ligand specificity.