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Dopamine transporter cysteine mutants: second extracellular loop cysteines are required for transporter expression
J B Wang1, A Moriwaki, G R Uhl
1Division of Intramural Research, National Institute on Drug Abuse, Baltimore, MD 21224.
Journal of Neurochemistry
|March 1, 1995
Summary
Cysteine residues in the dopamine transporter
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Thiol-modifying reagents suggest cysteine involvement in dopamine transporter (DAT) function.
- Identifying specific cysteine residues critical for DAT function is essential.
Purpose of the Study:
- To pinpoint DAT cysteines with functionally significant thiol groups.
- To elucidate the role of specific cysteines in DAT expression and function.
Main Methods:
- Site-directed mutagenesis to create six cysteine-to-alanine DAT mutants.
- Expression analysis using immunostaining of transfected COS cells.
- Functional assays including dopamine and MPP+ uptake, and cocaine analogue binding.
Main Results:
- Mutations at Cys180 and Cys189 in the second extracellular loop significantly reduced DAT expression.
- These mutants showed decreased membrane localization and increased perinuclear staining (Golgi apparatus).
- Mutations at other positions (90, 242, 305, 345) did not significantly affect DAT function or expression.
Conclusions:
- Cysteines in the DAT's second extracellular loop are crucial for proper transporter expression, potentially by aiding membrane insertion.
- These cysteines may be the targets for thiol-modifying reagents that alter wild-type DAT function.