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Characterization of a recombinant human dopamine transporter in multiple cell lines
A J Eshleman1, R L Neve, A Janowsky
1Research Service, Veterans Affairs Medical Center, Portland, Oregon, USA.
Summary
Researchers isolated a human dopamine transporter (hDAT) cDNA, revealing a key amino acid change. Transfected cells showed high-affinity dopamine uptake and binding, validating the hDAT as a functional target for drug development.
Area of Science:
- Neuroscience
- Molecular Biology
- Pharmacology
Background:
- The dopamine transporter (DAT) plays a crucial role in regulating dopaminergic neurotransmission.
- Understanding DAT function is vital for developing treatments for neurological and psychiatric disorders.
Purpose of the Study:
- To isolate and characterize a human dopamine transporter (hDAT) cDNA.
- To investigate the functional properties and drug-binding characteristics of the expressed hDAT.
Main Methods:
- Isolation of a 3.5-kilobase hDAT cDNA from a human substantia nigra library.
- Transfection of C6 glioma and COS-7 cells with hDAT cDNA.
- Dopamine uptake assays and radioligand binding studies using [3H]DA, [3H]CFT, and [3H]GBR-12935.
Main Results:
- The isolated hDAT cDNA contained two nucleotide differences from published sequences, including an Arg-344 to Methionine substitution.
- Transfected cells (C6-hDAT and Cos7-hDAT) exhibited high-affinity dopamine uptake (Km = 1.2-1.5 microM).
- Radioligand binding studies with [3H]CFT and [3H]GBR-12935 confirmed high-affinity binding sites on Cos7-hDAT cells, with drug inhibition profiles correlating strongly with uptake inhibition.
Conclusions:
- The isolated hDAT cDNA encodes a functional dopamine transporter.
- The characterized hDAT exhibits high affinity for dopamine uptake and radioligands.
- The findings support the utility of this hDAT construct for studying transporter function and screening potential therapeutic agents.