Ethanol-sensitive sites on the human dopamine transporter

Area of Science:

• Neuroscience
• Molecular Biology
• Pharmacology

Background:

• Ethanol is known to influence neurotransmitter transporter activity.
• Previous research indicated ethanol enhances dopamine transporter (DAT) function and cell surface expression.
• The precise molecular mechanisms underlying ethanol's modulation of DAT remain unclear.

Purpose of the Study:

• To investigate the molecular sites within the dopamine transporter (DAT) responsible for ethanol's modulatory effects.
• To compare the effects of ethanol on DAT and the related norepinephrine transporter (NET).
• To identify specific amino acid residues critical for ethanol potentiation of DAT activity.

Main Methods:

• Expression of human DAT and NET in Xenopus oocytes and HeLa cells.
• Construction and analysis of DAT-NET chimeras to map ethanol-sensitive regions.
• Site-directed mutagenesis of specific residues within the DAT second intracellular loop.
• Assessment of [3H]dopamine uptake to quantify transporter function.

Main Results:

• Ethanol potentiated DAT function but inhibited NET function in expressed systems.
• A 76-amino acid region spanning transmembrane domains 2 and 3 of DAT was identified as crucial for ethanol sensitivity.
• Mutations G130T and I137F in the second intracellular loop of DAT abolished ethanol potentiation.
• Mutations F123Y and L138F had no significant effect on ethanol potentiation.

Conclusions:

• Specific amino acid residues within the second intracellular loop of the dopamine transporter are critical for ethanol's modulatory effects.
• The findings identify novel sites involved in ethanol's interaction with DAT, distinct from its interaction with NET.
• This research provides a molecular basis for understanding how ethanol influences dopamine neurotransmission.