A rapid binding assay for solubilized dopamine transporters using [3H]WIN 35,428

Area of Science:

• Neuroscience
• Pharmacology
• Biochemistry

Background:

• Dopamine transporters (DAT) are crucial for regulating dopaminergic neurotransmission.
• Understanding DAT binding characteristics is essential for developing targeted therapeutics.
• Previous studies have utilized various radioligands to investigate DAT.

Purpose of the Study:

• To characterize the binding of the cocaine analog [3H]WIN 35,428 to digitonin-solubilized dopamine transporters from dog caudate nucleus.
• To compare the binding profile of [3H]WIN 35,428 with other known DAT ligands.
• To investigate the biochemical properties of solubilized DAT.

Main Methods:

• Utilized [3H]WIN 35,428 as a radioligand for dopamine transporters.
• Employed a charcoal separation assay to distinguish bound from free ligand.
• Performed competition binding studies with various dopaminergic and serotonergic agents.
• Chromatographed solubilized extracts using wheat germ agglutinin-Sepharose.

Main Results:

• Specific, saturable, high-affinity binding (Kd = 16 nM) of [3H]WIN 35,428 was observed in dog caudate nucleus extracts.
• Binding was inhibited by mazindol, GBR 12909, and (-)-cocaine, consistent with DAT.
• (-)-Cocaine showed higher potency compared to assays with [3H]GBR 12935, suggesting distinct binding site characteristics.
• Binding activity co-eluted with N-acetylglucosamine after wheat germ agglutinin-Sepharose chromatography.

Conclusions:

• [3H]WIN 35,428 effectively labels solubilized dopamine transporters from dog caudate nucleus.
• The binding characteristics are typical of DAT, with some differences noted compared to other ligands.
• Wheat germ agglutinin-Sepharose chromatography provides a method for purifying DAT based on its glycosylation pattern.