Dopamine transport sites selectively labeled by a novel photoaffinity probe: 125I-DEEP

The dopamine transporter was labeled using a photosensitive compound related to GBR-12909, 125I-1-[2-(diphenylmethoxy)ethyl]-4-[2- (4-azido-3-iodophenyl)ethyl]piperazine (125I-DEEP). 125I-DEEP bound reversibly and with high affinity to the dopamine transport protein in the absence of light and could be covalently attached to the protein following exposure to UV light. In rat striatal homogenates, 125I-DEEP was found to incorporate covalently into a protein with apparent molecular weight of 58,000 Da. The properties of this binding protein were characteristic of the dopamine transporter since covalent attachment could be inhibited by dopamine-uptake blockers with the proper pharmacological rank order of potencies. Covalent binding was also inhibited in a stereospecific manner by (+) and (-) cocaine, as well as other cocaine analogs. The protein was not found in the cerebellum. The dopamine transporter appears to exist in a glycosylated form since photoaffinity-labeled transport sites could adsorb to wheat germ-agglutinin and could be specifically eluted from the column by beta-N-acetylglucosamine.