Photoaffinity labeling of the dopamine reuptake carrier protein with 3-azido[3H]GBR-12935

S. P. Berger, R. E. Martenson, P. Laing, A. Thurkauf, B. DeCosta, K. C. Rice, S. M. Paul

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27 Scopus citations


A high affinity tritiated azido-diphenylpiperazine derivative, 3-azido[3H]GBR-12935, was synthesized as a potential photo-affinity probe of the dopamine transporter. Initially, the reversible binding of 3-azido[3H]GBR-12935 to crude synaptosomal membranes from the rat striatum was characterized. Specific binding was sodium dependent and inhibited by a variety of drugs that are known to potently inhibit dopamine uptake. Other neurotransmitter uptake inhibitors, as well as cis-flupenthixol, a potent inhibitor of [3H]GBR-12935 binding to piperazine binding sites, failed to inhibit specific binding at concentrations of ≤10 μM. A good correlation was observed between the relative potencies of these drugs in inhibiting dopamine uptake into synaptosomes and in inhibiting specific 3-azido[3H]GBR-12935 binding to rat striatal membranes (r = 0.95, p < 0.01). These data suggest that 3-azido[3H]GBR-12935, like other diphenylpiperazines such as [3H]GBR-12935 and [3H]GBR-12909, binds primarily to the dopamine transporter under defined assay conditions. After UV photolysis of crude synaptosomal membranes preincubated with 3-azido[3H]GBR-12935 (1-2 nM), a single radiolabeled polypeptide with an apparent molecular mass of 80 kDa was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Photoincorporation of 3-azido[3H]GBR-12935 into this polypeptide was inhibited selectively by compounds that inhibit the uptake of dopamine (but not other biogenic amines) and was completely dependent on the presence of Na+. No photolabeled proteins were observed when cerebellar membranes were substituted for striatal membranes. Essentially complete adsorption of the radiolabeled 80-kDa polypeptide to wheat germ agglutinin and elution with N-acetyl-D-glucosamine strongly suggest that the dopamine transporter polypeptide photolabeled by 3-azido[3H]GBR-12935 is glycosylated.

Original languageEnglish
Pages (from-to)429-435
Number of pages7
JournalMolecular pharmacology
Issue number4
StatePublished - Apr 1991


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