A rapid binding assay for solubilized dopamine transporters using [3H]WIN 35,428.
The cocaine analog [3H]WIN 35,428 was used to label digitonin-solubilized dopamine transporters from dog caudate nucleus. The assay consists of incubation of extracts with the ligand followed by separation of free from bound ligand by centrifugation after adding activated charcoal. Specific binding was observed in dog caudate but was absent in dog cerebellar extracts. Binding was linear with tissue, saturable, and of high affinity (Kd = 16 nM). In competition studies, soluble [3H]WIN 35,428 binding was inhibited strongly by mazindol, GBR 12909, and (-)-cocaine but only weakly by citalopram, desipramine, and (+)-cocaine; this is typical of binding to the dopamine transporter. Compared to assays using [3H]GBR 12935, (-)-cocaine was relatively more potent, suggesting that the cocaine and GBR 12935 binding sites are somewhat different. When soluble extract was chromatographed on a wheat germ agglutinin-Sepharose column, [3H]WIN 35,428 binding activity was eluted with N-acetylglucosamine in a manner similar to photoaffinity-labeled dopamine transporters.
Vaughan
R A
RA
Neuroscience Branch, NIDA Addiction Research Center, Baltimore, MD 21224.
Simantov
R
R
Lew
R
R
Kuhar
M J
MJ
D000327:Adsorption; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D001923:Brain Chemistry / Q000187:drug effects; D002352:Carrier Proteins / Q000032:analysis* / Q000493:pharmacokinetics; D002421:Caudate Nucleus / Q000187:drug effects / Q000378:metabolism; D002498:Centrifugation; D002606:Charcoal; D003042:Cocaine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004285:Dogs; D050483:Dopamine Plasma Membrane Transport Proteins; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007700:Kinetics; D008024:Ligands; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins / Q000032:analysis; D011485:Protein Binding; D013237:Stereoisomerism
D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D007202:Indicators and Reagents; D008024:Ligands; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D002606:Charcoal; C021279:(1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; D003042:Cocaine