Radiation inactivation studies of the dopamine reuptake transporter protein.
Using radiation inactivation, we have estimated the target size for the neuronal dopamine transporter protein. The specific binding of several radioligands previously shown to label the dopamine transporter was determined in an irradiated striatal membrane preparation. The apparent target size of the 1-[1-(2-[3H]benzo[b]thienyl)cyclohexyl]piperidine site was approximately 98 kDa. However, the apparent target size of the "cocaine binding site," as measured with the cocaine analogue 2 beta-[3H]carbomethoxy-3 beta-(4-fluorophenyl)tropane in the same assays, was approximately 140 kDa. Radiation inactivation of the binding of other ligands (GBR-12935 and mazindol) led to target size estimates in the same range (94 kDa and 133 kDa, respectively). All of these target sizes are significantly larger than the estimate of 70 kDa derived from the deduced amino acid sequence for the cloned dopamine reuptake transporter cDNA. Larger target sizes than expected have also been reported for ligand binding to the sodium-dependent serotonin transporter and glucose transporter. The estimated sizes for the ligand binding site(s) associated with the dopamine transporter protein are difficult to reconcile with a single transporter protein of 70 kDa. We conclude that the dopamine transporter protein is a homo- or hetero-oligomer when occupied in situ by uptake-blocking drugs like cocaine.
Laboratory of Psychiatry and Neurology, University of California, San Francisco/San Francisco Veterans Administration Medical Center 94121.
S P
SP
Berger
K
K
Farrell
D
D
Conant
E S
ES
Kempner
S M
SM
Paul
D000818:Animals; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000528:radiation effects*; D003342:Corpus Striatum / Q000378:metabolism / Q000528:radiation effects; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D008297:Male; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011485:Protein Binding; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D013574:Synaptosomes / Q000378:metabolism / Q000528:radiation effects
D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D004298:Dopamine