Dissociation of high-affinity cocaine analog binding and dopamine uptake inhibition at the dopamine transporter.
Cocaine initiates its euphoric effects by binding to the dopamine transporter (DAT), blocking uptake of synaptic dopamine. It has been hypothesized that the DAT transmembrane aspartic acid residue D79 forms an ionic interaction with charged nitrogen atoms in both dopamine and cocaine. We examined the consequences of novel and previously studied mutations of the D79 residue on DAT uptake of [3H]dopamine, DAT binding of the cocaine analog [3H]WIN 35,428, and drug inhibition of each process, all under identical conditions. The rat D79E DAT mutation decreased dopamine uptake Vmax by 7-fold and decreased dopamine turnover by 4-fold. Wild-type DAT displayed near-perfect agreement in the uptake and binding inhibition potencies for substrates, but cocaine and other nonsubstrate inhibitor drugs were approximately 3-fold less potent in uptake than in binding assays. Apparent affinities for substrates were unaffected by the D79E mutation unless the catechol moiety was modified. Strikingly, potencies for nonsubstrate inhibitors in uptake and binding assays matched for D79E DAT, because of a 3-fold lowering of binding affinities relative to WT DAT. The present findings reveal a complex role for D79 in determining substrate specificity and high-affinity binding of DAT inhibitors. We propose that at least two discrete inhibitor-binding DAT conformations or populations exist and that the DAT conformation/population responsible for inhibitor high-affinity binding is less responsible for dopamine uptake. The findings may be extensible to other psychostimulants and antidepressants that display discrepancies between binding affinity and monoamine uptake inhibition potency and may be relevant to development of a long-sought "cocaine antagonist".
Wang
Wenfei
W
Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York, USA.
Sonders
Mark S
MS
Ukairo
Okechukwu T
OT
Scott
Helen
H
Kloetzel
Megan K
MK
Surratt
Christopher K
CK
D000818:Animals; D001667:Binding, Competitive; D001692:Biological Transport; D019556:COS Cells; D003042:Cocaine / Q000031:analogs & derivatives / Q000494:pharmacology*; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology*; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins; D051381:Rats; D014162:Transfection
D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C493216:Slc6a3 protein, rat; D003042:Cocaine; D004298:Dopamine