The uptake inhibitors cocaine and benztropine differentially alter the conformation of the human dopamine transporter.
The binding affinity of the cocaine analog [(3)H]2 beta-carbomethoxy-3beta-(4-fluorophenyl) tropane (WIN) for the dopamine transporter (DAT) is increased by the reaction of Cys-90, at the extracellular end of the first transmembrane segment, with methanethiosulfonate (MTS) reagents. Cocaine enhances the reaction of Cys-90 with the sulfhydryl reagents, thereby augmenting the increase in binding. In contrast, cocaine decreases the reaction of Cys-135 and Cys-342, endogenous cysteines in cytoplasmic loops, with MTS reagents. Because this reaction inhibits [(3)H]WIN binding, cocaine protects against the loss of binding caused by reaction of these cysteines. In the present work, we compare the abilities of DAT inhibitors and substrates to affect the reaction of Cys-90, Cys-135, and Cys-342 with MTS ethyltrimethylammonium (MTSET). The results indicate that the different abilities of compounds to protect against the MTSET-induced inhibition of binding are attributable to differences in their abilities to attenuate the inhibitory effects of modification of Cys-135 and Cys-342 as well as to enhance the reaction with Cys-90 and the resulting potentiation of binding. The inhibitor benztropine was unique in its inability to protect Cys-135. Moreover, whereas cocaine, WIN, mazindol, and dopamine enhanced the reaction of Cys-90 with MTSET, benztropine had no effect on this reaction. These two features combine to give benztropine its weak potency in protecting ligand binding to wild-type DAT from MTSET. These results indicate that different inhibitors of DAT, such as cocaine and benztropine, produce different conformational changes in the transporter. There are differences in the psychomotor stimulant-like effects of these compounds, and it is possible that the different behavioral effects of these DAT inhibitors stem from their different molecular actions on DAT.
Reith
M E
ME
Department of Biomedical and Therapeutic Sciences, University of Illinois College of Medicine, Peoria, Illinois 61656, USA. MaartenR@uic.edu
Berfield
J L
JL
Wang
L C
LC
Ferrer
J V
JV
Javitch
J A
JA
D001590:Benztropine / Q000494:pharmacology*; D001665:Binding Sites; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003042:Cocaine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000494:pharmacology*; D003545:Cysteine; D004298:Dopamine / Q000494:pharmacology; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors / Q000493:pharmacokinetics / Q000494:pharmacology*; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D008454:Mazindol / Q000494:pharmacology; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D008698:Mesylates / Q000493:pharmacokinetics / Q000494:pharmacology; D008958:Models, Molecular; D009419:Nerve Tissue Proteins; D011487:Protein Conformation / Q000187:drug effects*
D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D007202:Indicators and Reagents; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D008698:Mesylates; D009419:Nerve Tissue Proteins; C493214:SLC6A3 protein, human; D001590:Benztropine; C074502:methanethiosulfonate; C021279:(1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; D008454:Mazindol; D003042:Cocaine; D003545:Cysteine; D004298:Dopamine