The uptake inhibitors cocaine and benztropine differentially alter the conformation of the human dopamine transporter

The binding affinity of the cocaine analog [H-3]2 beta -carbomethoxy-3 beta -(4-fluorophenyl) tropane (WIN) for the dopamine transporter (DAT) is incre ased by the reaction of Cys-90, at the extracellular end of the first trans membrane segment, with methanethiosulfonate (NITS) reagents. Cocaine enhanc es 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 NITS reag ents. Because this reaction inhibits [H-3]WIN binding, cocaine protects aga inst the loss of binding caused by reaction of these cysteines. In the pres ent work, we compare the abilities of DAT inhibitors and substrates to affe ct the reaction of Cys-90, Cys-135, and Cys-342 with MTS ethyltrimethyl-amm onium (MTSET). The results indicate that the different abilities of compoun ds to protect against the MTSET-induced inhibition of binding are attributa ble to differences in their abilities to attenuate the inhibitory effects o f modification of Cys-135 and Cys-342 as well as to enhance the reaction wi th Cys-90 and the resulting potentiation of binding. The inhibitor benztrop ine was unique in its inability to protect Cys-135. Moreover, whereas cocai ne, 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, s uch as cocaine and benztropine, produce different conformational changes in the transporter. There are differences in the psychomotor stimulant-like e ffects of these compounds, and it is possible that the different behavioral effects of these DAT inhibitors stem from their different molecular action s on DAT.