One of the major targets for cocaine in the central nervous system is the dopamine transporter (DAT), which clears dopamine from the extraneuronal space. Cocaine binds to DAT and inhibits reuptake, causing transmitter overflow and stimulation of downstream pathways that lead to euphoria and addiction. No pharmacological treatment for cocaine abuse currently exists, due in part to our dearth of knowledge regarding transporter structure and inhibitor binding mechanisms. To elucidate how cocaine interacts with DAT we utilized a synergistic approach involving irreversible labeling with the cocaine analogue 3β-(p-chlorophenyl)tropane-2β-carboxylic acid, 4′-azido-3′-iodophenylethyl ester (RTI82), computational modeling of ligand poses, and ligand protection analyses. Our results directly demonstrate RTI82 binding in a site and orientation that is consistent with many biochemical findings, and indicate specific mechanisms of transport inhibition. These findings have major relevance for the molecular basis of cocaine actions, and the approaches developed will be useful for examination of structurally and mechanistically distinct categories of uptake blockers.
