Development of medications to treat cocaine abuse is an important goal in addressing the national problem of drug abuse. In order to develop new drugs which prevent the binding of cocaine to its target, the dopamine transporter, it would be very useful to know where both cocaine and dopamine bind on the transporter protein. One approach to localization of the binding site for cocaine has been to explore the structural features of cocaine analogs which affect binding to the transporter. An extension of this approach has been to develop inhibitors which bind irreversibly to the transporter. This class of compounds reacts with the protein to form a covalent bond between the inhibitor and the protein. Knowledge of where this bond is formed, that is, which amino acid in the sequence is involved, allows the binding site to begin to be localized. The proposed work uses mass spectrometry to identify the sites of attachment of the irreversible ligands at the human dopamine transporter. With a range of irreversible inhibitors, including irreversible cocaine analogs, a map of the binding site can be constructed. In addition, analogs of dopamine, the substrate for the transporter, that irreversibly react with the transporter will be used to identify the substrate binding region. Thus, regions important in both inhibition and transport will be identified. The combined results will help to construct a picture of the ligand binding regions critical for human dopamine transporter function and inhibition. Without adequate structural information, it is not possible to understand the mechanism of this membrane bound protein when functioning normally or in response to acute or chronic drugs of abuse. Identification of domains involved in substrate and inhibitor binding is an important first step in understanding the mechanism of this and related transporters.
