Development of medications to treat cocaine abuse is an important goal. In order to develop new drugs which prevent binding of cocaine to its target, the dopamine transporter (DAT), 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 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. The objective then is to identify the site of covalent attachment. This is done by cleavage of the protein and following the protein fragment to which the ligand remains attached. Work by investigators at NIDA have used this method to begin to localize two photoaffinity ligands, [125I]RTI-82, a cocaine analog, and [125I]DEEP, a GBR analog to different domains of DAT. The proposed work is designed to use mass spectrometry to identify the sites of attachment of these ligands within the previously identified DAT fragments. In addition, the sites of attachment of two new photoaffinity ligands, which are substrates for DAT, will also be identified. The combined results will help to construct a picture of the ligand binding regions critical for DAT function and inhibition.
