Differential binding of tropane-based photoaffinity ligands on the dopamine transporter

Benztropine and its analogs are tropane ring-containing dopamine uptake inhibitors that produce behavioral effects markedly different from cocaine and other dopamine transporter blockers. We investigated the benztropine binding site on dopamine transporters by covalently attaching a benztropine-based photoaffinity ligand, [125I]N-[n-butyl-4-(4"'-azido-3"'-iodophenyl)]-4', 4"-difluoro-3alpha-(diphenylmethoxy)tropane ([125I]GA II 34), to the protein, followed by proteolytic and immunological peptide mapping. The maps were compared with those obtained for dopamine transporters photoaffinity labeled with a GBR 12935 analog, [125I]1-[2-(diphenylmethoxy)ethyl]-4-[2-(4-azido-3-iodophenyl)ethy l]p iperazine ([125I]DEEP), and a cocaine analog, [125I]3beta-(p-chlorophenyl)tropane-2beta-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([125I]RTI 82), which have been shown previously to interact with different regions of the primary sequence of the protein. [125I]GA II 34 became incorporated in a membrane-bound, 14 kDa fragment predicted to contain transmembrane domains 1 and 2. This is the same region of the protein that binds [125I]DEEP, whereas the binding site for [125I]RTI 82 occurs closer to the C terminal in a domain containing transmembrane helices 4-7. Thus, although benztropine and cocaine both contain tropane rings, their binding sites are distinct, suggesting that dopamine transport inhibition may occur by different mechanisms. These results support previously derived structure-activity relationships suggesting that benztropine and cocaine analogs bind to different domains on the dopamine transporter. These differing molecular interactions may lead to the distinctive behavioral profiles of these compounds in animal models of drug abuse and indicate promise for the development of benztropine-based molecules for cocaine substitution therapies.