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

Benztropine and its analogs are tropane ring-containing dopamine uptake inh ibitors that produce behavioral effects markedly different from cocaine and other dopamine transporter blockers. We investigated the benztropine bindi ng site on dopamine transporters by covalently attaching a benztropine-base d photoaffinity ligand, [I-125]N-[n-butyl-4-(4'''-azido-3'''-iodophenyl)]- 4',4 "-difluoro-3 alpha-(diphenylmethoxy)tropane ([I-125]GA II 34), to the protein, followed by proteolytic and immunological peptide mapping. The map s were compared with those obtained for dopamine transporters photoaffinity labeled with a GBR 12935 analog, [I-125]1-[2-(djphenylmethoxy)ethyl]-4-[2- (4-azido 3-iodophenyl)ethyl]piperazine ([I-125]DEEP), and a cocaine analog, [I-125]3 beta-(p-chlorophenyl)tropane-2 beta-carboxylic acid, 4'-azido-3'- iodophenylethyl ester ([I-125]RTI 82), which have been shown previously to interact with different regions of the primary sequence of the protein. [I- 125]GA II 34 became incorporated in a membrane-bound, 14 kDa fragment predi cted to contain transmembrane domains 1 and 2. This is the same region of t he protein that binds [I-125]DEEP, whereas the binding site for [I-125]RTI 82 occurs closer to the C terminal in a domain containing transmembrane hel ices 4-7. Thus, although benztropine and cocaine both contain tropane rings , their binding sites are distinct, suggesting that dopamine transport inhi bition may occur by different mechanisms. These results support previously derived structure-activity relationships suggesting that benztropine and co caine analogs bind to different domains on the dopamine transporter. These differing molecular interactions may lead to the distinctive behavioral pro files of these compounds in animal models of drug abuse and indicate promis e for the development of benztropine-based molecules for cocaine substituti on therapies.