The Journal of Neuroscience, January 15, 1999, 19(2):630–636 Differential Binding of Tropane-Based Photoaffinity Ligands on the Dopamine Transporter Roxanne A. Vaughan,1,3 Gregory E. Agoston,2 John R. Lever,4 and Amy Hauck Newman2 1Molecular Neurobiology Branch and 2Psychobiology Section, National Institute on Drug Abuse-Intramural Research Program, National Institutes of Health, Baltimore, Maryland 21224, and 3Neuroscience Division, Yerkes Regional Primate Center, and 4Department of Environmental Health Sciences, The Johns Hopkins University School of Public Health, Baltimore, Maryland 21205 Benztropine and its analogs are tropane ring–containing dopa- contain transmembrane domains 1 and 2. This is the same mine uptake inhibitors that produce behavioral effects markedly region of the protein that binds [ 125I]DEEP, whereas the binding different from cocaine and other dopamine transporter block- site for [ 125I]RTI 82 occurs closer to the C terminal in a domain ers. We investigated the benztropine binding site on dopamine containing transmembrane helices 4–7. Thus, although benz- transporters by covalently attaching a benztropine-based pho- tropine and cocaine both contain tropane rings, their binding toaffinity ligand, [ 125I]N-[n-butyl-4-(4--azido-3--iodophenyl)]- sites are distinct, suggesting that dopamine transport inhibition 49,40-difluoro-3a-(diphenylmethoxy)tropane ([ 125I]GA II 34), may occur by different mechanisms. These results support to the protein, followed by proteolytic and immunological pep- previously derived structure–activity relationships suggesting tide mapping. The maps were compared with those obtained that benztropine and cocaine analogs bind to different domains for dopamine transporters photoaffinity labeled with a GBR on the dopamine transporter. These differing molecular interac- 12935 analog, [ 125I]1-[2-(diphenylmethoxy)ethyl]-4-[2-(4-azido- tions may lead to the distinctive behavioral profiles of these 3-iodophenyl)ethyl]piperazine ([ 125I]DEEP), and a cocaine ana- compounds in animal models of drug abuse and indicate prom- log, [ 125I]3b-( p-chlorophenyl)tropane-2b-carboxylic acid, 49- ise for the development of benztropine-based molecules for azido-39-iodophenylethyl ester ([ 125I]RTI 82), which have been cocaine substitution therapies. shown previously to interact with different regions of the pri- Key words: cocaine; benztropine; dopamine transporter; mary sequence of the protein. [ 125I]GA II 34 became incorpo- photoaffinity label; dopamine uptake; proteolytic peptide map- rated in a membrane-bound, 14 kDa fragment predicted to ping; immunoprecipitation One of the major structural contributors to high-affinity binding been aided by the use of selective high-affinity probes and the of cocaine and its analogs to the dopamine transporter (DAT) is application of molecular and immunological techniques. These the tropane ring (Carroll et al., 1992a). Benztropine, a dopamine approaches can be combined to identify discrete binding sites and uptake inhibitor equipotent to cocaine, also contains a tropane elucidate molecular mechanisms underlying drug action. One of ring that is considered essential for its high-affinity binding. the primary central targets for cocaine action is the DAT where However, several studies have revealed that benztropine and its synaptic dopamine is cleared using the energy of electrochemical analogs display binding and behavioral profiles substantially dif- ion gradients to drive transport of extracellular dopamine into ferent from those of cocaine-like compounds (Meltzer et al., presynaptic terminals. When the DAT is blocked by cocaine, 1994, 1996, 1997; Newman et al., 1994, 1995; Agoston et al., dopamine levels are increased, and the resulting stimulation of 1997a; Katz et al., 1997; Kline et al., 1997), leading to the postsynaptic dopamine receptors is believed to be the primary suggestion that the two series of ligands interact at different sites neurochemical mechanism underlying the psychostimulant and on the protein (Newman et al., 1994, 1995). To investigate this reinforcing actions of the drug (Ritz et al., 1987; Kuhar et al., hypothesis further, we have developed a benztropine-based pho- 1991). Although there is compelling evidence to support the toaffinity ligand, which binds irreversibly to the DAT, to identify pivotal role the dopamine transporter plays in the reinforcing its binding regions and compare them with those of cocaine actions of psychostimulant drugs, subtle uptake inhibitor binding (Agoston et al., 1997b). properties, at the molecular level, may be requisite to effect The characterization of molecular targets of drug action has neurochemical and behavioral endpoints. Many dopamine uptake blockers, including highly selective and Received June 30, 1998; revised Oct. 27, 1998; accepted Nov. 2, 1998. potent analogs of cocaine, GBR 12909, benztropine, mazindol, This work was supported by the Intramural Research Program of the National and methylphenidate, have been characterized extensively and Institute on Drug Abuse, National Institutes of Health, and by United States Public Health Service Grant DA 08870 to J.R.L. G.E.A. was fully funded by a National are being evaluated preclinically as potential treatments for co- Institutes of Health Intramural Research Training Award fellowship. We thank Dr. caine abuse (for review, see Carroll et al., 1997). Despite the large Barry Hoffer for his support and editorial assistance. Correspondence should be addressed to Dr. Amy Hauck Newman, Psychobiology amount of pharmacological and behavioral data that has been Section, National Institute on Drug Abuse-Intramural Research Program, National collected on these structurally diverse compounds, the molecular Institutes of Health, 5500 Nathan Shock Drive, Baltimore, MD 21224. mechanism by which they bind to the protein and block transport Dr. Vaughan’s present address: Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine, Grand Forks, ND 58202. is not known. For example, it is currently unclear whether or not Copyright © 1999 Society for Neuroscience 0270-6474/99/190630-07$05.00/0 these compounds bind at single or multiple sites or whether they Vaughan et al. • Photoaffinity Ligands at the Dopamine Transporter J. Neurosci., January 15, 1999, 19(2):630–636 631 interfere with the same or different aspects of substrate translo- M sucrose), using a Brinkman Polytron homogenizer. The homogenate 3 cation (Berger et al., 1990; Meiergerd and Schenk, 1994; Pristupa was centrifuged at 20,000 g for 12 min, the supernatant was discarded, and the homogenization and centrifugation were repeated. The resulting et al., 1994; Reith et al., 1996). Although some studies have membranes were suspended in SP at 5–10 mg/ml original wet weight 125 125 indicated that many of these compounds access a common bind- followed by addition of 5 nM [ I]DEEP or [ I]RTI 82 or 40 nM ing domain (Froimowitz, 1993; Froimowitz et al., 1997), more [ 125I]GA II 34. Samples were incubated for 1 hr at 0°C and irradiated extensive structure–activity relationships (SAR) have identified with UV light for 45 sec, and the membranes were washed twice by molecular requirements indicating that some ligands may be centrifugation. For subsequent gel purification, membranes were solubi- lized with SDS-PAGE sample buffer (62.5 mM Tris, pH 6.8, 0.5% SDS, accessing different domains (Meltzer et al., 1994, 1996, 1997; 10% glycerol, and 10 mM dithiothreitol) at 20 mg/ml original wet weight. Newman et al., 1994, 1995; Agoston et al., 1997a). Elucidating the For subsequent in situ proteolysis, the unsolubilized membranes were nature of these ligand–protein interactions is essential for under- suspended in 50 mM Tris-HCl, pH 8.0, at 50 mg/ml original wet weight. standing the molecular basis of DAT action and for the develop- Proteolytic peptide mapping. Peptide mapping of photoaffinity-labeled DAT was done with two different procedures, (1) proteolysis after gel ment of potential cocaine pharmacotherapies. purification and (2) in situ proteolysis of membrane suspensions. For Herein, we report the discovery of a novel photoaffinity ligand, comparison of DATs labeled with different photoaffinity ligands, samples based on benztropine, and the identification of its recognition site were simultaneously prepared and analyzed exactly in parallel. For gel on the dopamine transporter via the use of proteolytic and im- purification, solubilized photolabeled membranes were electrophoresed munological peptide mapping. Furthermore, despite its tropane on 8% gels, and DATs were identified by autoradiography. The 80 kDa region of the gel containing the DAT was excised, and the protein was ring pharmacophore, this probe does not covalently bond to the passively eluted from the gel with 0.1 M ammonium bicarbonate, pH 8.0, same transmembrane domain region and thus may not be access- containing 0.1% SDS. Aliquots were treated with 2–2000 mg/ml trypsin ing the same binding site as cocaine. This study confirms the for 1 hr, followed by addition of sample buffer and electrophoresis on divergent SAR in these classes of compounds and may be relevant 14% SDS-PAGE gels. For in situ proteolysis, photolabeled membrane suspensions were treated with trypsin, followed by separation of mem- to their distinctive pharmacological profiles in animal models of branes and supernatants and immunoprecipitation of the membrane cocaine abuse. samples. This permits determination of the proximity of label incorpo- ration to transmembrane helices,