Michael J Tomlinson1, Danielle Krout1, Akula Bala Pramod1, John R Lever2, Amy Hauck Newman3, L Keith Henry4, Roxanne A Vaughan5. 1. Department of Biomedical Sciences, University of North Dakota School of Medicine and Health Sciences, 1301 North Columbia Road, Grand Forks, ND 58202, United States. 2. Research Service, Harry S. Truman Memorial Veterans' Hospital, Columbia, MO, 65201, United States; Department of Radiology and Radiopharmaceutical Sciences Institute, University of Missouri, Columbia, MO 65211, United States. 3. Medicinal Chemistry Section, National Institute on Drug Abuse - Intramural Research Program, Baltimore, MD 21224, United States. 4. Department of Biomedical Sciences, University of North Dakota School of Medicine and Health Sciences, 1301 North Columbia Road, Grand Forks, ND 58202, United States. Electronic address: keith.henry@med.und.edu. 5. Department of Biomedical Sciences, University of North Dakota School of Medicine and Health Sciences, 1301 North Columbia Road, Grand Forks, ND 58202, United States. Electronic address: roxanne.vaughan@med.und.edu.
Abstract
The dopamine transporter (DAT) is a neuronal membrane protein that is responsible for reuptake of dopamine (DA) from the synapse and functions as a major determinant in control of DA neurotransmission. Cocaine and many psychostimulant drugs bind to DAT and block reuptake, inducing DA overflow that forms the neurochemical basis for euphoria and addiction. Paradoxically, however, some ligands such as benztropine (BZT) bind to DAT and inhibit reuptake but do not produce these effects, and it has been hypothesized that differential mechanisms of binding may stabilize specific transporter conformations that affect downstream neurochemical or behavioral outcomes. To investigate the binding mechanisms of BZT on DAT we used the photoaffinity BZT analog [125I]N-[n-butyl-4-(4‴-azido-3‴-iodophenyl)]-4',4″-difluoro-3α-(diphenylmethoxy)tropane ([125I]GA II 34) to identify the site of cross-linking and predict the binding pose relative to that of previously-examined cocaine photoaffinity analogs. Biochemical findings show that adduction of [125I]GA II 34 occurs at residues Asp79 or Leu80 in TM1, with molecular modeling supporting adduction to Leu80 and a pharmacophore pose in the central S1 site similar to that of cocaine and cocaine analogs. Substituted cysteine accessibility method protection analyses verified these findings, but identified some differences in structural stabilization relative to cocaine that may relate to BZT neurochemical outcomes.
The dopamine transporter (n class="Gene">DAT) is a neuronal membrane protein that is responsible for reuptake of dopamine (DA) from the synapse and functions as a major determinant in control of DA neurotransmission. Cocaine and many psychostimulant drugs bind to DAT and block reuptake, inducing DA overflow that forms the neurochemical basis for euphoria and addiction. Paradoxically, however, some ligands such as benztropine (BZT) bind to DAT and inhibit reuptake but do not produce these effects, and it has been hypothesized that differential mechanisms of binding may stabilize specific transporter conformations that affect downstream neurochemical or behavioral outcomes. To investigate the binding mechanisms of BZT on DAT we used the photoaffinity BZT analog [125I]N-[n-butyl-4-(4‴-azido-3‴-iodophenyl)]-4',4″-difluoro-3α-(diphenylmethoxy)tropane ([125I]GA II 34) to identify the site of cross-linking and predict the binding pose relative to that of previously-examined cocaine photoaffinity analogs. Biochemical findings show that adduction of [125I]GA II 34 occurs at residues Asp79 or Leu80 in TM1, with molecular modeling supporting adduction to Leu80 and a pharmacophore pose in the central S1 site similar to that of cocaine and cocaine analogs. Substituted cysteine accessibility method protection analyses verified these findings, but identified some differences in structural stabilization relative to cocaine that may relate to BZT neurochemical outcomes.
Authors: Barbara Nightingale; Christina M Dersch; Terrence L Boos; Elisabeth Greiner; William J Calhoun; Arthur E Jacobson; Kenner C Rice; Richard B Rothman Journal: J Pharmacol Exp Ther Date: 2005-04-28 Impact factor: 4.030