Cocaine initiates its euphoric effects in the brain by binding to the dopamine transporter (DAT), blocking uptake of synaptic dopamine. No specific DAT-ligand contacts have been identified to date, but it is widely held (owing to a previous report) that the DAT transmembrane (TM) 1 aspartic acid residue (D79) forms an ionic interaction with charged nitrogen atoms in both dopamine and cocaine that governs recognition of the ligand. Alternatively, the D79 residue may contribute to a ligand aromatic binding pocket, an ion permeation pore that influences ligand binding, or may simply form intramolecular contacts that retain DAT infrastructure. Toward the goal of elaborating on the nature of DAT substrate and inhibtor binding sites, it is critical to determine whether the """"""""ion pair"""""""" model for DAT-ligand interactions is credible. The objective of this proposal is to address the validity of the ion pair model, while also assessing alternative potential contributions of the D79 residue to DAT structure and function. D79 DAT mutants that modify side chain size, charge or hydrogen bonding potential will be pharmacologically characterized with a diverse collection of DAT substrates and inhibitors. Other, cysteine-substituted, DAT mutants will test the accessibility of a given position in the DAT polypeptide to cysteine-specific alkylating agents, and whether such alkylation events influence DAT function. By measuring accessibility of an introduced DAT TM 1 (or vicinity) cysteine residue (variably located) in the presence and absence of substrates or inhibitors, the spatial relationship between D79 and DAT substrates and inhibitors will be explored. Because the TM 1 aspartate has been implicated as a contributor to the substrate permeation pore, the substituted cysteine accessibility method will also be used to elaborate on the secondary structure and cell membrane orientation of TM 1 in the vicinity of D79, for which there are multiple models. The proposed experiments should significantly enhance understanding of the TM 1 aspartate residue role in DAT function, as well as advance structure-function studies on the plasma membrane norepinephrine and serotonin transporters which share this residue. Clarification of the role of this residue in recognition of dopamine and cocaine may forward rational design of therapeutics that block cocaine action without significantly interfering with dopamine uptake. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
3R15DA016604-01S2
Application #
7249685
Study Section
Molecular, Cellular and Developmental Neurosciences 2 (MDCN)
Program Officer
Rapaka, Rao
Project Start
2003-08-01
Project End
2006-07-31
Budget Start
2003-08-01
Budget End
2006-07-31
Support Year
1
Fiscal Year
2006
Total Cost
$39,236
Indirect Cost
Name
Duquesne University
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
004501193
City
Pittsburgh
State
PA
Country
United States
Zip Code
15282
Lapinsky, David J; Aggarwal, Shaili; Nolan, Tammy L et al. (2012) (±)-2-(N-tert-Butylamino)-3'-[(125)I]-iodo-4'-azidopropiophenone: a dopamine transporter and nicotinic acetylcholine receptor photoaffinity ligand based on bupropion (Wellbutrin, Zyban). Bioorg Med Chem Lett 22:523-6
Lapinsky, David J; Yarravarapu, Nageswari; Nolan, Tammy L et al. (2012) Evolution of a Compact Photoprobe for the Dopamine Transporter Based on (±)-threo-Methylphenidate. ACS Med Chem Lett 3:378-382
Lapinsky, David J; Velagaleti, Ranganadh; Yarravarapu, Nageswari et al. (2011) Azido-iodo-N-benzyl derivatives of threo-methylphenidate (Ritalin, Concerta): Rational design, synthesis, pharmacological evaluation, and dopamine transporter photoaffinity labeling. Bioorg Med Chem 19:504-12
Indarte, Martín; Liu, Yi; Madura, Jeffry D et al. (2010) Receptor-Based Discovery of a Plasmalemmal Monoamine Transporter Inhibitor via High Throughput Docking and Pharmacophore Modeling. ACS Chem Neurosci 1:223-233
Lapinsky, David J; Aggarwal, Shaili; Huang, Yurong et al. (2009) A novel photoaffinity ligand for the dopamine transporter based on pyrovalerone. Bioorg Med Chem 17:3770-4
Indarte, Martin; Madura, Jeffry D; Surratt, Christopher K (2008) Dopamine transporter comparative molecular modeling and binding site prediction using the LeuT(Aa) leucine transporter as a template. Proteins 70:1033-46
Ukairo, Okechukwu T; Ramanujapuram, Suneetha; Surratt, Christopher K (2007) Fluctuation of the dopamine uptake inhibition potency of cocaine, but not amphetamine, at mammalian cells expressing the dopamine transporter. Brain Res 1131:68-76
Ukairo, Okechukwu T; Bondi, Corry D; Newman, Amy Hauck et al. (2005) Recognition of benztropine by the dopamine transporter (DAT) differs from that of the classical dopamine uptake inhibitors cocaine, methylphenidate, and mazindol as a function of a DAT transmembrane 1 aspartic acid residue. J Pharmacol Exp Ther 314:575-83
Surratt, Christopher K; Ukairo, Okechukwu T; Ramanujapuram, Suneetha (2005) Recognition of psychostimulants, antidepressants, and other inhibitors of synaptic neurotransmitter uptake by the plasma membrane monoamine transporters. AAPS J 7:E739-51