Dopamine Transporter Mutants with Cocaine Resistance and Normal Dopamine Uptake Provide Targets for Cocaine Antagonism

Abstract

Cocaine's blockade of dopamine reuptake by brain dopamine transporters (DAT) is a central feature of current understanding of cocaine reward and addiction. Empirical screening of smallmolecule chemical libraries has thus far failed to provide successful cocaine blockers that allow dopamine reuptake in the presence of cocaine and provide cocaine "antagonism". We have approached this problem by assessing expression, dopamine uptake, and cocaine analog affinities of 56 DAT mutants in residues located in or near transmembrane domains likely to play significant roles in cocaine recognition and dopamine uptake. A phenylalanine-to-alanine mutant in putative DAT transmembrane domain 3, F154A, retains normal dopamine uptake, lowers cocaine affinity 10-fold, and reduces cocaine stereospecificity. Such mutants provide windows into DAT structures that could serve as targets for selective cocaine blockers and document how combined strategies of mutagenesis and small molecule screening may improve our abilities to identify and design compounds with such selective properties.The dopamine transporter (DAT) is a putative 12-transmembrane domain (TM) protein that takes up dopamine into neurons of brain pathways that contribute to behavioral reward (Ranaldi et al., 1999;Redgrave et al., 1999). Cocaine blockade of dopamine uptake by the DAT expressed by these neurons has been identified as a crucial component for cocaine reward, suggesting that selective blockade of cocaine recognition in this pathway could have therapeutic importance for development of anticocaine medication Villemagne et al., 1999).To improve understanding of the ways in which DAT recognizes cocaine and dopamine, hundreds of cocaine analogs have been synthesized and hundreds of DAT mutant and chimeric molecules constructed and characterized (Kitayama et al

Cocaine Affinity Decreased by Mutations of Aromatic Residue Phenylalanine 105 in the Transmembrane Domain 2 of Dopamine Transporter

“…Ritz et al (1987), Kozak (1991), and Ritz and Kuhar (1993) mutated a large number of aromatic resides and prolines located in or near the putative transmembrane domains and found that some of the mutations decreased binding affinity to cocaine analog CFT without significantly affecting dopamine uptake. More recently they reported that changing F154 in TM3 to alanine retains normal DA uptake but lowers cocaine affinity by 10-fold (Lin and Uhl, 2002). Using a similar approach, Chen et al (2001) found that mutant D68N in hDAT exhibited transport activity that was nearly that of wt hDAT and 4-fold lowered sensitivity to cocaine.…”

Irreversible binding of a novel phenylisothiocyanate tropane analog to monoamine transporters in rat brain

“…The amino acid residues in DAT responsible for cocaine binding have been identified by techniques such as site-directed mutagenesis [19][20][21][22][23] which, while important, can be problematic because a change in a single amino acid can alter the protein's conformational structure and affect the interpretation of results. An alternative approach uses irreversible ligands as probes for cocaine binding sites in DAT.…”

Computational and Biochemical Docking of the Irreversible Cocaine Analog RTI 82 Directly Demonstrates Ligand Positioning in the Dopamine Transporter Central Substrate-binding Site

“…Binding of substrate, two Na ϩ (Na1 and Na2) ions, and one Cl Ϫ ion to their respective sites triggers conformational changes leading to closure of an extracellular gate that occludes S1, followed by opening of the intracellular gate and generation of an inwardly facing form that releases solutes to the cytoplasm (14,(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). The S1 site is highly conserved in mammalian transporters, and mutagenesis of many DAT residues near this site, including Phe-76, Asp-79, Val-152, Phe-155, Tyr-156, Asn-157, Phe-319, Val-327, and Ser-421, reduces dopamine transport (31)(32)(33)(34)(35)(36)(37)(38), supporting the participation of these amino acids in substrate recognition or translocation. In particular, Asp-79 in TM1 of DAT and the homologous Asp residues in NET and SERT coordinate the positive charge on monoamine substrates and play an essential role in transport (6,12,13,15,39,40).…”