Dopamine transporter tryptophan mutants highlight candidate dopamine- and cocaine-selective domains.
Cocaine blocks the normal role of the dopamine transporter (DAT) in terminating dopamine signaling and in restricting its spatial spread through molecular interactions that remain largely obscure. Cocaine analog structure-activity studies suggest roles for cationic and hydrophobic interactions between DAT, dopamine, cocaine, and the sodium and chloride ions whose gradients power uptake processes. Tryptophan residues lying in putative DAT transmembrane domains could contribute to both aromatic and cationic interactions between DAT and dopamine or cocaine. We thus produced mutant DATs with alanine substitutions for tryptophans lying in or near putative DAT transmembrane domains. We have focused analyses on mutations that exert selective influences on affinities for dopamine or the cocaine analog CFT [(-)-2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane]. Substitutions W162A, W255A, and W310A reduced dopamine uptake affinities. 5W266A, 12W555A, and 12W561A each reduced dopamine superficial recognition affinities by more than 3-fold and all retained affinity for CFT. W406A, W496A and W523A each reduced CFT affinity, and W84A increased CFT affinity. None of these four mutations decreased dopamine uptake affinity. These data, current provisional DAT structural models, and results from parallel studies of other mutants identify candidate dopamine-selective DAT domains for transmembrane dopamine permeation and regions in which mutations selectively lower CFT affinities. Tryptophan residues may contribute more extensively to these selective domains than other previously studied DAT amino acids. These sites provide tempting targets for selective blockers of cocaine recognition by DAT.
Lin
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Molecular Neurobiology Branch, National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health, Baltimore, Maryland, USA.
Wang
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Uhl
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D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001667:Binding, Competitive; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002412:Cations / Q000378:metabolism; D003042:Cocaine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D007150:Immunohistochemistry; D007477:Ions / Q000378:metabolism; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009419:Nerve Tissue Proteins; D011487:Protein Conformation; D011869:Radioligand Assay; D015398:Signal Transduction / Q000187:drug effects; D014316:Tritium; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D002412:Cations; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D007477:Ions; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D014316:Tritium; C021279:(1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; D014364:Tryptophan; D003042:Cocaine; D000409:Alanine; D004298:Dopamine