title: Dopamine transporter cysteine mutants: second extracellular loop cysteines are required for transporter expression.
abstract: Studies with thiol-modifying reagents have suggested that cysteines might play important roles in the function of the dopamine transporter (DAT). To identify DAT cysteines with important thiol groups, we have studied six mutant dopamine transporters in which cysteines were replaced by alanines. Substitutions of cysteines assigned to the DAT's second putative extracellular loop--positions 180 and 189--dramatically decreased the expression of the mutant transporters. Substitutions at positions 90, 242, 305, and 345 had no significant effect in decreasing dopamine uptake, MPP+ uptake, or cocaine analogue binding. Immunostaining COS cells transfected with Cys180 and Cys189 to Ala mutants revealed reduced membrane staining and prominent staining in perinuclear regions consistent with Golgi apparatus. These results suggest that cysteines i the DAT second extracellular loop may provide sulfide residues crucial to full transporter expression, at least in part, through interference with membrane insertion. Conceivably, they might also provide the targets for the influences of thiol-modifying reagents in modifying the function of the wild-type DAT expressed in striatal membranes.
authors.affiliation: Division of Intramural Research, National Institute on Drug Abuse, Baltimore, MD 21224.
authors.forename: J B
authors.initials: JB
authors.lastname: Wang
authors.forename: A
authors.initials: A
authors.lastname: Moriwaki
authors.forename: G R
authors.initials: GR
authors.lastname: Uhl
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D003042:Cocaine / Q000031:analogs & derivatives / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004298:Dopamine / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship
chemical_list: D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C021279:(1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; D003042:Cocaine; D003545:Cysteine; D004298:Dopamine