title: Attention deficit/hyperactivity disorder-derived coding variation in the dopamine transporter disrupts microdomain targeting and trafficking regulation.
abstract: Attention deficit/hyperactivity disorder (ADHD) is the most commonly diagnosed disorder of school-age children. Although genetic and brain-imaging studies suggest a contribution of altered dopamine (DA) signaling in ADHD, evidence of signaling perturbations contributing to risk is largely circumstantial. The presynaptic, cocaine- and amphetamine (AMPH)-sensitive DA transporter (DAT) constrains DA availability at presynaptic and postsynaptic receptors following vesicular release and is targeted by the most commonly prescribed ADHD therapeutics. Using polymorphism discovery approaches with an ADHD cohort, we identified a hDAT (human DAT) coding variant, R615C, located in the distal C terminus of the transporter, a region previously implicated in constitutive and regulated transporter trafficking. Here, we demonstrate that, whereas wild-type DAT proteins traffic in a highly regulated manner, DAT 615C proteins recycle constitutively and demonstrate insensitivity to the endocytic effects of AMPH and PKC (protein kinase C) activation. The disrupted regulation of DAT 615C parallels a redistribution of the transporter variant away from GM1 ganglioside- and flotillin1-enriched membranes, and is accompanied by altered CaMKII (calcium/calmodulin-dependent protein kinase II) and flotillin-1 interactions. Using C-terminal peptides derived from wild-type DAT and the R615C variant, we establish that the DAT 615C C terminus can act dominantly to preclude AMPH regulation of wild-type DAT. Mutagenesis of DAT C-terminal sequences suggests that phosphorylation of T613 may be important in sorting DAT between constitutive and regulated pathways. Together, our studies support a coupling of DAT microdomain localization with transporter regulation and provide evidence of perturbed DAT activity and DA signaling as a risk determinant for ADHD.
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-8548, USA.
authors.forename: Dhananjay
authors.initials: D
authors.lastname: Sakrikar
authors.forename: Michelle S
authors.initials: MS
authors.lastname: Mazei-Robison
authors.forename: Marc A
authors.initials: MA
authors.lastname: Mergy
authors.forename: Nathan W
authors.initials: NW
authors.lastname: Richtand
authors.forename: Qiao
authors.initials: Q
authors.lastname: Han
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Hamilton
authors.forename: Erica
authors.initials: E
authors.lastname: Bowton
authors.forename: Aurelio
authors.initials: A
authors.lastname: Galli
authors.forename: Jeremy
authors.initials: J
authors.lastname: Veenstra-Vanderweele
authors.forename: Michael
authors.initials: M
authors.lastname: Gill
authors.forename: Randy D
authors.initials: RD
authors.lastname: Blakely
mesh_terms: D000293:Adolescent; D000661:Amphetamine / Q000494:pharmacology; D000704:Analysis of Variance; D001289:Attention Deficit Disorder with Hyperactivity / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001596:Benzylamines / Q000494:pharmacology; D019921:Biotinylation; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism; D002461:Cell Line, Transformed; D002648:Child; D002675:Child, Preschool; D002772:Cholera Toxin / Q000378:metabolism; D015331:Cohort Studies; D004298:Dopamine / Q000378:metabolism / Q000494:pharmacology; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004563:Electrochemistry; D005260:Female; D006801:Humans; D047468:Immunoprecipitation; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D021962:Membrane Microdomains / Q000187:drug effects / Q000235:genetics*; D008565:Membrane Proteins / Q000378:metabolism; D010879:Piperazines / Q000494:pharmacology; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D013449:Sulfonamides / Q000494:pharmacology; D014162:Transfection / Q000379:methods; D014316:Tritium / Q000378:metabolism; D000096926:Benzenesulfonamides
chemical_list: D000661:Amphetamine; D001426:Bacterial Proteins; D001596:Benzylamines; D002118:Calcium; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D002772:Cholera Toxin; D004298:Dopamine; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D008164:Luminescent Proteins; D008565:Membrane Proteins; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D014316:Tritium; C106354:flotillins; C054562:yellow fluorescent protein, Bacteria; C072105:KN 93; D000096926:Benzenesulfonamides; C043425:vanoxerine