Attention deficit/hyperactivity disorder-derived coding variation in the dopamine transporter disrupts microdomain targeting and trafficking regulation.

Sakrikar D, Mazei-Robison MS, Mergy MA, Richtand NW, Han Q, Hamilton PJ, Bowton E, Galli A, Veenstra-Vanderweele J, Gill M, Blakely RD
J Neurosci. 2012 32 (16): 5385-97

PMID: 22514303 · PMCID: PMC3342037 · DOI:10.1523/JNEUROSCI.6033-11.2012

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.

MeSH Terms (33)

Adolescent Amphetamine Analysis of Variance Attention Deficit Disorder with Hyperactivity Bacterial Proteins Benzylamines Biotinylation Calcium Calcium-Calmodulin-Dependent Protein Kinase Type 2 Cell Line, Transformed Child Child, Preschool Cholera Toxin Cohort Studies Dopamine Dopamine Plasma Membrane Transport Proteins Dopamine Uptake Inhibitors Dose-Response Relationship, Drug Electrochemistry Female Humans Immunoprecipitation Luminescent Proteins Male Membrane Microdomains Membrane Proteins Piperazines Polymorphism, Single Nucleotide Protein Kinase Inhibitors Protein Transport Sulfonamides Transfection Tritium

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