Paying attention to the details: rare genetic variation in the dopamine transporter and ADHD

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 to 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 pre- and post-synaptic receptors following vesicular release and is targeted by the most commonly prescribed ADHD therapeutics. Using polymorphism discovery approaches with an ADHD cohort, we identified human DAT (hDAT) coding variants, A559V located in the transmembrane domain 12, a region thought to play a role in DAT oligomerization, and R615C, located in the transporter’s distal C-terminus, a region previously implicated in constitutive and regulated transporter trafficking. The A559V variant exhibits an anomalous DA efflux sustained by the actions of cell surface dopamine D2 receptor and hyperphosphorylation as well as altered AMPH-mediated trafficking. Detailed characterization of the R615C (DAT 615C) variant demonstrates that whereas wildtype DAT proteins traffic in a highly regulated manner, DAT 615C proteins recycle constitutively. Moreover, DAT 615C exhibits insensitivity to the endocytic effects of AMPH and protein kinase C (PKC) 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 calcium/calmodulin-dependent protein kinase II (CaMKII) and flotillin-1 interactions. Using C-terminal peptides derived from wildtype DAT and the R615C variant, we provide evidence that the DAT 615C C-terminus can act dominantly to preclude AMPH regulation of wildtype DAT. Mutagenesis of DAT C-terminal sequences suggest that phosphorylation of T613 may be important in sorting DAT between constitutive and regulated pathways. Together, our studies reinforce the utility of rare gene variant studies in neuropsychiatric disorders, they support a coupling of DAT microdomain localization with transporter regulation and they provide evidence of perturbed DAT-mediated DA signaling as a risk determinant for ADHD.