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 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 a human DAT (hDAT) coding variant, R615C, located in the transporter’s distal C-terminus, a region previously implicated in constitutive and regulated transporter trafficking. Here we demonstrate that whereas wildtype DAT proteins traffic in a highly regulated manner, DAT 615C proteins recycle constitutively, and demonstrate 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 establish 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 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.

Nonoisotopic Assay for the Presynaptic Choline Transporter Reveals Capacity for Allosteric Modulation of Choline Uptake

“…We recently identified a mutant dopamine transporter (DAT) derived from a subject with Attention Deficit/Hyperactivity Disorder (ADHD) that exhibits an altered ability to adopt normal protein associations and trafficking itineraries. 56 These findings raise the possibility that alterations mimicking the changes observed with the CHT LV- AA mutation may contribute to disorders linked to compromised cholinergic signaling. Since PMA reduces choline transport capacity in parallel with enhanced transporter endocytosis with either WT CHT or CHT LV-AA cells, it seems likely that the sequences that mediate PKCdependent transporter down-regulation lie distal to the LV dileucine motif.…”

The rare DAT coding variant Val559 perturbs DA neuron function, changes behavior, and alters in vivo responses to psychostimulants

“…Often, such variation is confined to a single, and sometimes small, pedigree, compelling the demonstration of functional perturbations in vitro and in vivo to make conclusions as to possible disease associations. Our efforts to date have uncovered multiple rare SLC6A3 coding variants in screens of ADHD subjects, with two of these, A559V and R615C, demonstrating altered function after heterologous expression (58,77). Here, we present the first opportunity, to our knowledge, to explore the impact of diseaseassociated, rare DAT variation in vivo.…”

Membrane-permeable C-terminal Dopamine Transporter Peptides Attenuate Amphetamine-evoked Dopamine Release*

“…The peptides had no effect on DA uptake and DAT surface levels, according to uptake and surface biotinylation experiments, respectively, consistent with no effect of the peptides on transporter turnover rate and transporter trafficking. A recent study analyzed the effect of a corresponding DAT peptide, and this peptide also showed no effect on DA uptake in the WT transporter (38).…”