Anomalous Dopamine Release Associated with a Human Dopamine Transporter Coding Variant

Abstract

Dopamine (DA) signaling at synapses is tightly coordinated through opposing mechanisms of vesicular fusion-mediated DA release and transporter-mediated DA clearance. Altered brain DA signaling is suspected to underlie multiple brain disorders, including schizophrenia, Parkinson's disease, bipolar disorder, and attention-deficit hyperactivity disorder (ADHD). We identified a pedigree containing two male children diagnosed with ADHD who share a rare human DA transporter (DAT; SLC6A3) coding variant, Ala559Val. Among Ͼ1000 control and affected subjects, the Val559 variant has only been isolated once previously, in a female subject with bipolar disorder. Although hDAT Ala559Val supports normal DAT protein and cell surface expression, as well as normal DA uptake, the variant exhibits anomalous DA efflux from DA-loaded cells. We also demonstrate that hDAT Ala599Val exhibits increased sensitivity to intracellular Na ϩ , but not intracellular DA, and displays exaggerated DA efflux at depolarized potentials. Remarkably, the two most common ADHD medications, amphetamine and methylphenidate, both block hDAT Ala559Val-mediated DA efflux, whereas these drugs have opposite actions at wild-type hDAT. Our findings reveal that DA efflux, typically associated with amphetamine-like psychostimulants, can be produced through a heritable change in hDAT structure. Because multiple gene products are known to coordinate to support amphetamine-mediated DA efflux, the properties of hDAT Ala559Val may have broader significance in identifying a new mechanism through which DA signaling disorders arise. Additionally, they suggest that block of inappropriate neurotransmitter efflux may be an unsuspected mechanism supporting the therapeutic actions of existing transporter-directed medications.

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

“…As noted in the prior publication, these mice reproduce normally and display no overt sensorimotor deficits. Our previous heterologous expression studies demonstrated no impact of the DAT Val559 substitution on total or cell surface expression, inward DA transport, or sensitivity to psychostimulants (8,58). Consistent with these findings, striatal extracts from DAT Val559 animals demonstrate normal transporter protein levels (Fig.…”

“…The key phenotypes found for DAT Val559 in transfected cells are ADE and a loss of AMPH-induced DA release (13,58). Evidence of ADE in vivo arises from both our striatal microdialysis studies that demonstrate a significant elevation of basal extracellular DA levels and our findings of elevated presynaptic D2 modulation of vesicular DA release that can be normalized with D2R antagonist (raclopride) treatments.…”

“…6F), suggesting that the enhanced darting speeds of DAT Val559 animals reflects a specific reaction to imminent handling. Our studies of AMPH action on DAT Val559-transfected cells demonstrated that the drug inhibits both DA uptake and ADE (58), but lacks the ability to produce DAT-mediated DA efflux (69). Moreover, our studies of AMPH effects on somatodendritic D2R-dependent IPSCs and on DA release in terminal fields revealed significantly blunted responses.…”

“…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.…”

Interactions of Cathinone NPS with Human Transporters and Receptors in Transfected Cells

“…Transporter-expressing cell lines allow the characterization of psychoactive compounds (Tatsumi et al, 1997) and are also a useful tool for discovering psychoactive therapeutic drugs (Bang-Andersen et al, 2011). Furthermore, in vitro experiments with transfected cells formed the basis for many genetic mutations that were later engineered in mice, which now serve for in vivo investigations of psychoactive drugs or as preclinical models of mental disorders (Henry et al, 2006;Mazei-Robison et al, 2008;Prasad et al, 2005). For example, in vitro experiments allowed the construction of a transgenic mouse model with a 5-hydroxytryptamine (5-HT [serotonin]) transporter (SERT) mutation for the in vivo assessment of SERT-mediated effects of antidepressants or cocaine (Prosser et al, 2014;Thompson et al, 2011) or to shed light on functional abnormalities of the DAT variant Val559, which is being investigated as a potential mouse model of attention-deficit hyperactivity disorder (Mergy et al, 2014).…”