Neuropsychiatric disease–associated genetic variants of the dopamine transporter display heterogeneous molecular phenotypes

Abstract

Genetic factors are known to significantly contribute to the etiology of psychiatric diseases such as attention deficit hyperactivity disorder (ADHD) and autism spectrum and bipolar disorders, but the underlying molecular processes remain largely elusive. The dopamine transporter (DAT) has received continuous attention as a potential risk factor for psychiatric disease, as it is critical for dopamine homeostasis and serves as principal target for ADHD medications. Constrain metrics for the DAT-encoding gene solute carrier family 6 member 3 (SLC6A3) indicate that missense mutations are under strong negative selection, pointing to pathophysiological outcomes when DAT function is compromised. Here, we systematically characterized six rare genetic variants of DAT (I312F, T356M, D421N, A559V, E602G, and R615C) identified in patients with neuropsychiatric disorders. We evaluated dopamine uptake and ligand interactions, along with ion coordination and electrophysiological properties, to elucidate functional phenotypes, and applied Zn 2+ exposure and a substituted cysteineaccessibility approach to identify shared structural changes. Three variants (I312F, T356M, and D421N) exhibited impaired dopamine uptake associated with changes in ligand binding, ion coordination, and distinct conformational disturbances. Remarkably, we found that all three variants displayed gain-offunction electrophysiological phenotypes. I312F mediated an increased uncoupled anion conductance previously suggested to modulate neuronal excitability. T356M and D421N both mediated a cocaine-sensitive leakage of cations, which for T356M, was potentiated by Zn 2+ , concurrent with partial functional rescue. Collectively, our findings support that gain of disruptive functions due to missense mutations in SLC6A3 may be key to understanding how dopaminergic dyshomeostasis arises in heterozygous carriers.A substantial proportion of the disease etiology of common psychiatric disorders, such as attention deficit hyperactivity disorder (ADHD), autism spectrum disorders (ASD), bipolar disorder, and schizophrenia is attributed genetic components (1). The underlying neurobiological mechanisms are not clear, but dopamine disturbances are believed to constitute a central component (2-7). The allelic spectrum of these dopamine-related disorders is rapidly expanding and comprises both common variants and rare structural or exonic mutations, including de novo variants (1,(8)(9)(10)(11)(12). Moreover, an interesting overlap in the genetic architecture of psychiatric disorders has been observed, and this pleiotrophy is seen for both common and rare variants (13)(14)(15)(16)(17)(18). Despite progress, much of the genetic component remains unaccounted for, and we also have the challenge ahead of translating most of the comprehensive genetic information into an understanding of underlying biological processes, and subsequently into clinically applicable knowledge. Rare variants may provide a unique handle for obtaining new disease insights as they are expected to h...

Autism-linked dopamine transporter mutation alters striatal dopamine neurotransmission and dopamine-dependent behaviors

“…This mutation, identified in an individual with ASD (for patient genetic information and clinical evaluation, see supplemental material for Hamilton et al;ref. 37), is positioned in the seventh transmembrane domain of DAT near the ion-binding site (38). In vitro work has demonstrated that this mutation drives reduced DA reuptake as well as anomalous DA efflux (37) and significantly reduces the affinity of the transporter for cocaine and methylphenidate (suggesting an important role for T356 in inhibitor binding) (38).…”

Dopamine transporter trafficking and Rit2 GTPase: Mechanism of action and in vivo impact

“…DAT is also the primary target for addictive and therapeutic psychostimulants, including amphetamine (AMPH), cocaine, and methylphenidate (Ritalin), which inhibit DAT as competitive substrates (AMPH) and antagonists (cocaine and methylphenidate) (Kristensen et al 2011). Genetic DAT deletions in mice and Drosophila melanogaster elevate extracellular DA concentrations and evoke hyperactivity (Gainetdinov et al 1998;Giros et al 1996;Kume et al 2005), and human DAT missense mutations have been reported in PD, ADHD and ASD patients (Mazei-Robison and Blakely 2005; Kurian et al 2009;Sakrikar et al 2012;Hamilton et al 2013;Ng et al 2014;Bowton et al 2014;Herborg et al 2018). Together, these studies underscore that DAT is critical to maintain DAergic homeostasis (Kristensen et al 2011).…”

Dopamine transporter trafficking and Rit2 GTPase: Mechanism of action and in vivo impact

“…psychostimulants, including AMPH, cocaine, and methylphenidate (Ritalin), which inhibit DAT as competitive substrates (AMPH) and antagonists (cocaine and methylphenidate) (9). Genetic DAT deletions in mice and Drosophila melanogaster elevate extracellular DA concentrations and evoke hyperactivity (10)(11)(12), and human DAT missense mutations have been reported in PD, ADHD and ASD patients (13)(14)(15)(16)(17)(18)(19). Together, these studies underscore that DAT is critical to maintain DAergic homeostasis (9).…”