Structural, functional, and behavioral insights of dopamine dysfunction revealed by a deletion in SLC6A3

Abstract

The human dopamine (DA) transporter (hDAT) mediates clearance of DA. Genetic variants in hDAT have been associated with DA dysfunction, a complication associated with several brain disorders, including autism spectrum disorder (ASD). Here, we investigated the structural and behavioral bases of an ASD-associated in-frame deletion in hDAT at N336 (∆N336). We uncovered that the deletion promoted a previously unobserved conformation of the intracellular gate of the transporter, likely representing the rate-limiting step of the transport process. It is defined by a “half-open and inward-facing” state (HOIF) of the intracellular gate that is stabilized by a network of interactions conserved phylogenetically, as we demonstrated in hDAT by Rosetta molecular modeling and fine-grained simulations, as well as in its bacterial homolog leucine transporter by electron paramagnetic resonance analysis and X-ray crystallography. The stabilization of the HOIF state is associated both with DA dysfunctions demonstrated in isolated brains of Drosophila melanogaster expressing hDAT ∆N336 and with abnormal behaviors observed at high-time resolution. These flies display increased fear, impaired social interactions, and locomotion traits we associate with DA dysfunction and the HOIF state. Together, our results describe how a genetic variation causes DA dysfunction and abnormal behaviors by stabilizing a HOIF state of the transporter.

Dopaminergic Ric GTPase activity impacts amphetamine sensitivity and sleep quality in a dopamine transporter-dependent manner inDrosophila melanogaster

“…Given our current findings that dDAT and Ric interact, and that DAergic Ric activity impacts DAT activity, surface expression, baseline DA-dependent behaviors, and AMPH sensitivity, it is likely that Drosophila is an orthologous model for studying the DAT/Rit2 interaction. Indeed, Drosophila has proven a powerful system to investigate AMPH-stimulated DAT efflux mechanisms 15,47,48,64 , as well as hDAT coding variants identified in ADHD and ASD 21,49,[68][69][70] . In the current study, Drosophila genetics enabled us to determine that Ric-mediated changes in behavior are, indeed, DAT-dependent, and not due to another Ric-mediated, DAT-independent mechanism.…”

A dominant-negative variant in the dopamine transporter PDZ-binding motif is linked to parkinsonism and neuropsychiatric disease

“…Rather, it reflects the absence of common risk variants. Several rare coding DAT variants with functional deficits both in vitro and in vivo have been identified in patients with psychiatric disorders (Bowton et al, 2014;Campbell et al, 2019;Cartier et al, 2015;Davis et al, 2018;DiCarlo et al, 2019;Gowrishankar et al, 2018;Grunhage et al, 2000;Hamilton et al, 2013;Hansen et al, 2014;Herborg et al, 2018;Mazei-Robison et al, 2005;Sakrikar et al, 2012;Stewart et al, 2019;Wu et al, 2015) but the causal link between these putative risk alleles and disease is still elusive as the family trees and cohort sizes have been too small for meaningful linkage or association analysis. We have, to the best or our knowledge, carried out the first association analysis of a coding DAT variant using large-scale exome data, and demonstrate a nominally significant association to bipolar disease.…”

“…DTDS typically presents in early infancy (Kurian, 1993;Kurian et al, 2011;; however, we recently identified an adult patient, who carries compound heterozygote missense mutations in SLC6A3 that only partially disrupt DAT function, and who suffers from an atypical form of DTDS with adult-onset of motor symptoms and comorbid neuropsychiatric disease (Hansen et al, 2014). The identification of this patient adds to several reports on heterozygous carriers of coding variants in SLC6A3 that have been diagnosed with neuropsychiatric disease including bipolar disorder, ADHD, and autism spectrum disorder (ASD) (Bowton et al, 2014;Campbell et al, 2019;Cartier et al, 2015;Grunhage et al, 2000;Hamilton et al, 2013;Mazei-Robison et al, 2005;Sakrikar et al, 2012). Most of these DAT variants show deficits in DAT properties and/or trafficking, and several cause behavioral changes in vivo, at least when homozygously expressed (Campbell et al, 2019;Cartier et al, 2015;DiCarlo et al, 2019;Hamilton et al, 2013;Hansen et al, 2014;Herborg et al, 2018; Mazei- Robison et al, 2005;Mergy et al, 2014;Sakrikar et al, 2012;Wu et al, 2015).…”

“…DA uptake and amperometric recordings of AMPH-induced efflux in isolated Drosophila brains were carried out as previously described (Campbell et al, 2019).…”

Structure and Gating Dynamics of Na+/Cl– Coupled Neurotransmitter Transporters

“…This is evident from the case of a congenital mutation in the dopamine transporter involving a single residue deletion ΔN336 in the IL3 of the transporter that results in a transport defective mutant leading to autism spectrum disorder. This deletion, induces the formation of a previously unobserved half-open inward-facing (HOIF) conformation that is transport inactive (Campbell et al, 2019).…”