Phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates the function of ion channels and transporters. Here, we demonstrate that PIP2 directly binds the human dopamine (DA) transporter (hDAT), a key regulator of DA homeostasis and a target of the psychostimulant amphetamine (AMPH). This binding occurs through electrostatic interactions with positively charged hDAT N-terminal residues and is shown to facilitate AMPH-induced, DAT-mediated DA efflux and the psychomotor properties of AMPH. Substitution of these residues with uncharged amino acids reduces hDAT-PIP2 interactions and AMPH-induced DA efflux, without altering the hDAT physiological function of DA uptake. We evaluated, for the first time, the significance of this interaction in vivo using locomotion as a behavioral assay in Drosophila melanogaster. Expression of mutated hDAT with reduced PIP2 interaction in Drosophila DA neurons impairs AMPH-induced locomotion without altering basal locomotion. We present the first demonstration of how PIP2 interactions with a membrane protein can regulate the behaviors of complex organisms.

BackgroundSyntaxin 1 (STX1) is a presynaptic plasma membrane protein that coordinates synaptic vesicle fusion. STX1 also regulates the function of neurotransmitter transporters, including the dopamine (DA) transporter (DAT). The DAT is a membrane protein that controls DA homeostasis through the high-affinity re-uptake of synaptically released DA.MethodsWe adopt newly developed animal models and state-of-the-art biophysical techniques to determine the contribution of the identified gene variants to impairments in DA neurotransmission observed in autism spectrum disorder (ASD).OutcomesHere, we characterize two independent autism-associated variants in the genes that encode STX1 and the DAT. We demonstrate that each variant dramatically alters DAT function. We identify molecular mechanisms that converge to inhibit reverse transport of DA and DA-associated behaviors. These mechanisms involve decreased phosphorylation of STX1 at Ser14 mediated by casein kinase 2 as well as a reduction in STX1/DAT interaction. These findings point to STX1/DAT interactions and STX1 phosphorylation as key regulators of DA homeostasis.InterpretationWe determine the molecular identity and the impact of these variants with the intent of defining DA dysfunction and associated behaviors as possible complications of ASD.

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Our laboratory recently characterized a novel autism spectrum disorder (ASD)-associated de novo missense mutation in the human dopamine transporter (hDAT) gene SLC6A3 (hDAT T356M). This hDAT variant exhibits dysfunctional forward and reverse transport properties that may contribute to DA dysfunction in ASD. Here, we report that Zn2+ reverses, at least in part, the functional deficits of ASD-associated hDAT variant T356M. These data suggest that the molecular mechanism targeted by Zn2+ to restore partial function in hDAT T356M may be a novel therapeutic target to rescue functional deficits in hDAT variants associated with ASD.

Parkinson disease (PD) is a progressive, neurodegenerative disorder affecting over 6.1 million people worldwide. Although the cause of PD remains unclear, studies of highly penetrant mutations identified in early-onset familial parkinsonism have contributed to our understanding of the molecular mechanisms underlying disease pathology. Dopamine (DA) transporter (DAT) deficiency syndrome (DTDS) is a distinct type of infantile parkinsonism-dystonia that shares key clinical features with PD, including motor deficits (progressive bradykinesia, tremor, hypomimia) and altered DA neurotransmission. Here, we define structural, functional, and behavioral consequences of a Cys substitution at R445 in human DAT (hDAT R445C), identified in a patient with DTDS. We found that this R445 substitution disrupts a phylogenetically conserved intracellular (IC) network of interactions that compromise the hDAT IC gate. This is demonstrated by both Rosetta molecular modeling and fine-grained simulations using hDAT R445C, as well as EPR analysis and X-ray crystallography of the bacterial homolog leucine transporter. Notably, the disruption of this IC network of interactions supported a channel-like intermediate of hDAT and compromised hDAT function. We demonstrate that Drosophila melanogaster expressing hDAT R445C show impaired hDAT activity, which is associated with DA dysfunction in isolated brains and with abnormal behaviors monitored at high-speed time resolution. We show that hDAT R445C Drosophila exhibit motor deficits, lack of motor coordination (i.e. flight coordination) and phenotypic heterogeneity in these behaviors that is typically associated with DTDS and PD. These behaviors are linked with altered dopaminergic signaling stemming from loss of DA neurons and decreased DA availability. We rescued flight coordination with chloroquine, a lysosomal inhibitor that enhanced DAT expression in a heterologous expression system. Together, these studies shed some light on how a DTDS-linked DAT mutation underlies DA dysfunction and, possibly, clinical phenotypes shared by DTDS and PD.

The Winter 2021 Webinar Audio Series (WAS) of the International Association of Medical Science Educators (IAMSE), titled, "USMLE Step-1 is Going to Pass/Fail, Now what do we do?" was broadcast live to audiences at academic institutions worldwide in five weekly webinars from January 7, 2021, to February 4, 2021. Recognized experts from various stakeholder groups discussed the impact of the decision to score the United States Medical Licensing Examination (USMLE) Step 1 exam Pass/Fail (P/F). The speakers identified challenges to their respective programs and explored creative ways to address potential consequences. Sessions included the perspectives of allopathic and osteopathic residency program directors, basic science faculty, undergraduate medical education curriculum designers, clinical educators, and programs for international medical students matriculating to the United States. On February 25, 2021, a bonus session provided cutting-edge updates from a co-chair of the Coalition for Physician Accountability Undergraduate Medical Education (UME) to Graduate Medical Education (GME) Review Committee (UGRC).

Parkinson's disease (PD) is a neurodegenerative disorder affecting over 6.1 million people worldwide. Studies of highly-penetrant mutations identified in early-onset familial parkinsonism have contributed to our understanding of the mechanisms underlying PD. Dopamine (DA) transporter (DAT) deficiency syndrome (DTDS) is a type of infantile parkinsonism-dystonia that shares clinical features with PD. Here, we define structural, functional, and behavioral consequences of a Cys substitution at R445 in human DAT (hDAT R445C), identified in a patient with DTDS. This R445 substitution disrupts a phylogenetically conserved intracellular (IC) network of interactions that compromise the hDAT IC gate. The disruption of this IC network supported a channel-like intermediate of hDAT and compromised hDAT function. Drosophila melanogaster expressing hDAT R445C show impaired hDAT activity, DA dysfunction in isolated brains and abnormal motor behaviors monitored at high-speed time resolution. These behaviors are linked with altered dopaminergic signaling, loss of DA neurons and decreased DA availability.

The studies by Hamilton and Campbell et al. describe the first autism spectrum disorder-associated de novo mutation in the human dopamine (DA) transporter (hDAT) gene. This mutation results in a Thr356 (highlighted in green) to Met (highlighted in gray) substitution in a region of the hDAT implicated in ion and substrate binding. The authors of this study report that this de novo mutation causes profound hDAT functional abnormalities. In Drosophila melanogaster, a behavioral assay was developed in order to mechanistically test the molecular discoveries in vivo. Flies expressing the mutant hDAT display anomalous locomotion (representative ambulatory vector shown above, in red). Graphical renderings by Andy Hamilton (CREAM creative, Boston, MA, USA; andy@thecreamcreative.com). For more information on this topic, please refer to the article by Hamilton et al. on pages 1315-1323.