Reverse Transport of the Dopamine Transporter in Neuropsychiatric Disease: Mechanisms of Regulation and Dysfunction
Belovich, Andrea Nicole
The dopamine (DA) transporter (DAT) is a presynaptic membrane protein that regulates dopaminergic neurotransmission by controlling DA levels at the synapse via active reuptake. The DAT is also capable of engaging in reverse transport of DA (DA efflux). The DAT can be induced to reverse transport upon exposure to the psychostimulant amphetamine (AMPH). Phosphatidylinositol (4,5)-bisphosphate (PIP2), a phospholipid that regulates the function of ion channels and transporters, directly binds the human DAT (hDAT) through electrostatic interactions with positively charged hDAT N-terminal residues. PIP2 is shown to facilitate AMPH-induced, DAT-mediated DA efflux independently of the hDAT physiological function of DA uptake. Expression of mutated hDAT with reduced PIP2 interaction in Drosophila DA neurons impairs AMPH-induced locomotion without altering basal locomotion. Furthermore, mutation of putative PIP2 binding sites in hDAT’s intracellular gating regions also impairs AMPH-induced DA efflux. Genetic variants with altered reverse transport of DA have been identified in individuals with Autism Spectrum Disorder (ASD). The de novo hDAT variant, hDAT T356M, preferentially adopts an outward-facing conformation and displays impaired DA uptake, anomalous DA efflux (ADE), and impaired AMPH-induced DA efflux in vitro. Expression of hDAT T356M in Drosophila results in basal hyperlocomotor activity and impaired AMPH-induced locomotion. The addition of Zn2+ can partially rescue the functional impairments of hDAT T356M, including AMPH-induced DA efflux. This contributes to the mechanistic understanding of how Zn2+-susceptible conformational states of the hDAT influence the hDAT’s ability to respond to AMPH. The rare, heritable variant, hDAT R51W, has impaired AMPH-induced DA efflux and locomotor response when expressed in Drosophila, but normal substrate uptake and surface expression. Syntaxin 1 (STX1) is a presynaptic plasma membrane protein that coordinates synaptic vesicle fusion. STX1 also interacts with the N-terminus of the hDAT, and is important for AMPH-induced DA efflux. When co-expressed with WT hDAT, the ASD-associated variant, STX1 R26Q, impairs the hDAT’s efflux response to AMPH. 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.