The reversal of dopamine transporter function is governed by plasma membrane interactions and disrupted by genetic variations
Hamilton, Peter James
Dopaminergic neurotransmission plays an important role in the regulation of cognitive, behavioral, and motor functions. Abnormalities in the dopamine (DA) system have been implicated in neuropsychiatric disorders including drug addiction, schizophrenia, attention deficit hyperactivity disorder, and autism spectrum disorder (ASD). The DA transporter (DAT) is a presynaptic protein that regulates DA neurotransmission by mediating the re-uptake of synaptically released DA. The DAT is a major molecular target of the psychostimulant amphetamine (AMPH), which causes an elevation in extracellular DA by inducing a reversal in DAT function. Since DA neurotransmission is heavily dictated by DAT function, understanding the regulators of DAT transport may be instrumental in understanding DA-related neuropsychiatric disorders. In this dissertation, I report multiple avenues of research that explore the regulatory events associated with DAT-mediated reverse transport of DA. First, I present that the phospholipid PIP2 directly binds, through electrostatic interactions, to positively charged DAT N-terminal residues. This interaction is required for robust AMPH-induced, DA efflux, yet does not affect the process of DA uptake. I also describe an ASD-associated de novo mutation in the DAT gene. This de novo mutation causes profound abnormalities in DAT function, including a persistent reverse transport of DA. Exposing the mutant DAT to Zn2+ partially reverses the functional deficits observed in the transporter. Lastly, I functionally characterize the effect of two separate gene variations associated with ASD, one in the DAT-interacting protein syntaxin 1 (STX1) and one in the DAT itself. I observe that the STX1 variant is hypo-phosphorylated at a key regulatory residue, resulting in a reduction in the capacity of the DAT to reverse transport DA. In parallel, I observe that the hDAT variant has reduced interactions with STX1, which also results in a reduction in the capacity of the DAT to transport DA in reverse. Collectively, these data outline multiple molecular and structural regulators of the reversal of DAT function and may contribute to a more complete understanding of the etiology of DA-related neuropsychiatric disorders.