SUMMARY Substance use disorder afflicts nearly one in seven people in the US, placing a heavy burden on healthcare resources, the economy, and individual quality of life. Currently, there is no effective medication available for cocaine use disorder (CUD), most likely because the neurobiology underlying the disease is complex and not completely understood. Dopaminergic neurotransmission in the mesolimbic circuit has been shown to play a critical role in CUD. Cocaine inhibits the activity of the dopamine transporter (DAT) to produce its behavioral effects. Regulation of the DAT, a principal regulator of dopamine signaling, occurs primarily through post- translational modifications, such as phosphorylation, which are triggered by presynaptic auto and hetero- receptor-linked signaling cascades. These modifications alter the interaction of cocaine with the DAT, leading to changes in the stimulating and rewarding effects of cocaine. However, to date, post-translational modifications have not been a focus of investigation in the CUD field. Our studies show that phosphorylation of the Threonine-53 residue of DAT, or phospho-T53-DAT (PT53-DAT), plays a pivotal role in regulating cocaine- directed behaviors. In this proposal, we explore this novel finding using cutting-edge techniques, including a knock-in mouse model with a phosphorylation-defective DAT-Thr53Ala mutant (DAT-A53), a viral-mediated brain region-specific blockade of DAT-T53 phosphorylation, and fiber photometry-based measurements of dopamine in freely moving mice using the fluorescent sensor, dLight. With these new models and tailored biochemical, neurochemical, and behavioral studies, the current proposal aims to fill a critical gap in our understanding of the central role of DAT phosphorylation in normal dopamine neurotransmission and in the presence of cocaine, including in live animals for the first time. As a key mechanism, we have discovered that the kappa-opioid receptor (KOR) regulates DAT via PT53-DAT. Our studies also show that KOR activation increases DAT activity through PT53-DAT, and when T53 is substituted with A53, the aversive effects of a KOR agonist are attenuated. Here, we will expand and test our overarching hypothesis “Cocaine induces addiction- like behaviors when the KOR phosphorylates DAT at T53 and alters DA dynamics”. Aim 1 will investigate the effect of T53 phosphorylation on KOR-mediated DAT upregulation, trafficking and protein-protein interactions. Aim 1 will also examine if these PT53-DAT-dependent effects are sex-, and brain-region specific. Aim 2 will examine the impact of PT53-DAT on extracellular DA dynamics and DA release and clearance modulation by KOR and cocaine in vivo and in brain slices. Aim 3 will determine whether PT53-DAT plays a role in KOR- and cocaine- associated behaviors. Outcomes from the proposed studies will provide novel insights into the mechanisms of CUD, open new horizons for examining phosphorylation of DAT-T53 as an underlying mecha...