Kappa opioid receptors regulate cocaine effects on nucleus accumbens dopamine through phosphorylation of dopamine transporter at the threonine 53 site

The endogenous peptide dynorphin (Dyn) and its target the kappa opioid receptor (KOR) play a crucial role in regulating factors related to stress and reward. The KOR is expressed in multiple cell types in the nucleus accumbens (NAc), including presynaptic dopamine (DA) terminals, where it inhibits DA release modulates the function of the DA transporter (DAT). The Dyn/KOR system is upregulated by exposure to drugs of abuse including the DAT inhibitor, cocaine, and their activity is integrally involved in negative affective states associated with withdrawal from substance abuse.

We aimed to better understand the impact of the Dyn/KOR system on presynaptic DA terminals and potential effects on DAT interactions with cocaine by measuring the impact of the KOR agonist U50,488 on electrically-evoked DA release and subsequent reuptake in NAc slices from C57BL6/J mice. We showed that superfusion of U50,488 inhibited DA release and markedly reduced cocaine-induced inhibition of DA reuptake, indicating tolerance to cocaine effects. We replicated this finding in the NAc of rhesus macaques using the DAT/NET inhibitor nomifensine, demonstrating that these mechanisms are conserved across DAT inhibitors and in non-human primates.

KOR activation results in phosphorylation of the Threonine-53 site on the DAT, a process thought to mediate its impact on DAT function. We tested whether this phosphorylation site is required for the KOR-mediated reduction cocaine effects. To tackle this question, we employed a knock-in mouse line with an Alanine-53 on the DAT (DAT-T53A), rendering that residue insensitive to phosphorylation. We show that DAT-T53A mice have enhanced DA release and uptake, and U50,488 has a reduced inhibitory effect on peak DA release. Remarkably, U50,488 no longer modified the effect of cocaine on uptake in these mice, demonstrating the dependence of this effect on phosphorylated Threonine-53 and highlighting a potential mechanism underlying cocaine tolerance.