PIP2 Stabilizes Dopamine Transporter Surface Expression

Dopamine (DA) signaling is central to movement, cognition, and reward pathways in the CNS and presynaptic DA reuptake mediated by the DA transporter (DAT) terminates DA signaling. DAT is potently inhibited by addictive psychostimulants cocaine and amphetamine (AMPH), and psychostimulant reward requires this inhibition. Thus, DAT availability is a critical factor for normal neurotransmission and reward, and mechanisms limiting DAT are likely to significantly impact these processes. Membrane trafficking acutely regulates DAT surface expression, and AMPH exposure and PKC activation reduce DAT surface expression by releasing an endocytic brake. However, the mechanisms constituting the DAT endocytic brake are undefined. A recent report found that phosphatidylinositol‐4,5‐bisphosphate (PIP2) interacts with DAT and facilitates AMPH‐stimulated DA efflux. Moreover, DAT/PIP2 interactions appeared to influence DAT surface stability. In the current study we directly tested whether PIP2 impacts DAT internalization. Acute PIP2 depletion decreased DA uptake and PKC activation did not further downregulate DAT, suggesting that PKC and PIP2 converge on a similar mechanism. Decreased DAT activity was accompanied by significantly increased DAT internalization rates, consistent with a requisite role for PIP2 in the DAT endocytic brake. Furthermore, DAT mutants lacking PIP2 binding displayed increased basal internalization rates. Ongoing experiments will test whether PIP2 depletion results in DAT surface losses and whether molecular components of the DAT endocytic brake work in concert with PIP2 to restrict DAT internalization.