Presynaptic Gq‐Coupled Receptor Activation Drives D2‐Dependent, Biphasic Dopamine Transporter Surface Trafficking
Authors: Kearney Patrick J. , Melikian Haley E. 
Published: June 2021

Dopamine (DA) neurotransmission is essential for movement, motivation, and reward, and DAergic dysfunction is implicated in multiple neuropsychiatric disorders including attention deficit hyperactivity disorder (ADHD), schizophrenia, Parkinson's disease, and addiction. The presynaptic DA transporter (DAT) clears extracellular DA following evoked release, and thereby temporally and spatially constrains DAergic signaling. Addictive and therapeutic psychostimulants, such as cocaine, amphetamine and methylphenidate (Ritalin), competitively inhibit DA uptake, and their binding to DAT is required for reward in rodents. Moreover, multiple DAT coding variants have been reported in ADHD and Autism Spectrum Disorder patients, underscoring DAT's importance in maintaining DA homeostasis. Given the central control that DAT exerts on DAergic signaling, molecular mechanisms that modulate DAT function and availability are vital and may reveal potential therapeutic interventions to modulate DAergic neurotransmission. DAT constitutively internalizes and recycles to the plasma membrane, and DAT surface levels rapidly diminish in response to acute protein kinase C (PKC) activation with phorbol esters. However, it is unclear whether PKC activation via an upstream, presynaptic Gq‐coupled receptor impacts DAT in bona fide DAergic terminals. Moreover, it is completely unknown how the complex signaling cascades co‐activated with PKC, in response to Gq stimulation, are integrated to control synaptic DAT surface expression and function. Here, we leveraged the Tet‐off system to conditionally express the chemogenetic DREADD (Designer Receptors Exclusively Activated by Designer Drugs), hM3Dq in Pitx3‐tTA mouse DAergic neurons, and tested the consequences of presynaptic Gq activation on DAT surface expression in ex vivo striatal slices. hM3Dq activation drove biphasic DAT surface trafficking, with a significant increase in DAT surface expression within 5 minutes in both the dorsal and ventral striatum. In the dorsal striatum, DAT rapidly returned to baseline at 10 and 30 minutes post‐stimulation. In contrast, in ventral striatum, DAT levels decreased below baseline levels 10 and 30 minutes post Gq‐stimulation. Pharmacological and DA depletion studies revealed that Gq‐mediated DAT insertion was dependent upon D2 receptor (D2R) activation and DA release. Activating the endogenous Gq‐coupled muscarinic receptor 5 (M5) with the positive allosteric modulator VU0238429 resulted in similar increases in DAT surface expression within 5 minutes in mouse slices. These studies suggest that DAT surface expression is rapidly modulated in response to presynaptic autoreceptor activation, and that presynaptic Gq activation sculpts DAT surface expression through multimodal signaling mechanisms. Ongoing studies will leverage pharmacological and genetic approaches to directly probe the mechanisms required for Gq‐mediated biphasic DAT trafficking, and test whether PKC‐stimulated DAT internalization is a retrieval mechanism to reset DAT surface levels following D2R‐mediated DAT insertion.Support or Funding InformationSupported by NIH R01‐DA035224 to H.E.M.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.