Regulated Trafficking of GABA Transporters, GAT1 and GAT3, in Response to Methamphetamine Through the Trace Amine Associated Receptor, TAAR1

Abstract ID 92456 Poster Board 118 GABA dysregulation has been implicated in a variety of conditions, from epilepsy to schizophrenia. The main GABA transporters in the CNS, GAT1 and GAT3, are responsible for the maintenance of inhibition throughout the brain by clearing GABA released during neurotransmission. Although the location of GAT1 and GAT3 varies based on the brain region of interest, GAT1 localizes mainly on neurons and periodically on astrocytes, while GAT3 localizes largely on peri-synaptic astrocytes. Previous work from this laboratory has demonstrated that psychostimulants such as methamphetamine induce the internalization of the catecholamine transporters DAT and NET and the glutamate transporter EAAT3. This process involves methamphetamine stimulation of the intracellular G-protein coupled receptor TAAR1 and activation of the small GTPase RhoA that facilitates internalization of the transporters. To examine whether GABA transporters are also internalized through a TAAR1/RhoA-dependent mechanism, we measured the uptake of radiolabeled GABA into mouse primary striatal mixed cultures. In these cultures, psychostimulant co-application had no measurable effect on GABA uptake, consistent with their reported lack of affinity for GABA transporters. However, pre-treatment with methamphetamine led to a significant decrease in GABA uptake. Inhibitors of RhoA, RhoA kinase (ROCK), and G-13-mediated GPCR signaling all prevented the methamphetamine-induced loss of GABA uptake, suggesting that RhoA-mediated internalization of GABA transporters is responsible for the reduction in GABA uptake. ELISA analysis of the intracellular GABA concentration of mouse primary striatal mixed cultures after treatment with NNC 711 (GAT1 inhibitor) and SNAP 5114 (GAT3 inhibitor) demonstrated the presence of both GAT1 and GAT3 in these cultures, as well as a significant role for both GAT1 and GAT3 in maintaining intracellular GABA content. This finding also suggests that TAAR1 is functional in astrocytes in addition to neurons, which has not been extensively explored. Recombinant expression systems for GFP-hGAT1 and GFP-hGAT3 were used to characterize GAT1 and GAT3 activity in HEK293 cells. Parallel radiolabeled uptake time course experiments revealed that GFP-hGAT1 and GFP-hGAT3 reach saturation after approximately 2 hours and have affinities of about 50 uM. TIRF microscopy investigating the internalization of transfected GFP-hGAT1 and GFP-hGAT3 demonstrate that both transporters are removed from the cell membrane in response to methamphetamine, but that GAT3 internalizes more rapidly. This suggests that GAT1 and GAT3 respond differently to regulation via a TAAR1/RhoA-dependent mechanism. Further work using biotinylation of surface proteins is being undertaken to support that both carriers can be internalized through a RhoA dependent mechanism. Our findings show that methamphetamine can decrease GAT activity through TAAR1-mediated activation of RhoA signaling. Thus, the TAAR1 pathway may be an important therapeutic target for a wide variety of conditions and neurodegenerative diseases. GAT inhibitors are currently used as anticonvulsants and off-label anxiolytic treatments, despite subsequent adverse side effects. These findings suggest alternative methods for modulating GABA transport via TAAR1 which could prove to be vital for reevaluating current treatments. This work was supported by the Intramural Research Program at the National Institute of Mental Health (NIMH).