Intracellular trafficking of the neuronal glutamate transporter EAAC1

The extracellular concentrations of the excitatory amino acid glutamate are controlled by a family of transporters that ensure appropriate signal-to-noise ratio as well as prevent glutamate-induced excitotoxicity. Expression of the neuronal glutamate transporter EAAC1 is enriched in the hippocampus and cortex, where the transporter is localized to the somatodendritic body of postsynaptic excitatory cells, and the presynaptic terminals of inhibitory cells. EAAC1 appears to limit the spillover of glutamate at excitatory synapses and provide precursor for the synthesis of γ-aminobutyric acid (GABA) in inhibitory cells. In the C6 glioma model system, treatment with either phorbol ester (PMA) or platelet-derived growth factor (PDGF), stimulates a rapid redistribution of EAAC1 from the large intracellular pool, resulting in a net increase in cell surface expression of the transporter. The mechanism(s) underlying these regulated redistribution events are unknown. A variety of biotinylation strategies were employed to test the hypothesis that PMA and PDGF increase cell surface expression of EAAC1 by altering the kinetics of transporter trafficking. Here we report that there is robust constitutive trafficking of the transporter, with a maximum half-life at the cell surface of 5–7 minutes in both C6 glioma and primary cortical neurons. Treatment with PMA or PDGF accelerates the rate of delivery of EAAC1 to the cell surface, and PMA also inhibits endoyctosis. A dominant-negative mutant of the recycling endosome-associated Rab GTPase Rab11 caused a dose-dependent decrease in cell surface EAAC1 that was associated with inhibition of both constitutive and PMA-stimulated trafficking. Similarly, a dominant-negative mutant of the exocytotic t-SNARE, SNAP23, caused a dose-dependent decrease in cell surface expression of EAAC1 that was due to a slower rate of delivery of transporter to the plasma membrane. These results indicate that EAAC1 is subject to rapid constitutive trafficking that may be regulated by both PMA and PDGF treatment. A Rab11-dependent trafficking compartment appears to be required for both the constitutive and PMA-stimulated trafficking of EAAC1. In addition, the delivery and insertion of transporter to the cell surface appears to require a SNAP23-containing SNARE complex