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J. Biol. Chem., Vol. 279, Issue 33, 34505-34513, August 13, 2004
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Rapid Trafficking of the Neuronal Glutamate Transporter, EAAC1
EVIDENCE FOR DISTINCT TRAFFICKING PATHWAYS DIFFERENTIALLY REGULATED BY PROTEIN KINASE C AND PLATELET-DERIVED GROWTH FACTOR*
From the Departments of Pediatrics and Pharmacology, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, Pennsylvania 19104-4318
The neuronal glutamate transporter, EAAC1, appears to both limit spillover between excitatory synapses and provide precursor for the synthesis of the inhibitory neurotransmitter, 
-aminobutyric acid. There is evidence for a large intracellular pool of EAAC1 from which transporter is redistributed to the cell surface following activation of protein kinase C (PKC) or platelet-derived growth factor (PDGF) receptor by seemingly independent pathways. A variety of biotinylation strategies were employed to measure trafficking of EAAC1 to and from the plasma membrane and to examine the effects of phorbol ester and PDGF on these events. Biotinylation of cell surface protein under trafficking-permissive conditions (37 °C) resulted in a 2-fold increase in the amount of biotinylated EAAC1 within 15 min in C6 glioma and in primary neuronal cultures, suggesting that EAAC1 has a half-life of 
5–7 min for residence at the plasma membrane. Both phorbol ester and PDGF increased the amount of transporter labeled under these conditions. Using a reversible biotinylation strategy, a similarly rapid internalization of EAAC1 was observed in C6 glioma. Phorbol ester, but not PDGF, blocked this measure of internalization. Incubation at 18 °C, which blocks some forms of intracellular membrane trafficking, inhibited PKC- and PDGF-dependent redistribution of EAAC1 but had no effect on basal trafficking of EAAC1. These studies suggest that both PKC and PDGF accelerate delivery of EAAC1 to the cell surface and that PKC has an additional effect on endocytosis. The data also suggest that basal and regulated pools of EAAC1 exist in distinct compartments.
Received for publication, April 12, 2004 , and in revised form, June 4, 2004.
* This work was supported in part by National Institutes of Health Grant NS39011. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by Neuropsychopharmacology Training Grant 5-T32-MH014654.
Recipient of an American Heart Association postdoctoral fellowship.
¶ To whom correspondence should be addressed: 502N Abramson Pediatric Research Bldg., 3615 Civic Center Blvd., Philadelphia, PA 19104-4318. Tel.: 215-590-2205; Fax: 215-590-3779; E-mail: Robinson{at}pharm.med.upenn.edu.
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