Constitutive endocytosis and recycling of the neuronal glutamate transporter, excitatory amino acid carrier 1 Marco I. Gonza ´lez, Bala T. S. Susarla, Keith M. Fournier, Amanda L. Sheldon and Michael B. Robinson Departments of Pediatrics and Pharmacology, Children’s Hospital of Philadelphia, University of Pennsylvania, Philadelphia, Pennsylvania, USA Abstract The neuronal glutamate transporter, excitatory amino acid carrier 1 (EAAC1), has a diverse array of physiologic and metabolic functions. There is evidence that there is a relatively large intracellular pool of EAAC1 both in vivo and in vitro, that EAAC1 cycles on and off the plasma membrane, and that EAAC1 cell surface expression can be rapidly regulated by intracellular signals. Despite the possible relevance of EAAC1 trafficking to both physiologic and pathologic processes, the cellular machinery involved has not been defined. In the present study, we found that agents that disrupt clathrin- dependent endocytosis or plasma membrane cholesterol increased steady-state levels of biotinylated EAAC1 in C6 glioma cells and primary neuronal cultures. Acute depletion of cholesterol increased the V max for EAAC1-mediated activity and had no effect on Na + -dependent glycine transport in the same system. These agents also impaired endocytosis as measured using a reversible biotinylating reagent. Co- expression with dominant-negative variants of dynamin or the clathrin adaptor, epidermal growth factor receptor pathway substrate clone 15, increased the steady-state levels of bio- tinylated myc-EAAC1. EAAC1 immunoreactivity was found in a subcellular fraction enriched in early endosome antigen 1 (EEA1) isolated by differential centrifugation and partially co- localized with EEA1. Co-expression of a dominant-negative variant of Rab11 (Rab11 S25N) reduced steady-state levels of biotinylated myc-EAAC1 and slowed constitutive delivery of myc-EAAC1 to the plasma membrane. Together, these observations suggest that EAAC1 is constitutively internalized via a clathrin- and dynamin-dependent pathway into early endosomes and that EAAC1 is trafficked back to the cell surface via the endocytic recycling compartment in a Rab11- dependent mechanism. As one defines the machinery re- quired for constitutive trafficking of EAAC1, it may be possible to determine how intracellular signals regulate EAAC1 cell surface expression. Keywords: EAAC1, endocytosis, recycling, transporter. J. Neurochem. (2007) 103, 1917–1931. The Na + -dependent glutamate transporter excitatory amino acid carrier 1 (EAAC1) appears to have a diverse array of physiologic and metabolic functions in the mammalian central nervous system. Knockdown of EAAC1 expression reduces glutamate uptake, causes moderate neurodegenera- tion, and an absence seizure-like phenotype (Rothstein et al. 1996). Mice deleted of EAAC1 develop an age-related neurodegenerative process associated with decreased levels of glutathione (Aoyama et al. 2006). These effects were attributed to a lack of cysteine, an EAAC1 substrate, required for the synthesis of glutathione. EAAC1-mediated glutamate uptake is also a source of glutamate for de novo synthesis of GABA by GABAergic neurons (Sepkuty et al. 2002; Mathews and Diamond 2003; Liang et al. 2006), suggesting an important role for EAAC1 in metabolic processes. Protein trafficking plays a central role in the regulation of EAAC1 cell surface expression and function. There is evidence that EAAC1 constitutively recycles on and off the plasma membrane with a half-life on the plasma membrane of 5–7 min (Fournier et al. 2004). The steady-state levels of EAAC1 at the plasma membrane are increased in C6 glioma cells, cultured neurons, or hippocampal slices by a variety of signaling pathways including activation of protein kinase C (PKC), protein kinase A, platelet-derived growth factor (PDGF) or neurotensin receptors (Davis et al. 1998; Gonza ´lez et al. 2002; Levenson et al. 2002; Najimi et al. 2002; Guillet et al. 2005). A substantial amount of EAAC1 immunoreactivity is observed in intracellular compartments in vivo (Rothstein et al. 1994; Conti et al. 1998; He et al. Received March 26, 2007; revised manuscript received July 24, 2007; accepted July 24, 2007. Address correspondence and reprint requests to Dr Michael B. Robinson, 502N Abramson Pediatric Research Building, 3615 Civic Center Boulevard, Philadelphia, PA 19104-4318, USA. E-mail: Robinson@pharm.med.upenn.edu Abbreviations used: DAT, dopamine transporter; EAAC1, excitatory amino acid carrier 1; EEA1, early endosome antigen 1; Eps15, epidermal growth factor receptor pathway substrate clone 15; ERC, endocytic recycling compartment; GABA, c-aminobutyric acid; GAT-1, c-amin- obutyric acid transporter 1; GLUT4, glucose transporter 4; LTP, long- term potentiation; MesNa, 2-mercaptoethanesulfonic acid sodium salt; MbCD, methyl-b-cyclodextrin; NMDA, N-methyl-D-aspartate; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate. Journal of Neurochemistry , 2007, 103, 1917–1931 doi:10.1111/j.1471-4159.2007.04881.x Ó 2007 The Authors Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 1917–1931 1917