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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 biotinylated 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 required for constitutive trafficking of EAAC1, it may be possible to determine how intracellular signals regulate EAAC1 cell surface expression.

A family of high-affinity transporters controls the extracellular concentration of glutamate in the brain, ensuring appropriate excitatory signaling and preventing excitotoxicity. There is evidence that one of the neuronal glutamate transporters, EAAC1, is rapidly recycled on and off the plasma membrane with a half-life of no more than 5-7 min in both C6 glioma cells and cortical neurons. Syntaxin 1A has been implicated in the trafficking of several neurotransmitter transporters and in the regulation of EAAC1, but it has not been determined if this SNARE protein is required for EAAC1 trafficking. Expression of two different sets of SNARE proteins was examined in C6 glioma with Western blotting. These cells did not express syntaxin 1A, vesicle-associated membrane protein-1 (VAMP1), or synaptosomal-associated protein of 25 kDa (SNAP-25), but did express a family of SNARE proteins that has been implicated in glucose transporter trafficking, including syntaxin 4, vesicle-associated membrane protein-2 (VAMP2), and synaptosomal-associated protein of 23 kDa (SNAP-23). cDNAs encoding variants of SNAP-23 were co-transfected with Myc-tagged EAAC1 to determine if SNAP-23 function was required for maintenance of EAAC1 surface expression. Expression of a dominant-negative variant of SNAP-23 that lacks a domain required for SNARE complex assembly decreased the fraction of EAAC1 found on the cell surface and decreased total EAAC1 expression, while two control constructs had no effect. The dominant-negative variant of SNAP-23 also slowed the rate of EAAC1 delivery to the plasma membrane. These data strongly suggest that syntaxin 1A is not required for EAAC1 trafficking and provide evidence that SNAP-23 is required for constitutive recycling of EAAC1.

Glutamate transporters may exist as homomultimers, but little is known about the mechanisms that ensure proper assembly and surface expression. In the present study, we investigated the mechanisms that contribute to posttranslational processing of the GLT-1 subtype of glutamate transporter. An extracellular leucine-based motif was identified that after mutation to alanine (6L/6A GLT-1) prevented export of GLT-1 from the endoplasmic reticulum (ER) to the plasma membrane and displayed a glycosylation pattern characteristic of “immature” transporter. This 6L/6A variant had a selective dominant-negative effect on wild-type GLT-1 expression and formed coimmunoprecipitable complexes with GLT-1. Mutation of two downstream arginine residues to alanine partially restored maturation and functional activity of the 6L/6A variant. The fact that this additional mutation rescued maturation of GLT-1 essentially excludes the possibility that the 6L/6A mutant variant is not appropriately processed because of simple misfolding. When the domain containing these motifs was introduced into a topologically similar location in the interleukin 2α receptor subunit (Tac protein), the mutations had a similar effect on protein maturation. Topological models place at least the leucine-based motif in an extracellular domain, which would face the lumen of the ER during assembly. On the basis of these data, we suggest that an evolutionarily conserved arginine-based motif functions as an ER retention signal and a lumenal leucine motif is required for suppression of this signal. Interestingly, a high percentage of variably spliced GLT-1 mRNAs lacking parts of this domain are found in the CNS, suggesting that GLT-1 expression may be regulated during assembly.

The neuronal glutamate transporter, EAAC1, appears to both limit spillover between excitatory synapses and provide precursor for the synthesis of the inhibitory neurotransmitter, gamma-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 degrees 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 approximately 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 degrees 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.