The activity of glutamate transporters is essential for the temporal and spatial regulation of the neurotransmitter concentration in the synaptic cleft which is critical for proper neuronal signaling. Because of their role in controlling extracellular glutamate concentrations, dysfunctional glutamate transporters have been implicated in several neurodegenerative diseases and psychiatric disorders. Therefore, investigating the mechanism of substrate transport by these transporters is essential in understanding their behavior when they malfunction. A bacterial glutamate transporter homologue has been successfully crystallized revealing the molecular architecture of glutamate transporters. However, many important questions remain unanswered. In this thesis, I will address the role of D439 in the binding of Na+, and I will identify other electrogenic steps that contribute to the total electrogenicity of the transporter cycle. The role of D439 in the binding of Na+ to the transporter was explored previously in this lab. While it was proposed that the effect of D439 in Na+ binding is indirect, the results described in this thesis provides added support to this work. Here, I will show that the D439 mutation changed the pharmacology of EAAC1 such that THA was converted from a transported substrate to a competitive inhibitor. I will also show that Na+ binding to the substrate-bound mutant transporter occurred with the same affinity as that of Na+ to the substrate-bound wild-type transporter. Therefore, based on these results, D439 is not directly involved in the binding of Na+ to the substrate-bound transporter, but that its effect is rather indirect through changing the substrate binding properties. Na+ binding steps to the empty transporter and to the glutamate-bound EAAC1 contribute only 20% of the total electrogenicity of the glutamate transporter reactions cycle. While K+-induced relocation has been proposed to be electrogenic, there is no experimental evidence that supports it. In this work, I will show that the K+-induced relocation of the empty transporter is electrogenic. Moreover, the results in this work show that the K+-dependent steps are slower than the steps associated with the Na+/glutamate translocation suggesting that the K+-induced relocation determines the transporter?s properties at steady state.

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Autism is a lifelong neurodevelopmental disorder. The etiology of autism still remains unclear due to the heterogeneous and complex nature of the disorder, however synergistic actions between genetic components and environmental factors have been suggested. Acetaminophen (APAP) is one of the most popular over-the-counter drugs that possess antipyretic and analgesic effects. It is considered a relatively safe and effective within therapeutic doses. Recently, early exposure to APAP has been suggested to be one of the underlying cause of autism. Children are often prescribed APAP to lessen fever or irritability after vaccination during the first year, and APAP may adversely affect the normal brain development. In order to better understand the association with APAP and autism, we used an inbred mouse strain BTBR T+tf/J (BTBR). BTBR exhibits behavioral deficits that mimic the core behavioral deficits of human autism. In the study, investigated 1) if BTBR mice showed differences in thiol biochemistry and EAAT3 levels in brain compared with C57BL/6J (C57) mice, 2) if early exposure to APAP induced behavioral changes worsening the autistic phenotypes of BTBR in adolescence, and 3) if APAP exposure in neonatal mice induced possible toxicity at various doses. As a result, we observed that BTBR mice have significantly lower plasma sulfate levels and EAAT expression levels in the frontal cortex compared to C57 mice. Surprisingly, neonatal therapeutic dose of APAP administration did not induce behavioral changes in both C57 and BTBR in adolescence. However, we showed that a supratheraputic dose of APAP significantly elevated levels of oxidative stress marker in the brain. Overall, the results suggested that BTBR mice would be a useful mouse model to investigate effects of various environmental factors that have been associated with autism. In addition, early exposure to APAP at supratherapeutic doses may negatively affect normal brain development.

No / The L-glutamate transporter GLT-1 is an abundant central nervous system (CNS) membrane protein of the excitatory amino acid transporter (EAAT) family that controls extracellular L-glutamate levels and is important in limiting excitotoxic neuronal death. Using reverse transcription-polymerase chain reaction, we have determined that four mRNAs encoding GLT-1 exist in mouse brain, with the potential to encode four GLT-1 isoforms that differ in their N and C termini. We expressed all four isoforms (termed MAST-KREK, MPK-KREK, MAST-DIETCI, and MPK-DIETCI according to amino acid sequence) in a range of cell lines and primary astrocytes and show that each isoform can reach the cell surface. In transfected human embryonic kidney (HEK) 293 or COS-7 cells, all four isoforms support high-affinity sodium-dependent L-glutamate uptake with identical pharmacological and kinetic properties. Inserting a viral epitope (tagged with V5, hemagglutinin, or FLAG) into the second extracellular domain of each isoform allowed coimmunoprecipitation and time-resolved Forster resonance energy transfer (tr-FRET) studies using transfected HEK-293 cells. Here we show for the first time that each of the four isoforms is able to combine to form homomeric and heteromeric assemblies, each of which is expressed at the cell surface of primary astrocytes. After activation of protein kinase C by phorbol ester, V5-tagged GLT-1 is rapidly removed from the cell surface of HEK-293 cells and degraded. This study provides direct biochemical evidence for oligomeric assembly of GLT-1 and reports the development of novel tools to provide insight into the trafficking of GLT-1.

No / Drugs which upregulate astrocyte glutamate transport may be useful neuroprotective compounds by preventing excitotoxicity. We set up a new system to identify potential neuroprotective drugs which act through GLT-1. Primary mouse striatal astrocytes grown in the presence of the growth-factor supplement G5 express high levels of the functional glutamate transporter, GLT-1 (also known as EAAT2) as assessed by Western blotting and (3)H-glutamate uptake assay, and levels decline following growth factor withdrawal. The GLT-1 transcriptional enhancer dexamethasone (0.1 or 1 muM) was able to prevent loss of GLT-1 levels and activity following growth factor withdrawal. In contrast, ceftriaxone, a compound previously reported to enhance GLT-1 expression, failed to regulate GLT-1 in this system. The neuroprotective compound riluzole (100 muM) upregulated GLT-1 levels and activity, through a mechanism that was not dependent on blockade of voltage-sensitive ion channels, since zonasimide (1 mM) did not regulate GLT-1. Finally, CDP-choline (10 muM-1 mM), a compound which promotes association of GLT-1/EAAT2 with lipid rafts was unable to prevent GLT-1 loss under these conditions. This observation extends the known pharmacological actions of riluzole, and suggests that this compound may exert its neuroprotective effects through an astrocyte-dependent mechanism.