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Glutamate plays pivotal roles in maintaining cellular homeostasis. In the nervous system, glutamate can function as an excitatory neurotransmitter. Excessive stimulation of glutamatergic receptors can lead to neuronal cell death, a process known as excitotoxicity. The rapid removal of glutamate from the synaptic cleft is accomplished by the Na+/K+-coupled excitatory amino acid transporters (EAATs). The expression and function of the EAATs can be modulated by intracellular signaling cascades and by protein-protein interactions. Novel mechanisms by which glutamate transporters are regulated in neurons were examined in this dissertation. Isolation and characterization of the murine orthologue to GTRAP3-18 revealed that it is a highly conserved gene since its amino acid sequence and genomic organization are highly conserved amongst the vertebrates. GTRAP3-18 mRNA and protein are expressed in neuron-rich regions of the central nervous system as well as in peripheral tissues such as the kidney and liver. The expression profile closely matches the published expression profile for EAAT3, its interacting partner. Mouse GTRAP3-18 negatively modulates mouse EAAT3-mediated glutamate uptake into transfected cells. Methyl-β-cyclodextrin (MeβCD) reduces Na+-dependent, EAAT3-mediated glutamate uptake into cells and increases GTRAP3-18 protein expression. Chronic treatment of HEK293 cells and primary neuron cultures with MeβCD results in the intracellular accumulation of EAAT3 but does not affect the subcellular distribution of GTRAP3-18. Intracerebroventricular administration of MeβCD induces spontaneous, recurrent seizures and neuronal cell death in the adult mouse. These studies demonstrate a novel means of regulating glutamate transporter function and the expression of glutamate transporter regulatory proteins using the macrocyclic polysaccharide MeβCD. Transient depletion of membrane cholesterol with MeβCD significantly reduces EAAT2- as well as EAAT1/EAAT3-mediated glutamate transport function. This decrease in glutamate uptake is due, in part, to MeβCD-induced reduction in the trafficking of EAATs to the plasma membrane from the trans-Golgi network. Cholesterol depletion by MeβCD also has a direct effect on the glutamate transporters expressed on the plasma membrane by disrupting the clustered localization of EAATs on the plasma membrane. Biochemical, immunohistochemical and pharmacological approaches were used to show that the EAATs are partially localized onto cholesterol-rich microdomains on the plasma membrane. EAAT2 was more strongly associated with these cholesterol-rich microdomains than the other EAATs expressed in the brain. Finally, the functional importance of association of the EAATs with these cholesterol-rich microdomains was demonstrated by measuring glutamate transport activity in vesicles derived from these cholesterol-rich microdomains. Taken together, these studies demonstrate novel mechanisms by the expression, localization and function of the excitatory amino acid neurotransmitters can be regulated. Modulation of the membrane localization of glutamate transporters can dramatically affect the clearance of glutamate from the synapse which could result in neuronal cell death. Glutamate plays pivotal roles in maintaining cellular homeostasis. In the nervous system, glutamate can function as an excitatory neurotransmitter. Excessive stimulation of glutamatergic receptors can lead to neuronal cell death, a process known as excitotoxicity. The rapid removal of glutamate from the synaptic cleft is accomplished by the Na+/K+-coupled excitatory amino acid transporters (EAATs). The expression and function of the EAATs can be modulated by intracellular signaling cascades and by protein-protein interactions. Novel mechanisms by which glutamate transporters are regulated in neurons were examined in this dissertation. Isolation and characterization of the murine orthologue to GTRAP3-18 revealed that it is a highly conserved gene since its amino acid sequence and genomic organization are highly conserved amongst the vertebrates. GTRAP3-18 mRNA and protein are expressed in neuron-rich regions of the central nervous system as well as in peripheral tissues such as the kidney and liver. The expression profile closely matches the published expression profile for EAAT3, its interacting partner. Mouse GTRAP3-18 negatively modulates mouse EAAT3-mediated glutamate uptake into transfected cells. Methyl-β-cyclodextrin (MeβCD) reduces Na+-dependent, EAAT3-mediated glutamate uptake into cells and increases GTRAP3-18 protein expression. Chronic treatment of HEK293 cells and primary neuron cultures with MeβCD results in the intracellular accumulation of EAAT3 but does not affect the subcellular distribution of GTRAP3-18. Intracerebroventricular administration of MeβCD induces spontaneous, recurrent seizures and neuronal cell death in the adult mouse. These studies demonstrate a novel means of regulating glutamate transporter function and the expression of glutamate transporter regulatory proteins using the macrocyclic polysaccharide MeβCD. Transient depletion of membrane cholesterol with MeβCD significantly reduces EAAT2- as well as EAAT1/EAAT3-mediated glutamate transport function. This decrease in glutamate uptake is due, in part, to MeβCD-induced reduction in the trafficking of EAATs to the plasma membrane from the trans-Golgi network. Cholesterol depletion by MeβCD also has a direct effect on the glutamate transporters expressed on the plasma membrane by disrupting the clustered localization of EAATs on the plasma membrane. Biochemical, immunohistochemical and pharmacological approaches were used to show that the EAATs are partially localized onto cholesterol-rich microdomains on the plasma membrane. EAAT2 was more strongly associated with these cholesterol-rich microdomains than the other EAATs expressed in the brain. Finally, the functional importance of association of the EAATs with these cholesterol-rich microdomains was demonstrated by measuring glutamate transport activity in vesicles derived from these cholesterol-rich microdomains. Taken together, these studies demonstrate novel mechanisms by the expression, localization and function of the excitatory amino acid neurotransmitters can be regulated. Modulation of the membrane localization of glutamate transporters can dramatically affect the clearance of glutamate from the synapse which could result in neuronal cell death.