The objective of this proposal is to examine how the activity and cellular localization of the neuronal glutamate transporter subtype EAAC1 is regulated by second messenger pathways. It has become clear in recent years that glutamate acts not only as a neurotransmitter in the normal mammalian brain, but that elevated levels of glutamate are involved in epileptic seizures as well as the excitotoxic neuronal damage that occurs in stroke, acute head trauma, and neurodegenerative disease. EAAC1 is a neuron specific glutamate transporter which is present in high levels in the cortex and the hippocampus. Better understanding of th regulation of EAAC1 may help explain why these areas of the brain are more susceptible to excitotoxic damage and seizure activity. Using the C6 glioma cell line that endogenously expresses EAAC1, the contribution of second messenger pathways to both transport activity and cellular localization will be assessed. Changes in 3[H]- glutamate uptake activity after manipulating these pathways with activators and inhibitors will be performed. Changes in cellular distribution of the transporter may be an additional mechanism for rapidly increasing glutamate uptake, and will be assessed in parallel to uptake studies using biotinylation of cell surface proteins with subsequent Western analysis and confocal microscopy. To control for more subtle changes in cellular membrane physiology, electrophysiological recordings from voltage clamped cells treated with the same pharmacological agents will be obtained. To better relate the C6 glioma findings to in vivo function, the EAAC1 transport characteristics of neuron enriched rat hippocampal cultures will be analyzed to look for similarities or differences of regulation, and the effects of neuronal EAAC1 upregulation on glutamate toxicity in these cultures will be examined. It is hoped that a better understanding of EAAC1 regulation might provide clues as to how the brain copes with excessive glutamate release in pathological states, if transporter malfunction contributes to neurological damage, and how therapeutic strategies might be used to enhance transporter function in times of excitotoxic stress such as stroke, trauma or chronic disease.
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| Sims, K D; Robinson, M B (1999) Expression patterns and regulation of glutamate transporters in the developing and adult nervous system. Crit Rev Neurobiol 13:169-97 |
| Davis, K E; Straff, D J; Weinstein, E A et al. (1998) Multiple signaling pathways regulate cell surface expression and activity of the excitatory amino acid carrier 1 subtype of Glu transporter in C6 glioma. J Neurosci 18:2475-85 |
