Glutamate is the predominant rapid excitatory neurotransmitter in the mammalian CNS and is involved in several forms of plasticity in the developing and adult nervous system. Excessive activation of glutamate receptors contributes to brain damage observed in acute insults to the CNS, including ischemia and head trauma. The extracellular concentrations of glutamate are controlled by a family of sodium-dependent transporters. We and others have found that the activity of one of these transporters, called EAAC1, can be rapidly increased by either activation of protein kinase C (PKC) or the platelet-derived growth factor receptor. These affects are associated with a redistribution of EAAC1 from a subcellular compartment to the cell surface and are independent of synthesis of new transporters. Based on our preliminary studies, we would like to pursue the following four specific aims: 1) We propose to examine the signaling molecules that are required for the PDGF-dependent redistribution of EAAC1. 2) We have recently found that EAAC1 constitutively recycles between the plasma membrane and intracellular compartments with a half-life of approximately 5-7 min. We propose to determine if EAAC1 is internalized by a caveolin-dependent endocytosis pathway and begin to identify the subcellular compartments used for recycling of EAAC1. 3) We have found that EAAC1 forms complexes with protein interacting with C kinase (PICK1) and the alpha subtype of PKC. We propose to identify domains required for these interactions and to determine how these interactions affect EAAC1 trafficking. 4) The mechanisms that regulate EAAC1 trafficking and one of the glutamate receptors appear to be quite similar, and a signal that increases EAAC1 decreases GluR2. We propose to use a variety of approaches to determine if EAAC1 and ionotropic glutamate receptors are regulated in this apparently complementary fashion using other approaches to chemically induce either long term depression (LTD) or long term potentiation (LTP). We are intrigued by this regulation because EAAC1 is enriched in areas where synaptic transmission is highly plastic and in neurons that are exquisitely sensitive to excitotoxic insults. EAAC1 is localized on the post-synaptic membrane and is perisynaptic surrounding GluRs where it is ideally situated to fine-tune synaptic transmission. We suspect that understanding the regulation of EAAC1 could have implications for understanding glutamate toxicity and/or synaptic plasticity.
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