The actions of glutamate as both an excitatory neurotransmitter and a potent neurotoxin necessitate that its concentrations within CNS be carefully regulated. High affinity glutamate transporters are believed to play a key role in maintaining the balance between these physiological and pathological processes. The rapid clearance of glutamate through these uptake systems and into neurons and glia is postulated to contribute to signal termination, transmitter recycling, and the maintenance of extracellular glutamate levels below those that can induce excitotoxic damage. In view of these protective roles, it is ironic that the reversed action of these systems under pathological conditions may actually increase extracellular glutamate levels and contribute to the induction of excitotoxic neuronal injury. The goal of this proposal continues to be a detailed biochemical characterization of the excitatory amino acid uptake systems. The experiments are designed to elucidate the pharmacological and kinetic properties that distinguish each of the glutamate transporters. Particular emphasis is placed on the use of conformationally constrained glutamate analogues to define their individual pharmacophores and generate a library of selective substrates and inhibitors. Experiments will be carried out with cells lines stably expressing each of the four identified human sodium dependent transporter subtypes (EAAT1, EAAT2, EAAT3, and EAAT4). As considerable progress has already been made in delineating the specificity of the EAAT2 subtype, studies on this transporter will focus on the development of photoaffinity inhibitors. Studies of uptake system heterogeneity will also be extended to include two sodium independent systems: one that is present on synaptic vesicles and a chloride dependent glial system. The end result of these investigations will be a series of well defined inhibitors and substrates that can be used to selectively study and modify transporter activity. In a final series of experiment, these inhibitors will be used to vary uptake activity and elucidate the role of transport in both protecting neurons from excitotoxic injury and as a source of extracellular glutamate under pathological conditions. Overall, the results of this project will provide significant insight into the properties of the excitatory amino acid transport systems and their ability to regulate the physiological and pathological levels of glutamate in the CNS.
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