Glutamate is now recognized as both a major excitatory transmitter and a potent neurotoxin. In this respect, the regulation of its concentration is a central issue in the physiology and pathology of the CNS. High affinity transport is believed to be responsible for signal termination, the recycling of the transmitter, and the maintenance of glutamate levels in the extracellular space at concentrations below those that might induce excitotoxic injury. These activities underscore the protective capacity of the uptake systems. Paradoxically, recent evidence suggests they may also contribute to the excitotoxic process as a site of glutamate efflux under pathological conditions. The goal of this proposal is a detailed biochemical characterization of these excitatory amino acid transport systems. These experiments are aimed to elucidate the pharmacological and kinetic properties that distinguish each of the individual processes. Particular emphasis is placed on the sodium-dependent glutamate transporter. Recent research has led to the discovery of selective and conformationally restricted inhibitors that now allow an in-depth investigation of this system's heterogeneity, specificity, and mechanism of translocation. Experiments will also be performed in synaptosomes and cultured astrocytes to elucidate the potential for cell specific differences. Current evidence also indicates the existence of several other excitatory amino acid transporters that are distinct from the sodium-dependent system. Investigations will be directed toward the definition of the kinetic and pharmacological properties that are specific to each. To further define the distinctive properties of the transport systems structure/function experiments will also include molecular modeling and the synthesis of a second generation of analogues. The end result of these chemical and biochemical studies will be a series of very well defined transport inhibitors and substrates that will then be used to modulate uptake in vitro and in vivo. Preliminary data in vivo suggests that the inhibition of transport can modify the susceptibility of neurons to glutamate- mediated injury. Experiments will build on these results to define the role of transport in maintaining a viable extracellular environment and protecting neurons from excitotoxic injury. Overall, these experiments should provide significant insight into the properties of the excitatory amino acid transport systems and the role they may play in regulating the physiological and/or pathological levels of glutamate in the CNS.
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