Within the central nervous system extracellular and synaptic concentrations of most classical neurotransmitters are tightly regulated by specific, high affinity transporters that mediate the rapid reuptake into the presynaptic terminal and surrounding glial cells. Transporters for the excitatory amino acid neurotransmitters are positioned to have a major influence on both synaptic signalling and on the neurotoxic actions mediated by glutamate and aspartate, yet they are often ignored in many studies examining the role of these neurotransmitters in the CNS. The purpose of this proposal is to better define the physiological functions of excitatory amino acid transmitter transporters by 1) establishing the kinetic properties of cloned transporters that determine their capacity to take up and release glutamate, 2) identifying the structural and topological features required for transport, 3) determining their regional and cellular localization in human brain and 4) examining how signal transduction mechanisms act to modulate the reuptake process. Initial experiments will be directed at a detailed characterization of the kinetics, ion dependence, electrogenicity of three cloned excitatory amino acid transporters that have been expressed in oocytes and transfected cells. Alterations in transmembrane ion gradients, the driving forces for reuptake can have dramatic effects on the direction of transport, and thus, under a variety of conditions such as those which occur during ischemia, these changes can lead to net glutamate release through transporter reversal. The development of an electrophysiological assay for examining glutamate efflux and the ionic requirements of transport, as well as mammalian cell expression system to study the flux of radiolabeled substrates should provide insight into how the carriers function normally and how they may contribute to mechanisms of neuronal excitotoxicity. Additional goals of these studies will be to address the regional and cellular localization of carriers in the human CNS to further evaluate their potential contribution to neurodegenerative disease. Although the project is focused initially on three human carriers that have been cloned in the applicant's laboratory, it will be expanded to address the role of additional carrier subtypes and cDNAs encoding different glutamate transporters as they are identified. The importance of understanding the function, localization and regulation of different amino acid transporter subtypes is underscored by the many clinical and experimental studies which have implicated abnormal or inadequate transmitter reaccumulation in degenerative disorders such as ALS, Huntington's disease, ischemia-induced neurotoxicity, and Alzheimer's dementia.
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