The goal is to determine the structure-function relationship and the cellular and subcellular expression of high affinity glutamate transporters in the CNS. We will focus on the high affinity neuronal and glial glutamate transporters which play an important role during the glutamatergic transmission process by removing released glutamate from the synaptic cleft. This project is based on our recent success in the cloning by functional expression in Xenopus oocytes of the EAAC1 high affinity glutamate transporters from rabbit, rat and human. In order to develop a detailed understanding of the role of high affinity glutamate uptake in the transmission process these studies will also include the glial high affinity glutamate transporter GLT-1. EAAC1 message is strongly expressed in glutamatergic neurons throughout the CNS and also in some non-glutamatergic neurons. In order to study the cellular and subcellular distribution of EAAC1 and to determine whether it functions as a presynaptic uptake carrier in glutamatergic terminals we propose to prepare a panel of polyclonal antibodies against different hydrophilic regions of EAAC1 and to use them for immunocytochemistry at the light and electronmicroscopy levels. The functional and pharmacological properties of EAAC1 and GLT-1 expressed in Xenopus oocytes will be extensively characterized using electrophysiological methods. Voltage jump experiments will provide a detailed insight into the kinetics of glutamate transport. In addition, genetic engineering of EAAC1 and GLT-1 should permit the identification of individual residues which are involved in the binding and translocation of glutamate and which directly affect the rate constants of individual steps of the transport process. To further assess the physiological and pathophysiological significance of EAAC1 and also GLT-1 studies are designed to determine whether ischemia or hippocampal long-term potentiation alter the expression levels of EAAC1 and GLT-1. We also propose to evaluate the action of peroxide on glutamate uptake. The latter is important because both impaired glutamate uptake and oxygen free radicals were implicated in the pathogenesis of familial amyotrophic lateral sclerosis (ALS). The results from these studies may lead to new strategies for the treatment of ischemia and neurodegenerative diseases such as ALS by modulating the transport functions of EAAC1 and/or GLT-1.
