Given the variety and significance of the actions of the excitatory neurotransmitter L-glutamate in the CNS, it is not surprising that increasing attention is being paid to the transporters that sequester L-glutamate into neurons and glia and thereby regulate its concentration. The systems specifically recognized as playing the most pronounced role in regulating the concentrations of L-glutamate in the CNS are the high-affinity, Na+- dependent excitatory amino acid transporters (EAATs), of which at least five subtypes have been identified (EAAT1-5). The long term goal of this application continues to be a detailed biochemical understanding of the pharmacology, structure, and function of these EAAT subtypes. Central to our approach is the development and utilization of novel conformationally constrained glutamate analogues to define the individual pharmacophores for each of the transporters. Equally important, the resulting library of inhibitors and substrates can also be exploited as structural and functional probes with which to investigate the roles of transport in transmitter recycling, signal termination, synaptic spillover and excitotoxic protection. As varying degrees of progress have been made in characterizing each of the EAAT subtypes, the aims of this project are designed to address specific pharmacological, structural and functional questions appropriate to our current levels of understanding of each of the subtypes. Specifically, these aims include: AIM I Delineate the lipophilic pocket(s) associated with the substrate binding domain of EAAT2. AIM II Covalently modify and identify structural domains within EAAT2/GLT1 transporter protein. AIM III Develop novel glutamate analogues as pharmacological and functional probes of EAAT3. AIM IV Conduct SAR studies on EAAT 1, 4 and 5 using conformationally constrained analogues.The results of this project will provide significant insight into the properties of the excitatory amino acid transporters and their ability to regulate the physiological and pathological levels of glutamate in the CNS.