Excitatory amino acid transporters (EAATs) contribute to the regulation of central nervous system neurotransmission by rapidly removing synaptically-released glutamate from the extracellular space. The long-term goal of this proposal is to continue research towards the understanding of the fundamental principles by which these transporters work. Although progress has been made towards this goal by the functional characterization of several of the five cloned transporters (EAATs 1-5) and the recent development of a structural model of glutamate transporters based on the x-ray structure of the bacterial aspartate transporter, GltPh, important questions about the actual transport mechanism, the functional sites of cation interaction, and the function and regulation of the glutamate transporter subtype EAAT5 remain unresolved. These questions will be addressed by undertaking research towards three specific aims. Aim 1 will characterize the mechanism(s) underlying the separable movements of K+ and Na+/glutamate across the membrane. The hypothesis to be tested is that K+ outward movement, as well as the independently-occurring Na+/glutamate translocation (the actual step(s) of moving Na+ and glutamate across the membrane) are voltage-dependent, multi-step processes, occurring through intermediate(s) on the translocation pathway. Aim 2 will identify structural elements of mammalian glutamate transporters contributing to their interaction with cations. Two hypotheses, both related to different aspects of the mechanism of cation interaction with EAATs will be tested: a) Only one of the two cation binding sites of GltPh corresponds to a Na+ binding site of mammalian EAATs; b) cation binding site(s) exist on mammalian EAATs that are not present in the GltPh structure. Aim 3 will identify the mechanistic basis of EAAT5[unreadable]s low transport activity, and of the regulation of its cell surface expression. The hypothesis to be tested is that EAAT5 is a slowly-gated anion channel with little transport activity, in contrast to EAAT3 and EAAT4, which both readily transport glutamate. To approach these three aims, wild-type glutamate transporters and transporters with point mutations to cation binding sites predicted from empirical valence mapping will be expressed in HEK293 cells, followed by functional analysis using uptake assays and transport current recording with < 100 ms time resolution, allowing us to follow the dynamics of transport in real time. Understanding the molecular mechanism and the dynamics of glutamate transport and its regulation is important because these transporters not only contribute to controlling the time course of the excitatory neurotransmitter in the synaptic cleft, thus indirectly modulating glutamatergic transmission, but they can also directly regulate cell excitability through their anion channel function. Furthermore, malfunctioning glutamate transporters have been implicated in several diseases of the nervous system.