Differential Modulation of Human Glutamate Transporter Subtypes by Arachidonic Acid

Arachidonic acid has been proposed to be a messenger molecule released following synaptic activation of glutamate receptors and during ischemia. Here we demonstrate that micromolar levels of arachidonic acid inhibit glutamate uptake mediated by EAAT1, a human excitatory amino acid transporter widely expressed in brain and cerebellum, by reducing the maximal transport rate approximately 30%. In contrast, arachidonic acid increased transport mediated by EAAT2, a subtype abundantly expressed in forebrain and midbrain, by causing the apparent affinity for glutamate to increase more than 2-fold. The results demonstrate that the response of different glutamate transporter subtypes to arachidonic acid could influence synaptic transmission and modulate excitotoxicity via positive or negative feedback according to the transporter(s) present in a particular region

Electrogenic uptake of gamma-aminobutyric acid by a cloned transporter expressed in Xenopus oocytes

GAT-1, a gamma-aminobutyric acid (GABA) transporter cloned from rat brain, was expressed in Xenopus oocytes. Voltage-clamp measurements showed concentration-dependent, inward currents in response to GABA (K0.5 4.7 microM). The transport current required extracellular sodium and chloride ions; the Hill coefficient for chloride was 0.7, and that for sodium was 1.7. Correlation of current and [3H]GABA uptake measurements indicate that flux of one positive charge occurs per molecule of GABA transported. Membrane hyperpolarization from -40 to -100 mV increased the transport current approximately 3-fold. The results indicate that the transport of one molecule of GABA involves the co-transport of two sodium ions and one chloride ion

Constitutive Ion Fluxes and Substrate Binding Domains of Human Glutamate Transporters

Application of L-glutamate activates ionic currents in voltage-clamped Xenopus oocytes expressing cloned human excitatory amino acid transporters (EAATs). However, even in the absence of L-glutamate, the membrane conductance of oocytes expressing EAAT1 was significantly increased relative to oocytes expressing EAAT2 or control oocytes. Whereas transport mediated by EAAT2 is blocked by the non-transported competitive glutamate analog kainate (K = 14 μM), EAAT1 is relatively insensitive (K \u3e 3 mM). Substitution of a block of 76 residues from EAAT2 into EAAT1, in which 18 residues varied from EAAT1, conferred high affinity kainate binding to EAAT1, and application of kainate to oocytes expressing the chimeric transporter blocked a pre-existing monovalent cation conductance that displayed a permeability sequence K \u3e Na \u3e Li choline. The results identify a structural domain of glutamate transporters that influences kainate binding and demonstrate the presence of a constitutive ion-selective pore in the transporter

Excitatory Amino Acid Transporters of the Salamander Retina: Identification, Localization, and Function

The rapid re-uptake of extracellular glutamate mediated by a family of high-affinity glutamate transporter proteins is essential to continued glutamatergic signaling and neuronal viability, but the contributions of individual transporter subtypes toward cellular physiology are poorly understood. Because the physiology of glutamate transport in the salamander retina has been well described, we have examined the expression and function of glutamate transporter subtypes in this preparation. cDNAs encoding five distinct salamander excitatory amino acid transporter (sEAAT) subtypes were isolated, and their molecular properties and distributions of expression were compared. We report evidence that at least four distinct sEAAT subtypes are expressed in glial (Müller) cells. In addition, four of the five transporter subtypes are localized in neurons throughout the retina. The brightest immunostaining was seen in the synaptic regions of the inner and outer plexiform layers and in the outer nuclear layer. Using electrophysiological measurements in the Xenopus oocyte expression system, we also examined the pharmacology and ionic dependence of the four expressing transporter subtypes that make it possible to distinguish, on the basis of functional behavior, among the various subtypes. Although no simple correlation between transporter subtype and retinal cell physiology can be made, the diverse population of sEAAT transporter subtypes with unique localization and functional properties indicates that glutamate transporters play a wide variety of roles in retinal function and are likely to underlie both the uptake of glutamate by Müller cells and the glutamate-elicited chloride conductance involved in signal transduction by photoreceptors and bipolar cells

Functional comparisons of three glutamate transporter subtypes cloned from human motor cortex

Reuptake plays an important role in regulating synaptic and extracellular concentrations of glutamate. Three glutamate transporters expressed in human motor cortex, termed EAAT1, EAAT2, and EAAT3 (for excitatory amino acid transporter), have been characterized by their molecular cloning and functional expression. Each EAAT subtype mRNA was found in all human brain regions analyzed. The most prominent regional variation in message content was in cerebellum where EAAT1 expression predominated. EAAT1 and EAAT3 mRNAs were also expressed in various non- nervous tissues, whereas expression of EAAT2 was largely restricted to brain. The kinetic parameters and pharmacological characteristics of transport mediated by each EAAT subtype were determined in transfected mammalian cells by radio-label uptake and in microinjected oocytes by voltage-clamp measurements. The affinities of the EAAT subtypes for L- glutamate were similar, with Km determinations varying from 48 to 97 microM in the mammalian cell assay and from 18 to 28 microM in oocytes. Glutamate uptake inhibitors were used to compare the pharmacologies of the EAAT subtypes. The EAAT2 subtype was distinguishable from the EAAT1/EAAT3 subtypes by the potency of several inhibitors, but most notably by sensitivity to kainic acid (KA) and dihydrokainic acid (DHK). KA and DHK potently inhibited EAAT2 transport, but did not significantly affect transport by EAAT1/EAAT3. Using voltage-clamp measurements, most inhibitors were found to be substrates that elicited transport currents. In contrast, KA and DHK did not evoke currents and they were found to block EAAT2-mediated transport competitively. This selective interaction with the EAAT2 subtype could be a significant factor in KA neurotoxicity. These studies provide a foundation for understanding the role of glutamate transporters in human excitatory neurotransmission and in neuropathology

Cloning and expression of a human neutral amino acid transporter with structural similarity to the glutamate transporter gene family

A cDNA was isolated from human brain that encodes an amino acid sequence 34-39% identical to previously published glutamate transporter sequences. Injection of RNA transcribed from this cDNA into Xenopus oocytes resulted in expression of a transport activity with the properties of the neutral amino acid uptake system ASC. Superfusion of alanine, serine, and cysteine evoked sodium-dependent inward currents in voltage-clamped oocytes expressing the transporter. These currents were dose-dependent, stereospecific, and saturable, with Km values ranging from 29 to 88 microM. Northern blot analyses revealed ubiquitous expression of this gene, termed ASCT1, consistent with the general metabolic role ascribed to system ASC

Excitatory amino acid transporter 5, a retinal glutamate transporter coupled to a chloride conductance

Although a glutamate-gated chloride conductance with the properties of a sodium-dependent glutamate transporter has been described in vertebrate retinal photoreceptors and bipolar cells, the molecular species underlying this conductance has not yet been identified. We now report the cloning and functional characterization of a human excitatory amino acid transporter, EAAT5, expressed primarily in retina. Although EAAT5 shares the structural homologies of the EAAT gene family, one novel feature of the EAAT5 sequence is a carboxy-terminal motif identified previously in N-methyl-d-aspartate receptors and potassium channels and shown to confer interactions with a family of synaptic proteins that promote ion channel clustering. Functional properties of EAAT5 were examined in the Xenopus oocyte expression system by measuring radiolabeled glutamate flux and two-electrode voltage clamp recording. EAAT5-mediated l-glutamate uptake is sodium- and voltage-dependent and chloride-independent. Transporter currents elicited by glutamate are also sodium- and voltage-dependent, but ion substitution experiments suggest that this current is largely carried by chloride ions. These properties of EAAT5 are similar to the glutamate-elicited chloride conductances previously described in retinal neurons, suggesting that the EAAT5-associated chloride conductance may participate in visual processing

Excitatory Amino Acid Transporters of the Salamander Retina: Identification, Localization, and Function

The rapid re-uptake of extracellular glutamate mediated by a family of high-affinity glutamate transporter proteins is essential to continued glutamatergic signaling and neuronal viability, but the contributions of individual transporter subtypes toward cellular physiology are poorly understood. Because the physiology of glutamate transport in the salamander retina has been well described, we have examined the expression and function of glutamate transporter subtypes in this preparation. cDNAs encoding five distinct salamander excitatory amino acid transporter (sEAAT) subtypes were isolated, and their molecular properties and distributions of expression were compared. We report evidence that at least four distinct sEAAT subtypes are expressed in glial (Müller) cells. In addition, four of the five transporter subtypes are localized in neurons throughout the retina. The brightest immunostaining was seen in the synaptic regions of the inner and outer plexiform layers and in the outer nuclear layer. Using electrophysiological measurements in the Xenopus oocyte expression system, we also examined the pharmacology and ionic dependence of the four expressing transporter subtypes that make it possible to distinguish, on the basis of functional behavior, among the various subtypes. Although no simple correlation between transporter subtype and retinal cell physiology can be made, the diverse population of sEAAT transporter subtypes with unique localization and functional properties indicates that glutamate transporters play a wide variety of roles in retinal function and are likely to underlie both the uptake of glutamate by Müller cells and the glutamate-elicited chloride conductance involved in signal transduction by photoreceptors and bipolar cells