A Model for the Topology of Excitatory Amino Acid Transporters Determined by the Extracellular Accessibility of Substituted Cysteines

AbstractExcitatory amino acid transporters (EAATs) function as both substrate transporters and ligand-gated anion channels. Characterization of the transporter's general topology is the first requisite step in defining the structural bases for these distinct activities. While the first six hydrophobic domains can be readily modeled as conventional transmembrane segments, the organization of the C-terminal hydrophobic domains, which have been implicated in both substrate and ion interactions, has been controversial. Here, we report the results of a comprehensive evaluation of the C-terminal topology of EAAT1 determined by the chemical modification of introduced cysteine residues. Our data support a model in which two membrane-spanning domains flank a central region that is highly accessible to the extracellular milieu and contains at least one reentrant loop domain

A Sensitive Membrane-Targeted Biosensor for Monitoring Changes in Intracellular Chloride in Neuronal Processes

Background: Regulation of chloride gradients is a major mechanism by which excitability is regulated in neurons. Disruption of these gradients is implicated in various diseases, including cystic fibrosis, neuropathic pain and epilepsy. Relatively few studies have addressed chloride regulation in neuronal processes because probes capable of detecting changes in small compartments over a physiological range are limited. Methodology/Principal Findings: In this study, a palmitoylation sequence was added to a variant of the yellow fluorescent protein previously described as a sensitive chloride indicator (YFPQS) to target the protein to the plasma membrane (mbYFPQS) of cultured midbrain neurons. The reporter partitions to the cytoplasmic face of the cellular membranes, including the plasma membrane throughout the neurons and fluorescence is stable over 30-40 min of repeated excitation showing less than 10% decrease in mbYFPQS fluorescence compared to baseline. The mbYFPQS has similar chloride sensitivity (k 50 = 41 mM) but has a shifted pKa compared to the unpalmitoylated YFPQS variant (cytYFPQS) that remains in the cytoplasm when expressed in midbrain neurons. Changes in mbYFPQS fluorescence were induced by the GABA A agonist muscimol and were similar in the soma and processes of the midbrain neurons. Amphetamine also increased mbYFPQS fluorescence in a subpopulation of cultured midbrain neurons that was reversed by the selective dopamine transporter (DAT) inhibitor, GBR12909, indicating that mbYFPQS is sensitive enough to detect endogenous DAT activity in midbrain dopamine (DA) neurons. Conclusions/Significance: The mbYFPQS biosensor is a sensitive tool to study modulation of intracellular chloride levels in neuronal processes and is particularly advantageous for simultaneous whole-cell patch clamp and live-cell imaging experiments. © 2012 Watts et al

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

Amphetamine Modulates Excitatory Neurotransmission through Endocytosis of the Glutamate Transporter EAAT3 in Dopamine Neurons

SummaryAmphetamines modify the brain and alter behavior through mechanisms generally attributed to their ability to regulate extracellular dopamine concentrations. However, the actions of amphetamine are also linked to adaptations in glutamatergic signaling. We report here that when amphetamine enters dopamine neurons through the dopamine transporter, it stimulates endocytosis of an excitatory amino acid transporter, EAAT3, in dopamine neurons. Consistent with this decrease in surface EAAT3, amphetamine potentiates excitatory synaptic responses in dopamine neurons. We also show that the process of internalization is dynamin- and Rho-mediated and requires a unique sequence in the cytosolic C terminus of EAAT3. Introduction of a peptide based on this motif into dopamine neurons blocks the effects of amphetamine on EAAT3 internalization and its action on excitatory responses. These data indicate that the internalization of EAAT3 triggered by amphetamine increases glutamatergic signaling and thus contributes to the effects of amphetamine on neurotransmission

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

SCORE studies on the impact of drug treatment on morbidity due to <i>Schistosoma mansoni</i> and <i>Schistosoma haematobium</i> infection

The Schistosomiasis Consortium for Operational Research (SCORE) was funded in 2008 to improve the evidence base for control and elimination of schistosomiasis-better understanding of the systemic morbidities experienced by children in schistosomiasis-endemic areas and the response of these morbidities to treatment, being essential for updating WHO guidelines for mass drug administration (MDA) in endemic areas. This article summarizes the SCORE studies that aimed to gauge the impact of MDA-based treatment on schistosomiasis-related morbidities. Morbidity cohort studies were embedded in the SCORE's larger field studies of gaining control of schistosomiasis in Kenya and Tanzania. Following MDA, cohort children had less undernutrition, less portal vein dilation, and increased quality of life in Year 5 compared with baseline. We also conducted a pilot study of the Behavioral Assessment System for Children (BASC-2) in conjunction with the Kenya gaining control study, which demonstrated beneficial effects of treatment on classroom behavior. In addition, the SCORE's Rapid Answers Project performed systematic reviews of previously available data, providing two meta-analyses related to morbidity. The first documented children's infection-related deficits in school attendance and achievement and in formal tests of learning and memory. The second showed that greater reductions in egg output following drug treatment correlates significantly with reduced odds of most morbidities. Overall, these SCORE morbidity studies provided convincing evidence to support the use of MDA to improve the health of school-aged children in endemic areas. However, study findings also support the need to use enhanced metrics to fully assess and better control schistosomiasis-associated morbidity