A Na+-dependent electrogenic glutamate transporter current in voltage-clamped cells of corpora allata in the cricket Gryllus bimaculatus

Application of L-glutamate (1 mM) to corpora allata cells of the adult male cricket Gryllus bimaculatus caused a membrane depolarization of 5.9+/-0.3 mV (mean +/- SE) from a resting potential of -62.2+/-1.3 mV (n=57). The underlying mechanism for this depolarization was studied by applying the two-electrode voltage-clamp technique. Application of L-glutamate (1 mM) elicited an inward current that peaked at 8.1+/-0.7 nA (n = 73) at a holding potential of-50 mV. Both L- and D-aspartate also induced an inward current of almost the same amplitude as L-glutamate, whereas D-glutamate failed to induce an inward current. Glutamate receptor agonists, such as kainate, quisqualate, alpha-amino-3-hydroxy-5-methyl isoxazole-4-propionic acid, and N-methyl-D-aspartate, were ineffective in eliciting inward currents. The glutamate-induced inward current did not reverse even when the holding potential was set to +40 mV. The replacement of extracellular Na+ with choline+ eliminated the inward current. These results strongly suggest that the current induced by glutamate is mediated by a glutamate transporter rather than a glutamate receptor. We further examined the effects of 12 amino acid analogs which are known to be selective inhibitors of the mammalian excitatory amino acid transporters (EAATs) on the corpora allata transporter. From the effects of these inhibitors, we conclude that the glutamate transporter expressed in corpora allata cells of the cricket is similar to the high affinity glutamate transporters cloned from human brain, especially EAAT1 and EAAT3. Unlike mammalian transporters, however, serine-O-sulfate has the most potent action, suggesting the unique feature of the glutamate transporter expressed in the corpora allata.     

Na(+)-dependent glutamate transporters (EAAT1, EAAT2, and EAAT3) of the blood-brain barrier. A mechanism for glutamate removal.

Na(+)-dependent transporters for glutamate exist on astrocytes (EAAT1 and EAAT2) and neurons (EAAT3). These transporters presumably assist in keeping the glutamate concentration low in the extracellular fluid of brain. Recently, Na(+)-dependent glutamate transport was described on the abluminal membrane of the blood-brain barrier. To determine whether the above-mentioned transporters participate in glutamate transport of the blood-brain barrier, total RNA was extracted from bovine cerebral capillaries. cDNA for EAAT1, EAAT2, and EAAT3 was observed, indicating that mRNA was present. Western blot analysis demonstrated all three transporters were expressed on abluminal membranes, but none was detectable on luminal membranes of the blood-brain barrier. Measurement of transport kinetics demonstrated voltage dependence, K(+)-dependence, and an apparent K(m) of 14 microM (aggregate of the three transporters) at a transmembrane potential of -61 mV. Inhibition of glutamate transport was observed using inhibitors specific for EAAT2 (kainic acid and dihydrokainic acid) and EAAT3 (cysteine). The relative activity of the three transporters was found to be approximately 1:3:6 for EAAT1, EAAT2, and EAAT3, respectively. These transporters may assist in maintaining low glutamate concentrations in the extracellular fluid.     

Zn(2+) inhibits the anion conductance of the glutamate transporter EEAT4

Glutamate transport by the excitatory amino acid transporters (EAATs) is coupled to the co-transport of 3 Na(+) ions and 1 H(+) and the counter-transport of 1 K(+) ion, which ensures that extracellular glutamate concentrations are maintained in the submicromolar range. In addition to the coupled ion fluxes, glutamate transport activates an uncoupled anion conductance that does not influence the rate or direction of transport but may have the capacity to influence the excitability of the cell. Free Zn(2+) ions are often co-localized with glutamate in the central nervous system and have the capacity to modulate the dynamics of excitatory neurotransmission. In this study we demonstrate that Zn(2+) ions inhibit the uncoupled anion conductance and also reduce the affinity of L-aspartate for EAAT4. The molecular basis for this effect was investigated using site-directed mutagenesis. Two histidine residues in the extracellular loop between transmembrane domains three and four of EAAT4 appear to confer Zn(2+) inhibition of the anion conductance.     

Hetero-oligomerization of neuronal glutamate transporters

Excitatory amino acid transporters (EAATs) mediate the uptake of glutamate into neuronal and glial cells of the mammalian central nervous system. Two transporters expressed primarily in glia, EAAT1 and EAAT2, are crucial for glutamate homeostasis in the adult mammalian brain. Three neuronal transporters (EAAT3, EAAT4, and EAAT5) appear to have additional functions in regulating and processing cellular excitability. EAATs are assembled as trimers, and the existence of multiple isoforms raises the question of whether certain isoforms can form hetero-oligomers. Co-expression and pulldown experiments of various glutamate transporters showed that EAAT3 and EAAT4, but neither EAAT1 and EAAT2, nor EAAT2 and EAAT3 are capable of co-assembling into heterotrimers. To study the functional consequences of hetero-oligomerization, we co-expressed EAAT3 and the serine-dependent mutant R501C EAAT4 in HEK293 cells and Xenopus laevis oocytes and studied glutamate/serine transport and anion conduction using electrophysiological methods. Individual subunits transport glutamate independently of each other. Apparent substrate affinities are not affected by hetero-oligomerization. However, polarized localization in Madin-Darby canine kidney cells was different for homo- and hetero-oligomers. EAAT3 inserts exclusively into apical membranes of Madin-Darby canine kidney cells when expressed alone. Co-expression with EAAT4 results in additional appearance of basolateral EAAT3. Our results demonstrate the existence of heterotrimeric glutamate transporters and provide novel information about the physiological impact of EAAT oligomerization.     

Glutamate modifies ion conduction and voltage-dependent gating of excitatory amino acid transporter-associated anion channels

Excitatory amino acid transporters (EAATs) mediate two distinct transport processes, a stoichiometrically coupled transport of glutamate, Na+, K+, and H+, and a pore-mediated anion conductance. We studied the anion conductance associated with two mammalian EAAT isoforms, hEAAT2 and rEAAT4, using whole-cell patch clamp recording on transfected mammalian cells. Both isoforms exhibited constitutively active, multiply occupied anion pores that were functionally modified by various steps of the Glu/Na+/H+/K+ transport cycle. Permeability and conductivity ratios were distinct for cells dialyzed with Na(+)- or K(+)-based internal solution, and application of external glutamate altered anion permeability ratios and the concentration dependence of the anion influx. EAAT4 but not EAAT2 anion channels displayed voltage-dependent gating that was modified by glutamate. These results are incompatible with the notion that glutamate only increases the open probability of the anion pore associated with glutamate transporters and demonstrate unique gating mechanisms of EAAT-associated anion channels.     

Novel Dicarboxylate Selectivity in an Insect Glutamate Transporter Homolog

Mammals express seven transporters from the SLC1 (solute carrier 1) gene family, including five acidic amino acid transporters (EAAT1–5) and two neutral amino acid transporters (ASCT1–2). In contrast, insects of the order Diptera possess only two SLC1 genes. In this work we show that in the mosquito Culex quinquefasciatus, a carrier of West Nile virus, one of its two SLC1 EAAT-like genes encodes a transporter that displays an unusual selectivity for dicarboxylic acids over acidic amino acids. In eukaryotes, dicarboxylic acid uptake has been previously thought to be mediated exclusively by transporters outside the SLC1 family. The dicarboxylate selectivity was found to be associated with two residues in transmembrane domain 8, near the presumed substrate binding site. These residues appear to be conserved in all eukaryotic SLC1 transporters (Asp444 and Thr448, human EAAT3 numbering) with the exception of this novel C. quinquefasciatus transporter and an ortholog from the yellow fever mosquito Aedes aegypti, in which they are changed to Asn and Ala. In the prokaryotic EAAT-like SLC1 transporter DctA, a dicarboxylate transporter which was lost in the lineage leading to eukaryotes, the corresponding TMD8 residues are Ser and Ala. Functional analysis of engineered mutant mosquito and human transporters expressed in Xenopus laevis oocytes provide support for a model defining interactions of charged and polar transporter residues in TMD8 with α-amino acids and ions. Together with the phylogenetic evidence, the functional data suggest that a novel route of dicarboxylic acid uptake evolved in these mosquitos by mutations in an ancestral glutamate transporter gene.     

Enhancement of substrate-gated Cl- currents via rat glutamate transporter EAAT4 by PMA

Glutamate transporters (also called excitatory amino acid transporters, EAAT) are important in extracellular homeostasis of glutamate, a major excitatory neurotransmitter. EAAT4, a neuronally expressed EAAT in cerebellum, has a large portion (95% of the total L-aspartate-induced currents in human EAAT4) of substrate-gated Cl^– currents, a distinct feature of this EAAT. We cloned EAAT4 from rat cerebellum. This molecule was predicted to have eight putative transmembrane domains. L-Glutamate induced an inward current in oocytes expressing this EAAT4 at a holding potential –60 mV. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, significantly increased the magnitude of L-glutamate-induced currents but did not affect the apparent affinity of EAAT4 for L-glutamate. This PMA-enhanced current had a reversal potential –17 mV at extracellular Cl^– concentration ([Cl^–]_o) 104 mM with an 60-mV shift per 10-fold change in [Cl^–]_o, properties consistent with Cl^–-selective conductance. However, PMA did not change EAAT4 transport activity as measured by [³H]-L-glutamate. Thus PMA-enhanced Cl^– currents via EAAT4 were not thermodynamically coupled to substrate transport. These PMA-enhanced Cl^– currents were partially blocked by staurosporine, chelerythrine, and calphostin C, the three PKC inhibitors. Ro-31-8425, a PKC inhibitor that inhibits conventional PKC isozymes at low concentrations (nM level), partially inhibited the PMA-enhanced Cl^– currents only at a high concentration (1 µM). Intracellular injection of BAPTA, a Ca²⁺-chelating agent, did not affect the PMA-enhanced Cl^– currents. 4-Phorbol-12,13-didecanoate, an inactive analog of PMA, did not enhance glutamate-induced currents. These data suggest that PKC, possibly isozymes other than conventional ones, modulates the substrate-gated Cl^– currents via rat EAAT4. Our results also suggest that substrate-gated ion channel activity and glutamate transport activity, two EAAT4 properties that could modulate neuronal excitability, can be regulated independently. oocytes; protein kinase C     

Regulation of the glutamate transporter EAAT3 by mammalian target of rapamycin mTOR

The serine/threonine kinase mammalian target of rapamycin (mTOR) is stimulated by insulin, growth factors and nutrients and confers survival of several cell types. The kinase has previously been shown to stimulate amino acid uptake. In neurons, the cellular uptake of glutamate by the excitatory amino-acid transporters (EAATs) decreases excitation and thus confers protection against excitotoxicity. In epithelia, EAAT3 accomplishes transepithelial glutamate and aspartate transport. The present study explored, whether mTOR regulates EAAT3 (SLC1A1). To this end, cRNA encoding EAAT3 was injected into Xenopus oocytes with or without cRNA encoding mTOR and the glutamate induced current (I(glu)), a measure of glutamate transport, determined by dual electrode voltage clamp. Moreover, EAAT3 protein abundance was determined utilizing chemiluminescence. As a result, I(glu) was observed in Xenopus oocytes expressing EAAT3 but not in water injected oocytes. Coexpression of mTOR significantly increased I(glu), an effect reversed by rapamycin (100 nM). mTOR coexpression increased EAAT3 protein abundance in the cell membrane. The decay of I(glu) following inhibition of carrier insertion with brefeldin A in oocytes coexpressing EAAT3 with mTOR was similar in the presence and absence of rapamycin (100 nM). In conclusion, mTOR is a novel powerful regulator of EAAT3 and may thus contribute to protection against neuroexcitotoxicity.     

A Conserved Aspartate Determines Pore Properties of Anion Channels Associated with Excitatory Amino Acid Transporter 4 (EAAT4)

Excitatory amino acid transporter (EAAT) glutamate transporters function not only as secondary active glutamate transporters but also as anion channels. Recently, a conserved aspartic acid (Asp¹¹²) within the intracellular loop near to the end of transmembrane domain 2 was proposed as a major determinant of substrate-dependent gating of the anion channel associated with the glial glutamate transporter EAAT1. We studied the corresponding mutation (D117A) in another EAAT isoform, EAAT4, using heterologous expression in mammalian cells, whole cell patch clamp, and noise analysis. In EAAT4, D117A modifies unitary conductances, relative anion permeabilities, as well as gating of associated anion channels. EAAT4 anion channel gating is characterized by two voltage-dependent gating processes with inverse voltage dependence. In wild type EAAT4, external l-glutamate modifies the voltage dependence as well as the minimum open probabilities of both gates, resulting in concentration-dependent changes of the number of open channels. Not only transport substrates but also anions affect wild type EAAT4 channel gating. External anions increase the open probability and slow down relaxation constants of one gating process that is activated by depolarization. D117A abolishes the anion and glutamate dependence of EAAT4 anion currents and shifts the voltage dependence of EAAT4 anion channel activation by more than 200 mV to more positive potentials. D117A is the first reported mutation that changes the unitary conductance of an EAAT anion channel. The finding that mutating a pore-forming residue modifies gating illustrates the close linkage between pore conformation and voltage- and substrate-dependent gating in EAAT4 anion channels.     

Regulation of glial glutamate transporters by C-terminal domains

Excitatory amino acid transporter 2 (EAAT2) is a high affinity glutamate transporter predominantly expressed in astroglia. Human EAAT2 encompasses eight transmembrane domains and a 74-amino acid C-terminal domain that resides in the cytoplasm. We examined the role of this region by studying various C-terminal truncations and mutations using heterologous expression in mammalian cells, whole-cell patch clamp recording and confocal imaging. Removal of the complete C terminus (K498X EAAT2) results in loss of function because of intracellular retention of truncated proteins in the cytoplasm. However, a short stretch of amino acids (E500X EAAT2) within the C terminus results in correctly processed transporters. E500X reduced glutamate transport currents by 90%. Moreover, the voltage and substrate dependence of E500X EAAT2 anion currents was significantly altered. WT and mutant EAAT2 anion channels are modified by external Na(+) in the presence as well as in the absence of L-glutamate. Whereas Na(+) stimulates EAAT2 anion currents in the presence of L-glutamate, increased [Na(+)] reduces such currents without glutamate. In cells internally dialyzed with Na(+), WT, and truncated EAAT2 display comparable Na(+) dependence. With K(+) as main internal cation, E500X drastically increased the apparent dissociation constant for external Na(+). The effects of E500X can be represented by a kinetic model that allows translocation of the empty transporter from the outward- to the inward-facing conformation and stabilization of the inward-facing conformation by internal K(+). Our results demonstrate that the C terminus modifies the glutamate uptake cycle, possibly affecting the movements of the translocation domain of EAAT2 glutamate transporter.     

高亲和力谷氨酸转运体结构和功能的研究进展

真核生物高亲和力谷氨酸转运体(excitatory amino acid transporters,EAATs)分为GLAST(EAAT1)、GLT-1(EAAT2)、EAAC1(EAAT3)、EAAT4和EAAT5等5个亚型.高亲和力谷氨酸转运体结构学的研究,揭示了谷氨酸转运体的跨膜拓扑结构、真核和原核生物EAATs结构的差异,以及在底物转运过程中的一些底物和协同转运离子的结合位点.其功能学的研究发现,EAATs在参与突触的传递,避免兴奋性氨基酸的毒性效应中发挥重要作用,同时也参与了对学习、记忆以及运动行为的调控.结合我们既往的工作,就近几年EAATs的结构和功能研究做一综述.     

Mutations in transmembrane domains 5 and 7 of the human excitatory amino acid transporter 1 affect the substrate-activated anion channel

L-Glutamate is the predominant excitatory neurotransmitter in the brain, and its extracellular concentration is tightly controlled by the excitatory amino acid transporters (EAATs). The transport of 1 glutamate molecule is coupled to the cotransport of 3 Na+ and 1 H+ and the countertransport of 1 K+. In addition to substrate transport, the binding of glutamate and Na+ activates an anion current which is thermodynamically uncoupled from the transport process. We have identified three amino acid residues in EAAT1 (D272 in TM5, K384 and R385 in TM7) that influence the amplitude of the anion channel current relative to the transport current. Transporters containing the mutations R268A, D272A, D272K, K384A, K384D, R385A, and R385D were expressed in Xenopus laevis oocytes and their transport and anion channel functions measured using the two-electrode voltage clamp techniques. The D272, K384, and R385 mutant transporters showed no change in transport properties but have increased levels of anion channel activity compared to wild-type transporters. These results identify additional residues of the EAAT1 transporter that may contribute to the gating mechanism of the anion channel of glutamate transporters and also provide hints as to how substrate binding leads to channel activation.     

Preconditioning with cortical spreading depression decreases intraischemic cerebral glutamate levels and down-regulates excitatory amino acid transporters EAAT1 and EAAT2 from rat cerebal cortex plasma membranes

We previously reported a 50% reduction in cortical infarct volume following transient focal cerebral ischemia in rats preconditioned 3 days earlier with cortical spreading depression (CSD). The mechanism of the protective effect of prior CSD remains unknown. Recent studies demonstrate reversal of excitatory amino acid transporters (EAATs) to be a principal cause for elevated extracellular glutamate levels during cerebral ischemia. The present study measured the effect of CSD preconditioning on (a) intraischemic glutamate levels and (b) regulation of glutamate transporters within the ischemic cortex of the rat. Three days following either CSD or sham preconditioning, rats were subjected to 200 min of focal cerebral ischemia, and extracellular glutamate concentration was measured by in vivo microdialysis. Cortical glutamate exposure decreased 70% from 1,772.4 +/- 1,469.2 microM-min in sham-treated (n = 8) to 569.0 +/- 707.8 microM-min in CSD-treated (n = 13) rats (p <0.05). The effect of CSD preconditioning on glutamate transporter levels in plasma membranes (PMs) prepared from rat cerebral cortex was assessed by western blot analysis. Down-regulation of the glial glutamate transporter isoforms EAAT2 and EAAT1 from the PM fraction was observed at 1, 3, and 7 days but not at 0 or 21 days after CSD. Semiquantitative lane analysis showed a maximal decrease of 90% for EAAT2 and 50% for EAAT1 at 3 days post-CSD. The neuronal isoform EAAT3 was unaffected by CSD. This period of down-regulation coincides with the time frame reported for induced ischemic tolerance. These data are consistent with reversal of glutamate transporter function contributing to glutamate release during ischemia and suggest that down-regulation of these transporters may contribute to ischemic tolerance induced by CSD.     

Water and urea permeation pathways of the human excitatory amino acid transporter EAAT1

Glutamate transport is coupled to the co-transport of 3 Na(+) and 1 H(+) followed by the counter-transport of 1 K(+). In addition, glutamate and Na(+) binding to glutamate transporters generates an uncoupled anion conductance. The human glial glutamate transporter EAAT1 (excitatory amino acid transporter 1) also allows significant passive and active water transport, which suggests that water permeation through glutamate transporters may play an important role in glial cell homoeostasis. Urea also permeates EAAT1 and has been used to characterize the permeation properties of the transporter. We have previously identified a series of mutations that differentially affect either the glutamate transport process or the substrate-activated channel function of EAAT1. The water and urea permeation properties of wild-type EAAT1 and two mutant transporters were measured to identify which permeation pathway facilitates the movement of these molecules. We demonstrate that there is a significant rate of L-glutamate-stimulated passive and active water transport. Both the passive and active L-glutamate-stimulated water transport is most closely associated with the glutamate transport process. In contrast, L-glutamate-stimulated [(14)C]urea permeation is associated with the anion channel of the transporter. However, there is also likely to be a transporter-specific, but glutamate independent, flux of water via the anion channel.     

Excitatory amino acid transporters: keeping up with glutamate

Excitatory amino acid transporters (EAATs) are the primary regulators of extracellular glutamate concentrations in the central nervous system. Among the five known human EAAT subtypes, the glial carriers, EAAT1 and EAAT2 have the greatest impact on clearance of glutamate released during neurotransmission. Studies of carriers expressed on neurons, Purkinje cells and photoreceptor cells (EAAT3, EAAT4 and EAAT5, respectively) suggest more subtle roles for these subtypes in regulating excitability and signalling. The data suggest that EAA transporters may influence glutamatergic transmission by regulating the amount of glutamate available to activate pre- and post-synaptic metabotropic receptors and by altering neuronal excitability through a transporter-associated anion conductance that is activated by carrier substrates. Recent studies on structural, mechanistic and physiological aspects of carrier function in a variety of model systems and organisms have led to surprising insights into how excitatory amino acid transporters shape cellular communication in the nervous system.     

The glutamate transporter subtypes EAAT4 and EAATs 1-3 transport glutamate with dramatically different kinetics and voltage dependence but share a common uptake mechanism

Here, we report the application of glutamate concentration jumps and voltage jumps to determine the kinetics of rapid reaction steps of excitatory amino acid transporter subtype 4 (EAAT4) with a 100-micros time resolution. EAAT4 was expressed in HEK293 cells, and the electrogenic transport and anion currents were measured using the patch-clamp method. At steady state, EAAT4 was activated by glutamate and Na+ with high affinities of 0.6 microM and 8.4 mM, respectively, and showed kinetics consistent with sequential binding of Na(+)-glutamate-Na+. The steady-state cycle time of EAAT4 was estimated to be >300 ms (at -90 mV). Applying step changes to the transmembrane potential, V(m), of EAAT4-expressing cells resulted in the generation of transient anion currents (decaying with a tau of approximately 15 ms), indicating inhibition of steady-state EAAT4 activity at negative voltages (<-40 mV) and activation at positive V(m) (>0 mV). A similar inhibitory effect at V(m) < 0 mV was seen when the electrogenic glutamate transport current was monitored, resulting in a bell-shaped I-V(m) curve. Jumping the glutamate concentration to 100 muM generated biphasic, saturable transient transport and anion currents (K(m) approximately 5 microM) that decayed within 100 ms, indicating the existence of two separate electrogenic reaction steps. The fast electrogenic reaction was assigned to Na+ binding to EAAT4, whereas the second reaction is most likely associated with glutamate translocation. Together, these results suggest that glutamate uptake of EAAT4 is based on the same molecular mechanism as transport by the subtypes EAATs 1-3, but that its kinetics and voltage dependence are dramatically different from the other subtypes. EAAT4 kinetics appear to be optimized for high affinity binding of glutamate, but not rapid turnover. Therefore, we propose that EAAT4 is a high-affinity/low-capacity transport system, supplementing low-affinity/high-capacity synaptic glutamate uptake by the other subtypes.