EAAT5 glutamate transporter rapidly binds glutamate with micromolar affinity in mouse rods.

Light responses of rod photoreceptor cells in the retina are encoded by changes in synaptic glutamate release that is in turn shaped by reuptake involving EAAT5 plasma membrane glutamate transporters. Heterologously expressed EAAT5 activates too slowly upon glutamate binding to support significant uptake. We tested EAAT5 activation in mouse rods in vivo by stimulating glutamate transporter anion currents (IA(glu)) with UV flash photolysis of MNI-glutamate, varying flash intensity to vary glutamate levels. Responses to uncaging rose rapidly with time constants of 2-3 ms, similar to IA(glu) events arising from spontaneous release. Spontaneous release events and IA(glu) evoked by weak flashes also declined with similar time constants of 40-50 ms. Stronger flashes evoked responses that decayed more slowly. Time constants were twofold faster at 35°C, suggesting that they reflect transporter kinetics, not diffusion. Selective EAAT1 and EAAT2 inhibitors had no significant effect, suggesting IA(glu) in rods arises solely from EAAT5. We calibrated glutamate levels attained during flash photolysis by expressing a fluorescent glutamate sensor iGluSnFr in cultured epithelial cells. We compared fluorescence at different glutamate concentrations to fluorescence evoked by photolytic uncaging of MNI-glutamate. The relationship between flash intensity and glutamate yielded EC50 values for EAAT5 amplitude, decay time, and rise time of ∼10 µM. Micromolar affinity and rapid activation of EAAT5 in rods show it can rapidly bind synaptic glutamate. However, we also found that EAAT5 currents are saturated by the synchronous release of only a few vesicles, suggesting limited capacity and a role for glial uptake at higher release rates.

Glutamate Transporters EAAT2 and EAAT5 Differentially Shape Synaptic Transmission from Rod Bipolar Cell Terminals.

Excitatory amino acid transporters (EAATs) control visual signal transmission in the retina by rapidly removing glutamate released from photoreceptors and bipolar cells (BCs). Although it has been reported that EAAT2 and EAAT5 are expressed at presynaptic terminals of photoreceptors and some BCs in mammals, the distinct functions of these two glutamate transporters in retinal synaptic transmission, especially at a single synapse, remain elusive. In this study, we found that EAAT2 was expressed in all BC types while coexisting with EAAT5 in rod bipolar (RB) cells and several types of cone BCs from mice of either sex. Our immunohistochemical study, together with a recently published literature (Gehlen et al., 2021), showed that EAAT2 and EAAT5 were both located in RB axon terminals near release sites. Optogenetic, electrophysiological and pharmacological analyses, however, demonstrated that EAAT2 and EAAT5 regulated neurotransmission at RB→AII amacrine cell synapses in significantly different ways: EAAT5 dramatically affected both the peak amplitude and kinetics of postsynaptic responses in AIIs, whereas EAAT2 had either relatively small or opposite effects. By contrast, blockade of EAAT1/GLAST, which was exclusively expressed in Müller cells, showed no obvious effect on AII responses, indicating that glutamate uptake by Müller cells did not influence synaptic transmission from RB terminals. Furthermore, we found that temporal resolution at RB→AII synapses was reduced substantially by blockade of EAAT5 but not EAAT2. Taken together, our work reveals the distinct functions of EAAT2 and EAAT5 in signal transmission at RB ribbon synapses.

Excitatory Amino Acid Transporter EAAT5 Improves Temporal Resolution in the Retina.

Excitatory amino acid transporters (EAATs) remove glutamate from the synaptic cleft. In the retina, EAAT1 and EAAT2 are considered the major glutamate transporters. However, it has not yet been possible to determine how EAAT5 shapes the retinal light responses because of the lack of a selective EAAT5 blocker or EAAT5 knock-out (KO) animal model. In this study, EAAT5 was found to be expressed in a punctate manner close to release sites of glutamatergic synapses in the mouse retina. Light responses from retinae of wild-type (WT) and of a newly generated model with a targeted deletion of EAAT5 (EAAT5-/-) were recorded in vitro using multielectrode arrays (MEAs). Flicker resolution was considerably lower in EAAT5-/- retinae than in WT retinae. The close proximity to the glutamate release site makes EAAT5 an ideal tool to improve temporal information processing in the retina by controlling information transfer at glutamatergic synapses.

Light-evoked glutamate transporter EAAT5 activation coordinates with conventional feedback inhibition to control rod bipolar cell output.

In the retina, modulation of the amplitude of dim visual signals primarily occurs at axon terminals of rod bipolar cells (RBCs). GABA and glycine inhibitory neurotransmitter receptors and the excitatory amino acid transporter 5 (EAAT5) modulate the RBC output. EAATs clear glutamate from the synapse, but they also have a glutamate-gated chloride conductance. EAAT5 acts primarily as an inhibitory glutamate-gated chloride channel. The relative role of visually evoked EAAT5 inhibition compared with GABA and glycine inhibition has not been addressed. In this study, we determine the contribution of EAAT5-mediated inhibition onto RBCs in response to light stimuli in mouse retinal slices. We find differences and similarities in the two forms of inhibition. Our results show that GABA and glycine mediate nearly all lateral inhibition onto RBCs, as EAAT5 is solely a mediator of RBC feedback inhibition. We also find that EAAT5 and conventional GABA inhibition both contribute to feedback inhibition at all stimulus intensities. Finally, our in silico modeling compares and contrasts EAAT5-mediated to GABA- and glycine-mediated feedback inhibition. Both forms of inhibition have a substantial impact on synaptic transmission to the postsynaptic AII amacrine cell. Our results suggest that the late phase EAAT5 inhibition acts with the early phase conventional, reciprocal GABA inhibition to modulate the rod signaling pathway between rod bipolar cells and their downstream synaptic targets.NEW & NOTEWORTHY Excitatory amino acid transporter 5 (EAAT5) glutamate transporters have a chloride channel that is strongly activated by glutamate, which modulates excitatory signaling. We found that EAAT5 is a major contributor to feedback inhibition on rod bipolar cells. Inhibition to rod bipolar cells is also mediated by GABA and glycine. GABA and glycine mediate the early phase of feedback inhibition, and EAAT5 mediates a more delayed inhibition. Together, inhibitory transmitters and EAAT5 coordinate to mediate feedback inhibition, controlling neuronal output.

Human brain neurons express a novel splice variant of excitatory amino acid transporter 5 (hEAAT5v).

Excitatory amino acid transporter 5 (EAAT5) is a protein that is known to be alternately spliced and to be abundantly expressed in the retina by populations of neurons including photoreceptors and bipolar cells. EAAT5 acts as a slow glutamate transporter and also as glutamate-gated chloride channel, the chloride conductance being large enough for EAAT5 to serve functionally as an "inhibitory" glutamate receptor. However, there has been a long-standing view that the classically spliced form of EAAT5 is not abundant or widespread in the brain and so it has not been extensively investigated in the literature. We recently identified a human-specific splicing form of EAAT5 that was not expressed by rodents but was shown to be a functional glutamate transporter. We have examined the expression of this form of EAAT5, hEAAT5v at the mRNA, and protein level in human brain, and show that populations of human cortical pyramidal neurons and cerebellar Purkinje cells show significant expression of hEAAT5v. Accordingly, we infer that EAAT5 may well be a player in modulating neuronal function in the human brain and propose that its localization in both glutamatergic and GABAergic neurons could be compatible with a role in influencing intracellular chloride and thereby neuronal parameters such as membrane potential rather than acting as a presynaptic glutamate transporter.

Amino Acid Transporters and Exchangers from the SLC1A Family: Structure, Mechanism and Roles in Physiology and Cancer.

The Solute Carrier 1A (SLC1A) family includes two major mammalian transport systems-the alanine serine cysteine transporters (ASCT1-2) and the human glutamate transporters otherwise known as the excitatory amino acid transporters (EAAT1-5). The EAATs play a critical role in maintaining low synaptic concentrations of the major excitatory neurotransmitter glutamate, and hence they have been widely researched over a number of years. More recently, the neutral amino acid exchanger, ASCT2 has garnered attention for its important role in cancer biology and potential as a molecular target for cancer therapy. The nature of this role is still being explored, and several classes of ASCT2 inhibitors have been developed. However none have reached sufficient potency or selectivity for clinical use. Despite their distinct functions in biology, the members of the SLC1A family display structural and functional similarity. Since 2004, available structures of the archaeal homologues GltPh and GltTk have elucidated mechanisms of transport and inhibition common to the family. The recent determination of EAAT1 and ASCT2 structures may be of assistance in future efforts to design efficacious ASCT2 inhibitors. This review will focus on ASCT2, the present state of knowledge on its roles in tumour biology, and how structural biology is being used to progress the development of inhibitors.

Genetic inactivation of glutamate neurons in the rat sublaterodorsal tegmental nucleus recapitulates REM sleep behaviour disorder.

SEE SCHENCK AND MAHOWALD DOI101093/AWW329 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: Idiopathic REM sleep behaviour disorder is characterized by the enactment of violent dreams during paradoxical (REM) sleep in the absence of normal muscle atonia. Accumulating clinical and experimental data suggest that REM sleep behaviour disorder might be due to the neurodegeneration of glutamate neurons involved in paradoxical sleep and located within the pontine sublaterodorsal tegmental nucleus. The purpose of the present work was thus to functionally determine first, the role of glutamate sublaterodorsal tegmental nucleus neurons in paradoxical sleep and second, whether their genetic inactivation is sufficient for recapitulating REM sleep behaviour disorder in rats. For this goal, we first injected two retrograde tracers in the intralaminar thalamus and ventral medulla to disentangle neuronal circuits in which sublaterodorsal tegmental nucleus is involved; second we infused bilaterally in sublaterodorsal tegmental nucleus adeno-associated viruses carrying short hairpin RNAs targeting Slc17a6 mRNA [which encodes vesicular glutamate transporter 2 (vGluT2)] to chronically impair glutamate synaptic transmission in sublaterodorsal tegmental nucleus neurons. At the neuroanatomical level, sublaterodorsal tegmental nucleus neurons specifically activated during paradoxical sleep hypersomnia send descending efferents to glycine/GABA neurons within the ventral medulla, but not ascending projections to the intralaminar thalamus. These data suggest a crucial role of sublaterodorsal tegmental nucleus neurons rather in muscle atonia than in paradoxical sleep generation. In line with this hypothesis, 30 days after adeno-associated virus injections into sublaterodorsal tegmental nucleus rats display a decrease of 30% of paradoxical sleep daily quantities, and a significant increase of muscle tone during paradoxical sleep concomitant to a tremendous increase of abnormal motor dream-enacting behaviours. These animals display symptoms and behaviours during paradoxical sleep that closely mimic human REM sleep behaviour disorder. Altogether, our data demonstrate that glutamate sublaterodorsal tegmental nucleus neurons generate muscle atonia during paradoxical sleep likely through descending projections to glycine/GABA premotor neurons in the ventral medulla. Although playing a role in paradoxical sleep regulation, they are, however, not necessary for inducing the state itself. The present work further validates a potent new preclinical REM sleep behaviour disorder model that opens avenues for studying and treating this disabling sleep disorder, and advances potential regions implicated in prodromal stages of synucleinopathies such as Parkinson's disease.

Synaptic remodeling generates synchronous oscillations in the degenerated outer mouse retina.

During neuronal degenerative diseases, neuronal microcircuits undergo severe structural alterations, leading to remodeling of synaptic connectivity. The functional consequences of such remodeling are mostly unknown. For instance, in mutant rd1 mouse retina, a common model for Retinitis Pigmentosa, rod bipolar cells (RBCs) establish contacts with remnant cone photoreceptors (cones) as a consequence of rod photoreceptor cell death and the resulting lack of presynaptic input. To assess the functional connectivity in the remodeled, light-insensitive outer rd1 retina, we recorded spontaneous population activity in retinal wholemounts using Ca(2+) imaging and identified the participating cell types. Focusing on cones, RBCs and horizontal cells (HCs), we found that these cell types display spontaneous oscillatory activity and form synchronously active clusters. Overall activity was modulated by GABAergic inhibition from interneurons such as HCs and/or possibly interplexiform cells. Many of the activity clusters comprised both cones and RBCs. Opposite to what is expected from the intact (wild-type) cone-ON bipolar cell pathway, cone and RBC activity was positively correlated and, at least partially, mediated by glutamate transporters expressed on RBCs. Deletion of gap junctional coupling between cones reduced the number of clusters, indicating that electrical cone coupling plays a crucial role for generating the observed synchronized oscillations. In conclusion, degeneration-induced synaptic remodeling of the rd1 retina results in a complex self-sustained outer retinal oscillatory network, that complements (and potentially modulates) the recently described inner retinal oscillatory network consisting of amacrine, bipolar and ganglion cells.

Distribution of Na,K-ATPase α subunits in rat vestibular sensory epithelia.

The afferent encoding of vestibular stimuli depends on molecular mechanisms that regulate membrane potential, concentration gradients, and ion and neurotransmitter clearance at both afferent and efferent relays. In many cell types, the Na,K-ATPase (NKA) is essential for establishing hyperpolarized membrane potentials and mediating both primary and secondary active transport required for ion and neurotransmitter clearance. In vestibular sensory epithelia, a calyx nerve ending envelopes each type I hair cell, isolating it over most of its surface from support cells and posing special challenges for ion and neurotransmitter clearance. We used immunofluorescence and high-resolution confocal microscopy to examine the cellular and subcellular patterns of NKAα subunit expression within the sensory epithelia of semicircular canals as well as an otolith organ (the utricle). Results were similar for both kinds of vestibular organ. The neuronal NKAα3 subunit was detected in all afferent endings-both the calyx afferent endings on type I hair cells and bouton afferent endings on type II hair cells-but was not detected in efferent terminals. In contrast to previous results in the cochlea, the NKAα1 subunit was detected in hair cells (both type I and type II) but not in supporting cells. The expression of distinct NKAα subunits by vestibular hair cells and their afferent endings may be needed to support and shape the high rates of glutamatergic neurotransmission and spike initiation at the unusual type I-calyx synapse.

Pharmacological inhibitions of glutamate transporters EAAT1 and EAAT2 compromise glutamate transport in photoreceptor to ON-bipolar cell synapses.

To maintain reliable signal transmission across a synapse, free synaptic neurotransmitters must be removed from the cleft in a timely manner. In the first visual synapse, this critical task is mainly undertaken by glutamate transporters (EAATs). Here we study the differential roles of the EAAT1, EAAT2 and EAAT5 subtypes in glutamate (GLU) uptake at the photoreceptor-to-depolarizing bipolar cell synapse in intact dark-adapted retina. Various doses of EAAT blockers and/or GLU were injected into the eye before the electroretinogram (ERG) was measured. Their effectiveness and potency in inhibiting the ERG b-wave were studied to determine their relative contributions to the GLU clearing activity at the synapse. The results showed that EAAT1 and EAAT2 plays different roles. Selectively blocking glial EAAT1 alone using UCPH101 inhibited the b-wave 2-24h following injection, suggesting a dominating role of EAAT1 in the overall GLU clearing capacity in the synaptic cleft. Selectively blocking EAAT2 on photoreceptor terminals had no significant effect on the b-wave, but increased the potency of exogenous GLU in inhibiting the b-wave. These suggest that EAAT2 play a secondary yet significant role in the GLU reuptake activity at the rod and the cone output synapses. Additionally, we have verified our electrophysiological findings with double-label immunohistochemistry, and extend the literature on the spatial distribution of EAAT2 splice variants in the mouse retina.

Possible roles of glutamate transporter EAAT5 in mouse cone depolarizing bipolar cell light responses.

A remarkable feature of neuronal glutamate transporters (EAATs) is their dual functions of classical carriers and ligand-gated chloride (Cl(-)) channels. Cl(-) conductance is rapidly activated by glutamate in subtype EAAT5, which mediates light responses in depolarizing bipolar cells (DBC) in retinae of lower vertebrates. In this study, we examine whether EAAT5 also mediates the DBC light response in mouse. We took advantage of an infrared illuminated micro-injection system, and studied the effects of the EAAT blocker (TBOA) and a glutamate receptor agonist (LAP4) on the mouse electroretinogram (ERG) b-wave responses. Our results showed that TBOA and LAP4 shared similar temporal patterns of inhibition: both inhibited the ERG b-wave shortly after injection and recovered with similar time courses. TBOA inhibited the b-wave completely at mesopic light intensity with an IC50 value about 1 log unit higher than that of LAP4. The inhibitory effects of TBOA and LAP4 were found to be additive in the photopic range. Furthermore, TBOA alone inhibited the b-wave in the cone operative range in knockout mice lacking DBCRs at a low concentration that did not alter synaptic glutamate clearance activity. It also produced a stronger inhibition than that of LAP4 on the cone-driven b-wave measured with a double flash method in wildtype mice. These electrophysiological data suggest a significant role for EAAT5 in mediating cone-driven DBC light responses. Our immunohistochemistry data indicated the presence of postsynaptic EAAT5 on some DBCCs and some DBCRs, providing an anatomical basis for EAAT5's role in DBC light responses.

Functional properties of the retinal glutamate transporters GLT-1c and EAAT5.

In the mammalian retina, glutamate uptake is mediated by members of a family of glutamate transporters known as "excitatory amino acid transporters (EAATs)." Here we cloned and functionally characterized two retinal EAATs from mouse, the GLT-1/EAAT2 splice variant GLT-1c, and EAAT5. EAATs are glutamate transporters and anion-selective ion channels, and we used heterologous expression in mammalian cells, patch-clamp recordings and noise analysis to study and compare glutamate transport and anion channel properties of both EAAT isoforms. We found GLT-1c to be an effective glutamate transporter with high affinity for Na(+) and glutamate that resembles original GLT-1/EAAT2 in all tested functional aspects. EAAT5 exhibits glutamate transport rates too low to be accurately measured in our experimental system, with significantly lower affinities for Na(+) and glutamate than GLT-1c. Non-stationary noise analysis demonstrated that GLT-1c and EAAT5 also differ in single-channel current amplitudes of associated anion channels. Unitary current amplitudes of EAAT5 anion channels turned out to be approximately twice as high as single-channel amplitudes of GLT-1c. Moreover, at negative potentials open probabilities of EAAT5 anion channels were much larger than for GLT-1c. Our data illustrate unique functional properties of EAAT5, being a low-affinity and low-capacity glutamate transport system, with an anion channel optimized for anion conduction in the negative voltage range.

Excitatory amino acid transporter 5 is widely expressed in peripheral tissues.

It is routinely stated in the literature that Excitatory Amino Acid Transporter 5 (EAAT5) is a retina-specific glutamate transporter. EAAT5 is expressed by retinal photoreceptors and bipolar cells, where it serves as a slow transporter and as an inhibitory glutamate receptor, the latter role is due to the gating of a large chloride conductance. The dogma of an exclusively retinal distribution has arisen because Northern blot analyses have previously shown only modest hybridisation in non-retinal tissues. Others have re-interpreted this as indicating that EAAT5 was only present in retinal tissues. However, this view appears to be erroneous; recent evidence demonstrating abundant expression of EAAT5 in rat testis prompted us to re-examine this dogma. A new antibody was developed to an intracellular loop region of rat EAAT5. This new tool, in concert with RT-PCR and sequencing, demonstrated that EAAT5 is widely distributed at the mRNA and protein levels in many non-nervous tissues including liver, kidney, intestine, heart, lung, and skeletal muscle. We conclude that EAAT5 is a widely distributed protein. Whether it functions in all locations as a glutamate transporter, or mainly as a glutamate-gated chloride conductance, remains to be determined.

Glutamate transporters EAAT4 and EAAT5 are expressed in vestibular hair cells and calyx endings.

Glutamate is the neurotransmitter released from hair cells. Its clearance from the synaptic cleft can shape neurotransmission and prevent excitotoxicity. This may be particularly important in the inner ear and in other sensory organs where there is a continually high rate of neurotransmitter release. In the case of most cochlear and type II vestibular hair cells, clearance involves the diffusion of glutamate to supporting cells, where it is taken up by EAAT1 (GLAST), a glutamate transporter. A similar mechanism cannot work in vestibular type I hair cells as the presence of calyx endings separates supporting cells from hair-cell synapses. Because of this arrangement, it has been conjectured that a glutamate transporter must be present in the type I hair cell, the calyx ending, or both. Using whole-cell patch-clamp recordings, we demonstrate that a glutamate-activated anion current, attributable to a high-affinity glutamate transporter and blocked by DL-TBOA, is expressed in type I, but not in type II hair cells. Molecular investigations reveal that EAAT4 and EAAT5, two glutamate transporters that could underlie the anion current, are expressed in both type I and type II hair cells and in calyx endings. EAAT4 has been thought to be expressed almost exclusively in the cerebellum and EAAT5 in the retina. Our results show that these two transporters have a wider distribution in mice. This is the first demonstration of the presence of transporters in hair cells and provides one of the few examples of EAATs in presynaptic elements.

Alternate splicing and expression of the glutamate transporter EAAT5 in the rat retina.

Excitatory amino acid transporter 5 (EAAT5) is an unusual glutamate transporter that is expressed in the retina, where it is localised to two populations of glutamatergic neurons, namely the bipolar neurons and photoreceptors. EAAT5 exhibits two distinct properties, acting both as a slow glutamate transporter and as a glutamate-gated inhibitory receptor. The latter property is attributable to a co-associated chloride conductance. EAAT5 has previously been thought to exist only as a full-length form. We now demonstrate by PCR cloning and sequencing, the presence of five novel splice variant forms of EAAT5 which skip either partial or complete exons in the rat retina. Furthermore, we demonstrate that each of these variants is expressed at the protein level as assessed by Western blotting using splice-specific antibodies that we have generated. We conclude that EAAT5 exists in multiple spliced forms, and propose, based upon retention or absence of key structural features, that these variant forms may potentially exhibit distinct properties relative to the originally described form of EAAT5.

The discovery of slowness: low-capacity transport and slow anion channel gating by the glutamate transporter EAAT5.

Excitatory amino acid transporters (EAATs) control the glutamate concentration in the synaptic cleft by glial and neuronal glutamate uptake. Uphill glutamate transport is achieved by the co-/countertransport of Na(+) and other ions down their concentration gradients. Glutamate transporters also display an anion conductance that is activated by the binding of Na(+) and glutamate but is not thermodynamically coupled to the transport process. Of the five known glutamate transporter subtypes, the retina-specific subtype EAAT5 has the largest conductance relative to glutamate uptake activity. Our results suggest that EAAT5 behaves as a slow-gated anion channel with little glutamate transport activity. At steady state, EAAT5 was activated by glutamate, with a K(m)= 61 ± 11 µM. Binding of Na(+) to the empty transporter is associated with a K(m) = 229 ± 37 mM, and binding to the glutamate-bound form is associated with a K(m) = 76 ± 40 mM. Using laser-pulse photolysis of caged glutamate, we determined the pre-steady-state kinetics of the glutamate-induced anion current of EAAT5. This was characterized by two exponential components with time constants of 30 ± 1 ms and 200 ± 15 ms, which is an order of magnitude slower than those observed in other glutamate transporters. A voltage-jump analysis of the anion currents indicates that the slow activation behavior is caused by two slow, rate-limiting steps in the transport cycle, Na(+) binding to the empty transporter, and translocation of the fully loaded transporter. We propose a kinetic transport scheme that includes these two slow steps and can account for the experimentally observed data. Overall, our results suggest that EAAT5 may not act as a classical high-capacity glutamate transporter in the retina; rather, it may function as a slow-gated glutamate receptor and/or glutamate buffering system.

Expression of multiple glutamate transporter splice variants in the rodent testis.

Glutamate is a regulated molecule in the mammalian testis. Extracellular regulation of glutamate in the body is determined largely by the expression of plasmalemmal glutamate transporters. We have examined by PCR, western blotting and immunocytochemistry the expression of a panel of sodium-dependent plasmalemmal glutamate transporters in the rat testis. Proteins examined included: glutamate aspartate transporter (GLAST), glutamate transporter 1 (GLT1), excitatory amino acid carrier 1 (EAAC1), excitatory amino acid transporter 4 (EAAT4) and EAAT5. We demonstrate that many of the glutamate transporters in the testis are alternately spliced. GLAST is present as exon-3- and exon-9-skipping forms. GLT1 was similarly present as the alternately spliced forms GLT1b and GLT1c, whereas the abundant brain form (GLT1a) was detectable only at the mRNA level. EAAT5 was also strongly expressed, whereas EAAC1 and EAAT4 were absent. These patterns of expression were compared with the patterns of endogenous glutamate localization and with patterns of d-aspartate accumulation, as assessed by immunocytochemistry. The presence of multiple glutamate transporters in the testis, including unusually spliced forms, suggests that glutamate homeostasis may be critical in this organ. The apparent presence of many of these transporters in the testis and sperm may indicate a need for glutamate transport by such cells.

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.

Molecular cloning of canine excitatory amino acid transporter 5 and its detection in primary lens epithelial cells.

The full-length cDNA sequence of canine excitatory amino acid transporter (EAAT) 5 was determined in samples taken from the canine retina. The sequence was 2,467 bp long and was predicted to encode the 560 amino acid polypeptides. The deduced amino acid sequence of canine EAAT5 showed similarities of 91.8 and 92.7% to those of humans and rats, respectively. In canine, EAAT5 has a 49.4% identity with EAAT1, 43.7% with EAAT2, 46.4% with EAAT3, and 45.7% with EAAT4. RT-PCR analysis revealed EAAT5 expression in primary lens epithelial cell culture and the cerebellum, and Western blot analysis detected a single band of 60 kDa which confirmed EAAT5 protein expression in these cells. In addition, all subtypes of EAAT were detected in canine lens epithelial cells, indicating the pivotal role of EAATs in supplying glutamate, the precursor of antioxidant glutathione in the lens.

Excitatory amino acid transporter expression by astrocytes is neuroprotective against microglial excitotoxicity.

Glutamate-induced excitotoxicity is considered as a major cause of neurodegenerative disease. Excitatory amino acid transporters (EAATs) on glial cells are responsible for the homeostasis of extracellular glutamate in the central nervous system which may contribute to the prevention of excitotoxic neurodegeneration. However, the differential EAAT expression in astrocytes and microglia is not fully understood. In this study, we compared the expression of EAATs in astrocytes and microglia, and we assessed the neuroprotective and neurotoxic function of astrocytes and microglia by a co-culture system. RT-PCR analyses detected that astrocytes expressed each EAAT (EAAT1-5) whereas microglia did not express EAAT4. Western blot analyses demonstrated that astrocytes express a much larger amount of membrane-localized EAATs than microglia. Astrocytes prevented excito-neurotoxicity by the reduction of exogenous glutamate whereas microglia did not. Conversely, activated microglia released an excess of glutamate that induced excitotoxic neuronal death. Astrocytes rescued neurons from microglial glutamate-induced death in a ratio-dependent manner. Inhibition of EAATs abolished glutamate uptake and the neuroprotective effect of astrocytes, but it did not alter any microglial neurotoxic or neuroprotective effects. These results revealed that astrocytic EAATs can counteract microglial glutamate-induced neuronal death whereas microglial EAATs are inconsequential to neurotoxicity and neuroprotection.