- Email: [email protected]
Functions of glutamate transporters in the brain
Neuroscience Research 37 (2000) 15 – 19 www.elsevier.com/locate/neures
Update Article
Functions of glutamate transporters in the brain Kohichi Tanaka * Department of Molecular Neuroscience, Medical Research Institute, Tokyo Medical and Dental Uni6ersity, Bunkyo-Ku, Tokyo 113 -8519, Japan Received 21 December 1999; accepted 4 February 2000
Abstract L-glutamate is the primary excitatory neurotransmitter in the mammalian central nervous system and has also been implicated as a potent neurotoxin. To ensure a high signal-to-noise ratio during synaptic transmission and to prevent neuronal damage that might occur as a result of excessive activation of glutamate receptors, the extracellular glutamate concentration is tightly controlled by glutamate transporters in the plasma membrane of neurons and the surrounding glial cells. Five subtypes of glutamate transporters have been identified and characterized by molecular cloning. Recent studies of glutamate transporters using the genetic knockout strategy indicate that glial, but not neuronal, glutamate transporters play critical roles in maintaining the extracellular glutamate concentrations and are thereby essential for both normal synaptic transmission at the photoreceptor synapses and protection of neurons against glutamate excitotoxicity. This review summarizes the current knowledge on the properties and functional roles of glutamate transporters, focusing on the properties of the anion channel in the transporters, the unexpected localization of these transporters, their role in synaptic transmission and plasticity, and their involvement in the protection of neurons against excitotoxicity. © 2000 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
Keywords: Glutamate transporter; Ligand-gated chloride channel; Synaptic transmission; Excitotoxicity; Knockout mouse; Glial cell
1. Introduction L-glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. High-affinity glutamate transporters are believed to be essential for terminating synaptic transmission as well as for maintaining the extracellular glutamate concentration below neurotoxic levels. So far, five structurally distinct subtypes of glutamate transporters (GLT-1, GLAST, EAAC1, EAAT4, and EAAT5) have been identified and characterized by molecular cloning (Sims and Robinson, 1999). Recent studies of mice with targeted disruptions in each of the glutamate transporter genes demonstrated the roles of the different transporter subtypes in synaptic transmission and neurotoxicity. Here, I review the recent advances in our understanding of the unique channel-like properties of the glutamate transporters, the unexpected localization of these transporters, their roles in synaptic transmission and plastic-
* Tel.: +81-3-58035846; fax: + 81-3-58035843. E-mail address: [email protected] (K. Tanaka)
ity, and also neurotoxicity.
their
role
in
protection
against
2. The anion channel in glutamate transporters The transport of glutamate by glutamate transporters is thermodynamically coupled to the cotransport of at least two sodium ions, one proton and the countertransport of a potassium ion (Fig. 1) (Barbour, et al., 1988; Kanai, et al., 1995; Zerangue and Kavanaugh, 1996). Thus, at least one net positive charge enters the cell per glutamate transported. However, Wadiche et al. (1995a) found that the amount of charge that moves when glutamates are applied to cloned glutamate transporters expressed in oocytes is greater than that predicted by the stoichiometry just described (Wadiche et al., 1995a). This extra charge entry elicited during glutamate application arises from a thermodynamically uncoupled Cl− flux. Billups et al. (1996) found that while both the removal of K+ and acidification of the external pH greatly reduced glutamate transport, neither had any effect on the opening of the anion channel
0168-0102/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 0 0 ) 0 0 1 0 4 - 8
16
K. Tanaka / Neuroscience Research 37 (2000) 15–19
of glutamate transporters. Furthermore, zinc reduces glutamate transport by the retinal glial cell transporter, but potentiates opening of the transporter’s anion channel (Spiridon et al., 1998). These results suggest that the anion channel conductance can be activated independently of glutamate transport. Available data support the idea that glutamate transporters function both as transporters and as a ligand-gated chloride channel, but there has been no structural evidence to suggest whether glutamate and Cl− permeate the same pore or pass through different pathways in the transporter proteins and/or whether other proteins are necessary for the generation of different pathways associated with these transporters (Fig. 1). The relative proportion of the current generated by the charge movements coupled directly to glutamate movement or substrate-gated chloride conductance varies for each glutamate transporter subtype. The glial transporters GLT-1 and GLAST, and the neuronal transporter EAAC1, show relatively small chloride flux relative to the currents elicited by ion–coupled cotransport, while for the neuronal transporters, EAAT4 and EAAT5, the chloride current dominates the currents elicited by ion-coupled cotransport (Wadiche et al., 1995a; Arriza et al., 1997). Activation of anion conductance during glutamate uptake has been proposed to clamp the membrane potential at a relatively negative potential. This negative potential could dampen neuronal excitability and potentiate the reuptake of glutamate.
3. Postsynaptic glutamate transporters Conventionally, neuronal glutamate uptake has been thought of as occurring at presynaptic terminals. However, this concept has been challenged by recent studies. The neuronal glutamate transporters identified to date in the brain, EAAC1 and EAAT4, are not found in
axons or presynaptic terminals, but instead are expressed on the somata and dendrites of neurons (Rothstein et al., 1994; Yamada et al., 1996). Furthermore, EAAT4 is present in a GABAergic cell type, the cerebellar Purkinje cell (PC), and appears to be concentrated on the extrajunctional membrane of the PC spines in contact with the glutamate-releasing terminals of the parallel and climbing fibers (Tanaka et al., 1997a). The postsynaptic localization of the neuronal glutamate transporters and their expression in nonglutamatergic neurons suggest that they may also function in ways other than simply serving as sites of glutamate reuptake. 4. Role of glutamate transporters in synaptic transmission Recent studies in glutamate transporter-deficient mice have clarified the role of each glutamate transporter subtype in synaptic transmission. There was no significant difference in the decay phase of the nonNMDA and NMDA receptor components of the synaptic current at the hippocampal Schaffer collateral to pyramidal cell synapse between the wild-type and GLT-1 mutant mice (Tanaka et al., 1997b). These results indicate that GLT-1 does not determine the decay rate of the synaptic currents in the hippocampus. However, the peak concentration of synaptically released glutamate is increased and the glutamate concentration remains elevated in the synaptic cleft for longer periods in mutant mice (Tanaka et al., 1997b). Longterm potentiation (LTP) in GLT-1 mutants was significantly attenuated in the hippocampal CA1 region, whereas long-term depression (LTD) remained intact (Katagiri et al., 1999). When LTP was induced in the presence of low concentrations of D-APV, the attenuation of LTP in the mutants was overcome, indicating that the attenuated LTP in the mutants is due to
Fig. 1. Schematic models for the transport of glutamate and gating of the transporter’s chloride channel. During the transport cycle, two or three sodium ions and a proton are cotransported with each negatively charged glutamate molecule, and a potassium ion is counter-transported. Glutamate transporters can also function as ion channels permeable to Cl−. This channel could either be located in the same pore (A) or in a different portion of the transporter protein, possibly in association with another protein (B).
K. Tanaka / Neuroscience Research 37 (2000) 15–19
excessive activation of NMDA receptors. Consistent with these results, the ratio of the NMDA to AMPA component of the synaptic currents was significantly larger in mutant mice than in wild-type mice (Katagiri et al., 1999). The increased glutamate concentration in the synaptic cleft in the mutants activates NMDA receptors preferentially, since NMDA receptors have a high affinity for glutamate. Thus, GLT-1 plays an important role in LTP induction through regulation of the extracellular level of glutamate. The time constant for one cycle of glutamate translocation has been estimated to be 70 ms, which is significantly slower than the predicted time course of synaptically released glutamate (Wadiche et al., 1995b). Therefore, it has been hypothesized that glutamate binding to transporters, rather than its uptake likely dominates the synaptic concentration decay kinetics (Tong and Jahr, 1994). Mennerick et al. (1999) demonstrated, however, that glutamate translocation, rather than glutamate binding to the transporters, determined the time course of the glutamate concentration transient at the excitatory synapses in hippocampal microcultures. The results of Mennerick et al., do not address the very rapid phase of the time course of the glutamate concentration transient, observed by others (Tong and Jahr, 1994). Therefore, a possible explanation for the difference between the results of Mennerick et al., and the speculation of Tong and Jahr might be that the buffer property of glutamate transporters functions on the fastest time scales, and the translocation function on a slower time scale of glutamate clearance. It was previously reported that glutamate transporter blockers prolong the excitatory postsynaptic current (EPSC) at both parallel fiber (PF)- and climbing fiber (CF)-PC synapses (Takahashi et al., 1995). GLAST is strongly expressed in the Bergmann glial processes that ensheath these synapses and is considered to be responsible in large part for cerebellar glutamate transport (Lehre et al., 1995; Yamada et al., in press). However, the kinetics of both PF- and CF-EPSCs were normal in GLAST-mutant mice, suggesting that GLAST is not the dominant factor that determines the kinetics of EPSCs at these synapses (Watase et al., 1998). Furthermore, there was no significant difference in the inhibitory potency of the rapidly dissociating non-NMDA receptor antagonist PDA on the PF-EPSCs between the wild-type and GLAST-mutant mice, indicating that GLAST does not play a major role in the clearance of free glutamate from the synaptic clefts at these synapses (Watase et al., 1998). In the retina, GLAST is predominantly expressed in Muller cell processes that ensheath both photoreceptor-bipolar cell and bipolar cell-ganglion cell synapses. Although the GLAST-mutant mice had a normal electroretinogram a-wave, which reflects the activities of photoreceptors, the b-wave, which originates mainly in ON bipolar
17
cells, was attenuated by more than 50% as compared with that in the wild-type mice (Harada et al., 1998). These results suggest that GLAST is required for normal neurotransmission between photoreceptors and bipolar cells. Photoreceptors use graded membrane-potential changes, instead of action potentials, to transmit the response to light to bipolar cells (Juusola et al., 1996). At the onset of a bright light, photoreceptors are hyperpolarized and the release of glutamate from them is diminished. Transmission of this effect to bipolar cells requires that the glutamate in the synaptic cleft be removed by reuptake. Therefore, GLAST is not required for normal synaptic transmission at conventional synapses (i.e. PF and CF to PC synapses), but is essential for proper synaptic transmission at the photoreceptor-bipolar cell synapse. A role for postsynaptic glutamate transporters in PCs (EAAC1 and EAAT4) in the termination of synaptic transmission was suggested by Takahashi et al. (1996), who observed that when the rate of glutamate uptake by these transporters was reduced, by following inclusion of D-aspartate in the pipette used to whole-cell clamp the PC, the decay of the CF synaptic current was prolonged. In preliminary experiments (Hashimoto et al., unpublished), we have been unable to detect any significant difference in the kinetics of either CF- or PF-EPSCs between the wild-type and EAAT4-mutant mice. Therefore, it is likely that EAAC1 plays a decisive role in determining the decay rate of CF-EPSCs. Unfortunately, the CF-EPSCs in EAAC1 knockout mice have not yet been measured (Peghini et al., 1997).
5. Significance of glutamate transporters in neuropathologic conditions Although previous studies indicate that glutamate transporters directly regulate the extracellular glutamate concentration and limit excitotoxicity, until recently, little was known about the contributions of the different transporter subtypes in excitotoxic processes. GLT-1 and GLAST knockout mice show increased susceptibility to traumatic brain injury (Tanaka et al., 1997b; Watase et al., 1998), whereas EAAT4-mutant mice show no such vulnerability to brain injury (Maeno et al., unpublished). EAAC1-knockout mice show no apparent neurodegeneration even after 1 year, but the susceptibility to traumatic injury was not measured. Since excitotoxic damage may contribute to traumatic brain injury, these results suggest that glial glutamate transporters may function mainly to keep the glutamate concentration low in the extracellular space and to prevent excitotoxic brain damage. It has been proposed that glutamate transporter dysfunction in amyotrophic lateral sclerosis (ALS) may be a significant factor in the pathophysiology of the disease (Rothstein et al., 1992).
18
K. Tanaka / Neuroscience Research 37 (2000) 15–19
Rothstein and colleagues have suggested that the expression of two spliced forms of the GLT-1 RNA, resulting from retention of intronic sequences and deletion of one protein coding exon, is responsible for the reduction in GLT-1 protein expression and activity, sometimes observed in ALS brain and spinal cord samples (Lin et al., 1998). However, recent reports have shown that both cDNA variants are equally expressed in ALS patients and in controls (Nagai et al., 1998; Meyer et al., 1999). Therefore, the involvement of aberrantly spliced transcripts from the GLT-1 gene in ALS is unlikely to be a primary factor, and is more complex than previously recognized. For further information about the pathophysiological significance of glial glutamate transporters in epilepsy and noise-induced hearing loss, the reader is referred to our recent papers (Tanaka et al., 1997b; Watanabe et al., 1999; Hakuba et al., 2000).
6. Prospects for the future Much progress has been made in our understanding of the molecular aspects of glutamate transporters during the last 7 years, there still remain a number of unanswered questions regarding the role of glutamate transporters. First, do glutamate transporters play a significant role in limiting the extrasynaptic diffusion of glutamate, thereby minimizing cross-talks between neighboring excitatory synapses? Second, is the reversed operation of glutamate transporters a major contributor to the increase in extracellular glutamate concentration under conditions of ischemia? Third, what is the role of glutamate transporters in controlling the extracellular glutamate concentration during brain development? Fourth, what is the functional role of the uncoupled chloride conductance associated with EAAT4? GLAST-, GLT-1-, and EAAT4-mutant mice will provide excellent model systems for answering the questions raised above, and such efforts are currently in progress in our laboratory.
References Arriza, J.L., Eliasof, S., Kavanaugh, M.P., Amara, S.G., 1997. Excitatory amino acid transporter 5, a retinal glutamate transporter coupled to a chloride conductance. Proc. Natl. Acad. Sci. USA 94, 4155 – 4160. Barbour, B., Brew, H., Attwell, D., 1988. Electrogenic glutamate uptake in glial cells is activated by intracellular potassium. Nature 335, 433 – 435. Billups, B., Rossi, D., Attwell, D., 1996. Anion conductance behavior of the glutamate uptake carrier in salamander retinal glial cells. J. Neurosci. 16, 6722 – 6731. Harada, T., Harada, C., Watanabe, M., et al., 1998. Functions of the two glutamate transporters GLAST and GLT-1 in the retina. Proc. Natl. Acad. Sci. USA 95, 4663–4666.
Hakuba, N., Koga, K., Gyo, K., Usami, S., Tanaka, K., 2000. Exacerbation of noise-induced hearing loss in mice lacking the glutamate transporter GLAST. J. Neurosci. in press. Juusola, M., French, A.S., Uusitalo, R.O., Weckstrom, M., 1996. Information processing by graded-potential transmission through tonically active synapses. Trends Neurosci. 19, 292 – 297. Kanai, Y., Nussberger, S., Romero, M.F., Boron, W.F., Hebert, S.C., Hediger, M.A., 1995. Electrogenic properties of the epithelial and neuronal high-affinity glutamate transporter. J. Biol. Chem. 270, 16561 – 16568. Katagiri, H., Tanaka, K., Takahashi, T., Manabe, T., 1999. Reqirement of appropriate synaptic cleft glutamate concentrations for hippocampal LTP induction. Soc. Neurosci. Abstr. 25, 734. Lehre, K.P., Levy, L.M., Ottersen, O.P., Storm-Mathisen, J., Danbolt, N.C., 1995. Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocytochemical observations. J. Neurosci. 15, 1835 – 1853. Lin, C.L., Bristol, L.A., Jin, L., et al., 1998. Aberrant RNA processing in a neurodegenerative disease: the cause for absent EAAT2, a glutamate transporter, in amyotrophic lateral sclerosis. Neuron 20, 589 – 602. Mennerick, S., Shen, W., Xu, W., et al., 1999. Substrate turnover by transporters curtails synaptic glutamate transients. J. Neurosci. 19, 9242 – 9251. Meyer, T., Fromm, A., Munch, C., et al., 1999. The RNA of the glutamate transporter EAAT2 is variably spliced in amyotrophic lateral sclerosis and normal individuals. J. Neurol. Sci. 170, 45 – 50. Nagai, M., Abe, K., Okamoto, K., Itoyama, Y., 1998. Identification of alternative splicing forms of GLT-1 mRNA in the spinal cord of amyotrophic lateral sclerosis patients. Neurosci. Lett. 244, 165 – 168. Peghini, P., Janzen, J., Stoffel, W., 1997. Glutamate transporter EAAC-1-deficient mice develop dicarboxylic aminoaciduria and behavioral abnormalities but no neurodegeneration. EMBO J. 16, 3822 – 3832. Rothstein, J.D., Martin, L., Levey, A.I., et al., 1994. Localization of neuronal and glial glutamate transporters. Neuron 13, 713–725. Rothstein, J.D., Martin, L.J., Kuncl, R.W., 1992. Decreased glutamate transport by the brain and spinal cord in amyotrophic lateral sclerosis. N. Engl. J. Med. 326, 1464 – 1468. Sims, K.D., Robinson, M.B., 1999. Expression patterns and regulation of glutamate transporters in the developing and adult nervous system. Crit. Rev. Neurobiol. 13, 169 – 197. Spiridon, M., Kamm, D., Billups, B., Mobbs, P., Attwell, D., 1998. Modulation by zinc of the glutamate transporter in glial cells and cones isolated from the tiger salamander retina. J. Physiol. (Lond) 506, 363 – 376. Takahashi, M., Kovalchuk, Y., Attwell, D., 1995. Pre- and postsynaptic determinants of EPSC waveform at cerebellar climbing fiber and parallel fiber to Purkinje cell synapse. J. Neurosci. 15, 5693 – 5702. Takahashi, M., Sarantis, M., Attwell, D., 1996. Postsynaptic glutamate uptake in rat cerebellar Purkinje cells. J. Physiol. (Lond) 497, 523 – 530. Tanaka, J., Ichikawa, R., Watanabe, M., Tanaka, K., Inoue, Y., 1997a. Extra-junctional localization of glutamate transporter EAAT4 at excitatory Purkinje cell synapses. Neuroreport 8, 2461 – 2464. Tanaka, K., Watase, K., Manabe, T., et al., 1997b. Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science 276, 1699 – 1702. Tong, G., Jahr, C.E., 1994. Block of glutamate transporters potentiates postsynaptic excitation. Neuron 13, 1195 – 1203. Wadiche, J.I., Amara, S.G., Kavanaugh, M.P., 1995a. Ion fluxes associated with excitatory amino acid transport. Neuron 15, 721 – 728.
K. Tanaka / Neuroscience Research 37 (2000) 15–19 Wadiche, J.I., Arriza, J.L., Amara, S.G., Kavanaugh, M.P., 1995b. Kinetics of a human glutamate transporter. Neuron 14, 1019 – 1027. Watanabe, T., Morimoto, K., Hirao, T., Suwaki, H., Watase, K., Tanaka, K., 1999. Amygdala-kindled and pentylenetetrazole-induced seizures in glutamate transporter GLAST-deficient mice. Brain Res. 845, 92 – 96. Watase, K., Hashimoto, K., Kano, M., et al., 1998. Motor discoordination and increased susceptibility to cerebellar injury in GLAST mutant mice. Eur. J. Neurosci. 10, 976–988.
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Yamada, K., Fukaya, M., Shibata, T., et al., 2000. Dynamic transformation of Bergmann glial fibers proceeds in correlation with dendritic outgrowth and synapse formation of cerebellar Purkinje cells. J. Comp. Neurol. 418, 106 – 120. Yamada, K., Watanabe, M., Shibata, T., Tanaka, K., Wada, K., Inoue, Y., 1996. EAAT4 is a post-synaptic glutamate transporter at Purkinje cell synapses. Neuroreport 7, 2013 – 2017. Zerangue, N., Kavanaugh, M.P., 1996. Flux coupling in a neuronal glutamate transporter. Nature 383, 634 – 637.
Update Article
Functions of glutamate transporters in the brain Kohichi Tanaka * Department of Molecular Neuroscience, Medical Research Institute, Tokyo Medical and Dental Uni6ersity, Bunkyo-Ku, Tokyo 113 -8519, Japan Received 21 December 1999; accepted 4 February 2000
Abstract L-glutamate is the primary excitatory neurotransmitter in the mammalian central nervous system and has also been implicated as a potent neurotoxin. To ensure a high signal-to-noise ratio during synaptic transmission and to prevent neuronal damage that might occur as a result of excessive activation of glutamate receptors, the extracellular glutamate concentration is tightly controlled by glutamate transporters in the plasma membrane of neurons and the surrounding glial cells. Five subtypes of glutamate transporters have been identified and characterized by molecular cloning. Recent studies of glutamate transporters using the genetic knockout strategy indicate that glial, but not neuronal, glutamate transporters play critical roles in maintaining the extracellular glutamate concentrations and are thereby essential for both normal synaptic transmission at the photoreceptor synapses and protection of neurons against glutamate excitotoxicity. This review summarizes the current knowledge on the properties and functional roles of glutamate transporters, focusing on the properties of the anion channel in the transporters, the unexpected localization of these transporters, their role in synaptic transmission and plasticity, and their involvement in the protection of neurons against excitotoxicity. © 2000 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
Keywords: Glutamate transporter; Ligand-gated chloride channel; Synaptic transmission; Excitotoxicity; Knockout mouse; Glial cell
1. Introduction L-glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. High-affinity glutamate transporters are believed to be essential for terminating synaptic transmission as well as for maintaining the extracellular glutamate concentration below neurotoxic levels. So far, five structurally distinct subtypes of glutamate transporters (GLT-1, GLAST, EAAC1, EAAT4, and EAAT5) have been identified and characterized by molecular cloning (Sims and Robinson, 1999). Recent studies of mice with targeted disruptions in each of the glutamate transporter genes demonstrated the roles of the different transporter subtypes in synaptic transmission and neurotoxicity. Here, I review the recent advances in our understanding of the unique channel-like properties of the glutamate transporters, the unexpected localization of these transporters, their roles in synaptic transmission and plastic-
* Tel.: +81-3-58035846; fax: + 81-3-58035843. E-mail address: [email protected] (K. Tanaka)
ity, and also neurotoxicity.
their
role
in
protection
against
2. The anion channel in glutamate transporters The transport of glutamate by glutamate transporters is thermodynamically coupled to the cotransport of at least two sodium ions, one proton and the countertransport of a potassium ion (Fig. 1) (Barbour, et al., 1988; Kanai, et al., 1995; Zerangue and Kavanaugh, 1996). Thus, at least one net positive charge enters the cell per glutamate transported. However, Wadiche et al. (1995a) found that the amount of charge that moves when glutamates are applied to cloned glutamate transporters expressed in oocytes is greater than that predicted by the stoichiometry just described (Wadiche et al., 1995a). This extra charge entry elicited during glutamate application arises from a thermodynamically uncoupled Cl− flux. Billups et al. (1996) found that while both the removal of K+ and acidification of the external pH greatly reduced glutamate transport, neither had any effect on the opening of the anion channel
0168-0102/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 0 0 ) 0 0 1 0 4 - 8
16
K. Tanaka / Neuroscience Research 37 (2000) 15–19
of glutamate transporters. Furthermore, zinc reduces glutamate transport by the retinal glial cell transporter, but potentiates opening of the transporter’s anion channel (Spiridon et al., 1998). These results suggest that the anion channel conductance can be activated independently of glutamate transport. Available data support the idea that glutamate transporters function both as transporters and as a ligand-gated chloride channel, but there has been no structural evidence to suggest whether glutamate and Cl− permeate the same pore or pass through different pathways in the transporter proteins and/or whether other proteins are necessary for the generation of different pathways associated with these transporters (Fig. 1). The relative proportion of the current generated by the charge movements coupled directly to glutamate movement or substrate-gated chloride conductance varies for each glutamate transporter subtype. The glial transporters GLT-1 and GLAST, and the neuronal transporter EAAC1, show relatively small chloride flux relative to the currents elicited by ion–coupled cotransport, while for the neuronal transporters, EAAT4 and EAAT5, the chloride current dominates the currents elicited by ion-coupled cotransport (Wadiche et al., 1995a; Arriza et al., 1997). Activation of anion conductance during glutamate uptake has been proposed to clamp the membrane potential at a relatively negative potential. This negative potential could dampen neuronal excitability and potentiate the reuptake of glutamate.
3. Postsynaptic glutamate transporters Conventionally, neuronal glutamate uptake has been thought of as occurring at presynaptic terminals. However, this concept has been challenged by recent studies. The neuronal glutamate transporters identified to date in the brain, EAAC1 and EAAT4, are not found in
axons or presynaptic terminals, but instead are expressed on the somata and dendrites of neurons (Rothstein et al., 1994; Yamada et al., 1996). Furthermore, EAAT4 is present in a GABAergic cell type, the cerebellar Purkinje cell (PC), and appears to be concentrated on the extrajunctional membrane of the PC spines in contact with the glutamate-releasing terminals of the parallel and climbing fibers (Tanaka et al., 1997a). The postsynaptic localization of the neuronal glutamate transporters and their expression in nonglutamatergic neurons suggest that they may also function in ways other than simply serving as sites of glutamate reuptake. 4. Role of glutamate transporters in synaptic transmission Recent studies in glutamate transporter-deficient mice have clarified the role of each glutamate transporter subtype in synaptic transmission. There was no significant difference in the decay phase of the nonNMDA and NMDA receptor components of the synaptic current at the hippocampal Schaffer collateral to pyramidal cell synapse between the wild-type and GLT-1 mutant mice (Tanaka et al., 1997b). These results indicate that GLT-1 does not determine the decay rate of the synaptic currents in the hippocampus. However, the peak concentration of synaptically released glutamate is increased and the glutamate concentration remains elevated in the synaptic cleft for longer periods in mutant mice (Tanaka et al., 1997b). Longterm potentiation (LTP) in GLT-1 mutants was significantly attenuated in the hippocampal CA1 region, whereas long-term depression (LTD) remained intact (Katagiri et al., 1999). When LTP was induced in the presence of low concentrations of D-APV, the attenuation of LTP in the mutants was overcome, indicating that the attenuated LTP in the mutants is due to
Fig. 1. Schematic models for the transport of glutamate and gating of the transporter’s chloride channel. During the transport cycle, two or three sodium ions and a proton are cotransported with each negatively charged glutamate molecule, and a potassium ion is counter-transported. Glutamate transporters can also function as ion channels permeable to Cl−. This channel could either be located in the same pore (A) or in a different portion of the transporter protein, possibly in association with another protein (B).
K. Tanaka / Neuroscience Research 37 (2000) 15–19
excessive activation of NMDA receptors. Consistent with these results, the ratio of the NMDA to AMPA component of the synaptic currents was significantly larger in mutant mice than in wild-type mice (Katagiri et al., 1999). The increased glutamate concentration in the synaptic cleft in the mutants activates NMDA receptors preferentially, since NMDA receptors have a high affinity for glutamate. Thus, GLT-1 plays an important role in LTP induction through regulation of the extracellular level of glutamate. The time constant for one cycle of glutamate translocation has been estimated to be 70 ms, which is significantly slower than the predicted time course of synaptically released glutamate (Wadiche et al., 1995b). Therefore, it has been hypothesized that glutamate binding to transporters, rather than its uptake likely dominates the synaptic concentration decay kinetics (Tong and Jahr, 1994). Mennerick et al. (1999) demonstrated, however, that glutamate translocation, rather than glutamate binding to the transporters, determined the time course of the glutamate concentration transient at the excitatory synapses in hippocampal microcultures. The results of Mennerick et al., do not address the very rapid phase of the time course of the glutamate concentration transient, observed by others (Tong and Jahr, 1994). Therefore, a possible explanation for the difference between the results of Mennerick et al., and the speculation of Tong and Jahr might be that the buffer property of glutamate transporters functions on the fastest time scales, and the translocation function on a slower time scale of glutamate clearance. It was previously reported that glutamate transporter blockers prolong the excitatory postsynaptic current (EPSC) at both parallel fiber (PF)- and climbing fiber (CF)-PC synapses (Takahashi et al., 1995). GLAST is strongly expressed in the Bergmann glial processes that ensheath these synapses and is considered to be responsible in large part for cerebellar glutamate transport (Lehre et al., 1995; Yamada et al., in press). However, the kinetics of both PF- and CF-EPSCs were normal in GLAST-mutant mice, suggesting that GLAST is not the dominant factor that determines the kinetics of EPSCs at these synapses (Watase et al., 1998). Furthermore, there was no significant difference in the inhibitory potency of the rapidly dissociating non-NMDA receptor antagonist PDA on the PF-EPSCs between the wild-type and GLAST-mutant mice, indicating that GLAST does not play a major role in the clearance of free glutamate from the synaptic clefts at these synapses (Watase et al., 1998). In the retina, GLAST is predominantly expressed in Muller cell processes that ensheath both photoreceptor-bipolar cell and bipolar cell-ganglion cell synapses. Although the GLAST-mutant mice had a normal electroretinogram a-wave, which reflects the activities of photoreceptors, the b-wave, which originates mainly in ON bipolar
17
cells, was attenuated by more than 50% as compared with that in the wild-type mice (Harada et al., 1998). These results suggest that GLAST is required for normal neurotransmission between photoreceptors and bipolar cells. Photoreceptors use graded membrane-potential changes, instead of action potentials, to transmit the response to light to bipolar cells (Juusola et al., 1996). At the onset of a bright light, photoreceptors are hyperpolarized and the release of glutamate from them is diminished. Transmission of this effect to bipolar cells requires that the glutamate in the synaptic cleft be removed by reuptake. Therefore, GLAST is not required for normal synaptic transmission at conventional synapses (i.e. PF and CF to PC synapses), but is essential for proper synaptic transmission at the photoreceptor-bipolar cell synapse. A role for postsynaptic glutamate transporters in PCs (EAAC1 and EAAT4) in the termination of synaptic transmission was suggested by Takahashi et al. (1996), who observed that when the rate of glutamate uptake by these transporters was reduced, by following inclusion of D-aspartate in the pipette used to whole-cell clamp the PC, the decay of the CF synaptic current was prolonged. In preliminary experiments (Hashimoto et al., unpublished), we have been unable to detect any significant difference in the kinetics of either CF- or PF-EPSCs between the wild-type and EAAT4-mutant mice. Therefore, it is likely that EAAC1 plays a decisive role in determining the decay rate of CF-EPSCs. Unfortunately, the CF-EPSCs in EAAC1 knockout mice have not yet been measured (Peghini et al., 1997).
5. Significance of glutamate transporters in neuropathologic conditions Although previous studies indicate that glutamate transporters directly regulate the extracellular glutamate concentration and limit excitotoxicity, until recently, little was known about the contributions of the different transporter subtypes in excitotoxic processes. GLT-1 and GLAST knockout mice show increased susceptibility to traumatic brain injury (Tanaka et al., 1997b; Watase et al., 1998), whereas EAAT4-mutant mice show no such vulnerability to brain injury (Maeno et al., unpublished). EAAC1-knockout mice show no apparent neurodegeneration even after 1 year, but the susceptibility to traumatic injury was not measured. Since excitotoxic damage may contribute to traumatic brain injury, these results suggest that glial glutamate transporters may function mainly to keep the glutamate concentration low in the extracellular space and to prevent excitotoxic brain damage. It has been proposed that glutamate transporter dysfunction in amyotrophic lateral sclerosis (ALS) may be a significant factor in the pathophysiology of the disease (Rothstein et al., 1992).
18
K. Tanaka / Neuroscience Research 37 (2000) 15–19
Rothstein and colleagues have suggested that the expression of two spliced forms of the GLT-1 RNA, resulting from retention of intronic sequences and deletion of one protein coding exon, is responsible for the reduction in GLT-1 protein expression and activity, sometimes observed in ALS brain and spinal cord samples (Lin et al., 1998). However, recent reports have shown that both cDNA variants are equally expressed in ALS patients and in controls (Nagai et al., 1998; Meyer et al., 1999). Therefore, the involvement of aberrantly spliced transcripts from the GLT-1 gene in ALS is unlikely to be a primary factor, and is more complex than previously recognized. For further information about the pathophysiological significance of glial glutamate transporters in epilepsy and noise-induced hearing loss, the reader is referred to our recent papers (Tanaka et al., 1997b; Watanabe et al., 1999; Hakuba et al., 2000).
6. Prospects for the future Much progress has been made in our understanding of the molecular aspects of glutamate transporters during the last 7 years, there still remain a number of unanswered questions regarding the role of glutamate transporters. First, do glutamate transporters play a significant role in limiting the extrasynaptic diffusion of glutamate, thereby minimizing cross-talks between neighboring excitatory synapses? Second, is the reversed operation of glutamate transporters a major contributor to the increase in extracellular glutamate concentration under conditions of ischemia? Third, what is the role of glutamate transporters in controlling the extracellular glutamate concentration during brain development? Fourth, what is the functional role of the uncoupled chloride conductance associated with EAAT4? GLAST-, GLT-1-, and EAAT4-mutant mice will provide excellent model systems for answering the questions raised above, and such efforts are currently in progress in our laboratory.
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