The high affinity uptake system for excitatory amino acids in the brain

Progress in Neurobiology Vol. 44, pp. 377 to 396, 1994 Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved

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0301-00S2(94)E0029-G THE HIGH AFFINITY UPTAKE SYSTEM FOR EXCITATORY AMINO ACIDS IN THE BRAIN N I E L S C. D A N B O L T Anatomical Institute, University of Oslo, P.O. Box 1105, Blindern, N-0317 Oslo, Norway

CONTENTS I. Introduction 1.1. Uptake of glutamate from the extracellar fluid l.l. 1. "High affinity" uptake 1.1.2. "Low affinity" uptake 1.1.3. Other uptake systems 1.2. Glutamate uptake in non-neuronal tissues 1.3. Glutamate uptake in other organisms 1.4 Intracellular glutamate uptake 1.5. Glutamate-glutamine cycle 2. The brain plasma membrane high affinity sodium dependent glutamate transporter 2.1. The mechanism of transport 2.2. Substrate selectivity 2.3. Blocking of uptake 2.4. Purification of a glutamate transporter 2.5. Pharmacological demonstration of glutamate transporter heterogeneity 2.6. Cloning of three glutamate transporters 2.6.1. GLAST- 1 2.6.2. GLT-1 2.6.3. EAAC- 1 2.7. Topology of the cloned glutamate transporters 2.8. Neurotransmitter transporter families 2.8.1. Transporters in the plasma membrane 2.8.2. Transporters in synaptic vesicles 2.9. Anatomical localization 2.9.1. Earlier studies 2.9.2. Localization of the cloned glutamate transporters 2.10. Immunoprecipitation and reconstitution experiments 2.11. Regulation of glutamate uptake 2.12. Pathological importance 2.13. How glutamate transporters may be involved in pathology 2.13. I. Failure of transport 2.13.2. Reversal of transport 2.13.3. Excessive transport 2.13.4. Transport of toxic substances 3. Comments on the sodium dependent "binding" of excitatory amino acids 3.1. Binding studies have been very useful for studying receptors 3.2. Sodium dependent "binding" of excitatory amino acids 3.3. Localization of sodium dependent "binding" sites 3.4. The importance of distinguishing uptake from binding 4. Concluding remarks Acknowledgements References

I. I N T R O D U C T I O N The amino acid (S)-glutamate is considered to be the major mediator of excitatory signals in the mammalian central nervous system (Fonnum, 1984; Ottersen and Storm-Mathisen, 1984; Collingridge and Lester, 1989; Headley and Grillner, 1990). The brain contains large amounts of glutamate

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(about 1 0 m m o l / k g wet weight). Almost all the glutamate is located intracellularly, the nerve terminals having the highest concentration (Ottersen et al., 1992; Storm-Mathisen et al., 1992). The extraceUular concentration of glutamate is normally low (1-3 #M).The concentration gradient of glutamate across the plasma membranes is thus several thousand fold. 377

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Most neurons, and even glial cells (H6sli and H6sli, 1993), appear to have glutamate receptors. Glutamate is probably involved in most aspects of normal brain function including cognition, memory and learning. At least three separate classes of glutamate receptors, each with several subtypes, have recently been cloned (for review see: Barnes and Henley, 1992; Sommer and Seeburg, 1992; Schoepp and Conn, 1993; Seeburg, 1993; Hollmann and Heinemann, 1994). The metabotropic receptors are coupled to G-proteins and activation of them typically leads to intracellular effects such as changes in inositol phosphate or cyclic nucleotide metabolism. (1S,3R)-Aminocyclopentane dicarboxylic acid (trans-ACPD) is thought to be a selective agonist. The ionotropic receptors are ligand-gated ion channels and are subdivided into the NMDA receptor family and the AMPA/kainate receptor family. The AMPA/kainate type receptors mediate fast information transfer while the NMDA receptors are only operative when the post-synaptic membrane is already depolarized. The NMDA receptor channels conduct Ca 2÷ (and Na ÷), are slowly inactivated and have a high affinity for glutamate in contrast to the rapidly inactivating Na ÷ conducting AMPA/kainate receptor channels with low affinity for glutamate. The glutamate receptors are located on the surface of the cells expressing them. Therefore, it is the glutamate concentration in the surrounding extracellular fluid that determines the extent of receptor stimulation. It is of critical importance that the extracellular glutamate concentration is kept low. Firstly, this is required for a high signal to noise (background) ratio. Glutamate must be removed to allow the receptor stimulation to fluctuate with the release of glutamate from the nerve terminals. Secondly, excessive activation of glutamate receptors, e.g. caused by uncontrolled extracellular accumulation of glutamate, can lead to neuronal damage (McBean and Roberts, 1985; Choi and Rothman, 1990; Choi, 1992). Thirdly, for economic reasons it is necessary to conserve the glutamate released. This situation places special emphasis on the mechanisms controlling the extracellular levels of glutamate. 1.1. Uptake of Glutamate from the Extracellular Fluid

As there appear to be no enzymes extracellularly that can metabolize glutamate, glutamate must be removed from the fluid surrounding the receptors by cellular uptake systems (Balcar and Joynston, 1972; Logan and Snyder, 1972; Johnston, 1981). Both "low affinity" and "high affinity" uptake systems have been described (Logan and Snyder, 1971, 1972; Stallcup et al., 1979). 1.1.1. "High Affinity" Uptake

The "high affinity" uptake of glutamate is sodium dependent and is mediated by glutamate transporter proteins located both in the plasma membrane of glial cells (Schousboe, 1981; Wilkin et al., 1982; Danbolt et al., 1992; Levy et al., 1993a) and in neurons releasing glutamate as a transmitter (Divac et al., 1977; Storm-Mathisen and Iversen, 1979; Taxt and Storm-Matbisen, 1984; Garthwaite and Garthwaite,

1988; Gundersen et al., 1993; Nakamura et al., 1993). Whether it is the neurons or the glial cells which have the largest uptake activity is not known. The reported affinities (K,, values) of the glutamate uptake systems vary somewhat between different preparations. Using synaptosomes from rat cerebral cortex, Km values of around 20/~M have usually been reported (Johnston, 1981). Studying salamander (retinal) Miiller cells electrophysiolgically Barbour and collaborators (1991) generally found Km=8-20/~M. In plasma membrane vesicles (Kanner and Sharon, 1978; Roskoski et al., 1981) and reconstituted preparations (Danbolt et al., 1990; Pines and Kanner, 1990) Km values of 2-5 and 1 pM have been reported, respectively. One reason for these differences could be that the different assays preferentially detect different subpopulations of glutamate transporters with different affinities for glutamate. Other reasons could be technical: firstly, traces of endogenous glutamate in a preparation dilute the added labelled glutamate. When not recognized, this will result in an overestimation of Kin. Secondly, in reconstituted liposomes, the uptake may be linear with respect to time at the lower substrate concentrations, but not at the highest substrate concentrations. This may lead to an underestimation of Kin. In fact, using short incubation times (two seconds), the Km for glutamate uptake in reconstituted preparations was found to be in the order of 10 pM (D. Trotti and N.C. Danbolt, unpublished observations) which is in agreement with electrophysiological data. 1.1.2. "Low Affinity" Uptake The "low affinity" uptake exhibits Km values above 500/~M (Johnston, 1981) and, in contrast to the "high affinity" system, is described as sodium-independent (but see Takagaki, 1976) and sensitive to (R)-glutamate (Benjamin and Quastel, 1976). This uptake system has been suggested to supply brain cells with amino acids for metabolic purposes. One might think that this function is redundant and futile as the high affinity systems normally keep the extracellular concentration of these amino acids in the low micromolar range. However, the glutamate concentration in the narrow synaptic clefts during synaptic release is not known, but has been suggested to be in the order of 1 mM (Clements et al., 1992). As low affinity uptake is usually reported to have a higher limax than high affinity uptake, a possible function of the former is to help reduce the peak concentrations of glutamate. The low affinity system is not well characterized (Erecifiska and Silver, 1990) and its existence as a separate entity is controversial (Debler and Lajtha, 1987; Wheeler, 1987; Robinson et al., 1991, 1993). It could, for instance, represent low affinity interactions with transporters for other substances. Mabjeesh and Kanner (1989) claim that part of the low affÉnity GABA transport observed in membrane vesicle preparations is due to an artefact--namely inside-out vesicles. However, the same group (Pines and Kanner, 1990) has reported a sodium and potassium dependent system with relatively low affinity (Kin about 100/~M) for glutamate (in right-side-out plasma membrane vesicles).

Excitatory Amino Acids 1.1.3. Other Uptake Systems In addition to the glutamate uptake systems mentioned above, sodium independent, chloride dependent high affinity glutamate uptake has been described both in brain tissue and in cell cultures (for review see Erecifiska and Silver, 1990; Balcar and Li, 1992). Brose and coworkers (1990) claim that at least part of this transport in brain membranes is due to contamination with mitochondria. They have purified a protein with glutamate binding properties and shown immunocytochemically that this protein is located in the inner mitochondrial membrane of all tissues. However, another sodium independent uptake system for glutamate has been demonstrated in intact cells in culture (Waniewski and Martin, 1984; Cho and Bannai, 1990). Similar systems are described in a variety of cell types (Bannai, 1984; see references in: Cho and Bannai, 1990). This uptake is, in contrast to the sodium dependent uptake, inhibited by cystine, quisqualate, ibotenate, 2-aminoadipate (2-aminohexanedioic acid) and 2-aminopimelate (2-aminoheptanedioic acid), but not be aspartate and kainate (Murphy et aL, 1989; Bannai, 1986; Cho and Bannai, 1990; Miura et al., 1992). The transporter in human fibroblasts is an electroneutral cystine-glutamate exchanger that carries cystine into the cell in exchange for internal glutamate (Bannai, 1986). Since the physiological function of the transporter is to take up cystine at the expense of intracellular glutamate (Bannai, 1986), it may be more appropriate to refer to the carrier as a cystine transporter sensitive to glutamate rather than as a glutamate transporter. The cystine uptake is competitively inhibited by high external glutamate. Inhibition of cystine uptake may result in cell death due to oxidative stress, since cystine is required for glutathione synthesis (Murphy et al., 1989; Cho and Bannai, 1990). The uptake of cystine increases when cells are exposed to hydrogen peroxide (Miura et al., 1992). Oligodendrocytes are specially vulnerable to glutamate induced cystine depletion and this may be a mechanism causing periventricular white matter injuries in premature human infants (Oka et al., 1993). Sodium dependent high affinity heteroexchange mechanisms for glutamate and ascorbate (Griinewald, 1993) and for glutamate and GABA (Bonanno et al., 1993) have also been reported. Thus, a variety of both sodium dependent and independent glutamate transport systems exists, many of which are poorly characterized. 1.2. Glutamate Uptake in Non-neuronal Tissues Uptake systems for dicarboxylic amino acids are widely distributed in the body (Lerner, 1987; Christensen, 1984, 1990). Sodium and potassium dependent (S)-glutamate transport sensitive to (R)-aspartate and (S)-aspartate, but not to (R)-glutamate has been reported in liver (Sips et al., 1982), placenta (Moe and Smith, 1989; Hoeltzli et al., 1990), small intestine (Wingrove and Kimmich, 1988; Kanai and Hediger, 1992), kidney (Sacktor et al., 1981) and cultured fibroblasts (Balcar, 1992). One of the cloned sodium dependent high affinity glutamate transporters (Kanai and Hediger, 1992) was isolated from a rabbit

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small intestine cDNA library and in situ hybridization and Northern blotting suggest that it is expressed not only in the epithelium of the small intestine, but also in kidney, liver, and brain. In addition to the sodium dependent ,.ansporters, several sodium-independent systems are also described (Christensen, 1984). A system of nomenclature for membrane transport systems has been suggested (Bannai et al., 1984a, b). The one letter code proposed for dicarboxylic amino acid transporters is X. According to this scheme sodium dependent transport systems should be referred to by capital letters, while sodium independent systems should be referred to by lower case letters. The charge of the transported amino acid is indicated by + or - as superscript. The letters A, G or C as subscript indicate acceptance of aspartate, glutamate or cystine as preferred substrates. The brain sodium dependent high affinity transporter would thus be referred to as X-At, while the sodium independent cystine-glutamate exchanger would be called x- c (or

X-CG ). 1.3. Glutamate Uptake in Other Organisms Bacteria and other microorganisms express a variety of glutamate uptake mechanisms. In E. coli five different uptake systems have been identified, including cotransporters coupled to either H + or Na + (Schellenberg and Furlong, 1977; Deguchi et al., 1990; Wallace et al., 1990). Glutamate and dicarboxylate transporters have been described in other bacteria as well (e.g. Ronson et al., 1984; De Vrij et al., 1989; Ruhrmann and Kr~mer, 1992; Holtom et aL, 1993; Watson et al., 1993) and in fungi (Gupta et al., 1992; Rezhovfi et al., 1992). Corynebacterium glutamicum is best known for its ability to secrete large amounts of amino acids and is used for industrial amino acid production (Gutmann et al., 1992). Oocytes from the frog Xenopus laevis have also been reported to accumulate glutamate (Steffgen et al., 1991). Glutamate acts as a transmitter in the neuromuscular junction in arthropods (Gerschenfeld, 1973; Usherwood, 1981). Since the motor endplates are not sealed from the blood by glial cells (Osborne, 1975), the glutamate concentration in the haemolymph (insect equivalent of blood plasma) has to be kept low. This is achieved by Na+-dependent medium-affinity uptake systems (McLean and Caveney, 1993; Tomlin et al., 1993). The substrate selectivity of this uptake is similar to the N a + + K+-coupled high affinity uptake in the mammalian brain with the notable exception that (S)-trans-pyrrolidine-2,4-dicarboxylic acid is a poor inhibitor. 1.4. Intracellular Glutamate Uptake Once inside the cell, glutamate undergoes further redistribution (Erecifiska and Silver, 1990; Nicholls, 1993). It may be taken up by mitochondria (Dennis et aL, 1976; Minn and Gayet, 1977) either by a glutamate/OH- antiporter (which is formally equivalent to a glutamate/H + symporter) or by the glutamate/aspartate exchanger. The latter carrier is the best characterized (Krfimer and Palmieri, 1989) and has been purified in an active form using reconstitution of transport as an assay (Kr~imer et al.,

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1986). A majority of the enzymes for which glutamate is a substrate are located in mitochondria. In glutamatergic nerve terminals, glutamate is carried into synaptic vesicles for use as a transmitter. The vesicles accumulate glutamate by means of a glutamate transporter that is very different from those of the plasma membrane. The vesicular uptake is independent of sodium and potassium, has lower affinity (Km about 1 mM), is highly selective for (S)-glutamate, does not interact with aspartate and requires low concentrations of chloride (Naito and Ueda, 1983; Fykse et al., 1992). The chloride binding site is distinct from the substrate binding site and regulates transport activity. It is located on the cytoplasmatic side and is sensitive to 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) (Hartinger and Jahn, 1993). The vacuolar H+-ATPase, located in the vesicular membrane, pumps H ÷ into the vesicles. The obtained internal positive membrane potential drives the vesicular glutamate transporter (Naito and Ueda, 1985; Maycox et al., 1988; Moriyama et al., 1990; Tabb et al., 1992). Synaptic vesicles isolated from rat brain have been shown to contain high levels of glutamate (Riveros et al., 1986; Burger et al., 1989). In the absence of Mg 2+ and ATP to fuel the proton pump, glutamate will leak out of the vesicles down the concentration gradient by reversal of the vesicular glutamate transporter.

1.5. Glutamate-Glutamine Cycle The glutamate taken up by glial cells is rapidly, but ATP dependently, metabolized to glutamine by glutamine synthetase. Glutamine is exported from the glial cells to the extracellular fluid and is taken up by nerve endings which convert glutamine back to glutamate. Brain uptake of glutamine has been reviewed by Erecifiska and Silver (1990). Glutamine is a substrate for at least three different transport systems in the brain: the A-system (alanine-preferring), the L-system (leucine-preferring) and the ASC-system (alanine-, serine- and cysteine-preferring). A human brain ASC-transporter (Arriza et al., 1993; Shafqat et al., 1993) has recently been cloned, on the basis of sequence homology, to glutamate transporters, but glutamine is not a substrate for these.

2. THE BRAIN PLASMA MEMBRANE H I G H AFFINITY S O D I U M DEPENDENT GLUTAMATE TRANSPORTER Since glutamate is both highly toxic to neurons expressing glutamate receptors and very abundant in the brain, it is logical that the brain has formidable protective mechanisms against it. Under normal conditions intact brain tissue is remarkably resistant to *Synaptic plasma membrane vesiclesmust not be confused with synaptic vesicles. Synaptic plasma membrane vesicles are prepared by osmotic rupture of a synaptosome preparation and represents microsacks (saccules) of plasma membranes without cytoplasm. Synaptic vesicles, on the other hand, are the small vesicles storing the transmitter inside the nerve endings.

glutamate toxicity due to the actions of the plasma membrane high affinity glutamate transporters. From here on, the "brain plasma membrane high affinity sodium dependent glutamate transporter" will be referred to simply as the "glutamate transporter" as this is the quantitatively dominating glutamate uptake system in the brain. The importance of the glutamate uptake is illustrated by two recent publications. (1) Garthwaite and collaborators (1992) incubated slices of rat cerebellum with various concentrations of glutamate. The cells on the surface of the slices were highly vulnerable to glutamate while more than 30 times higher glutamate concentration was needed to damage cells deeper in the tissue slices. Even prolonged incubation with glutamate did not increase the sensitivity. The authors suggest that the lower sensitivity of the neurons inside the slices is chiefly due to the actions of the glutamate uptake system preventing glutamate from reaching the neurons. (2) Rosenberg et al. (1992) have studied neurons (from rat embryonic cerebral cortex) cultured together with either a few or many astrocytes. Fifty per cent of the neurons in astrocyte-poor cultures were killed after a 30 min exposure to 4/~M glutamate (followed by a 20-24 hr incubation in normal medium). In contrast, 205/~M glutamate was required to kill the same percentage of neurons in astrocyte-rich cultures. When glutamate uptake was blocked by removing sodium, the astrocyte-rich cultures became as sensitivie to glutamate as the astrocyte-poor cultures. N M D A and the (R)-enantiomer of glutamate both activate glutamate receptors, but are not taken up. The astrocyte-rich and astrocyte-poor cultures were equally sensitive to these drugs. Thus, the transport systems obscure the neurotoxic potential of glutamate. Previously Frandsen and Schousboe (1990) have shown that as little as 1/~M glutamate is sufficient to kill 50% of cerebral cortex neurons in culture, provided the uptake is blocked by (S)-aspartate-fl-hydroxamate.

2.1. The Mechanism of Transport Most of what is known about the transport mechanism and pharmacology of these transport systems originates from studies on crude preparations such as brain slices, crude homogenates, synaptosomes (pinched off nerve endings), cell cultures and plasma membrane vesicles.* Most of these results have later been confirmed with more refined techniques such as reconstitution of transport activity in artificial membranes (e.g. Danbolt et al., 1990; Pines and Kanner, 1990) or by electrophysiological techniques (e.g. Attwell et al., 1991; Barbour et al., 1991; Eliasof and Werblin, 1993; Bouvier et al., 1992). Potassium is required for net transport (Kanner and Sharon, 1978; Sarantis and Attwell, 1990). In the proposed model for the mechanism of glutamate transport (Kanner and Shuldiner, 1987), the transporter is described as a shuttle that either has to transport potassium, or sodium and glutamate. When sodium/glutamate is transported into the cell, either potassium or sodium/glutamate have to be transported out before the transporter again can transport into the cell. Thus, in the absence of potassium, net transport cannot occur, because the transporter has to

Excitatory Amino Acids carry sodium/glutamate out again. In the presence of internal potassium, net transport is possible because the transporter takes potassium out instead of sodium/glutamate. Thus it exchanges external glutamate/sodium with internal potassium. The transporters utilize the ion gradients of both sodium and potassium as energy sources for the transport process by allowing the influx of sodium ions and the efflux of potassium ions. As the transport is stimulated by an internally negative membrane potential, the number of sodium ions has to be such that net positive charge is moving inwards together with glutamate (Kanner and Schuldiner, 1987; Nicholls and Attwell, 1990). It has recently been shown that the glutamate transporter in salamander retinal glial cells also transports O H - ions (or HCO 3- ions), suggesting a carrier cycle in which two Na ÷ ions accompany each glutamate anion into the cell, while one K ÷ ion and one O H - ion (or HCO3- ion) are transported out (Bouvier et al., 1992). Rubidium (Kanner and Schuldiner, 1987) and caesium (Danbolt et al., 1990; Barbour et al., 1991) can substitute for potassium. The action of sodium appears to be specific in that so far no ion has been identified which can replace it. The stoichiometry of ion coupled glutamate transport is not a trivial parameter as the energy consumption of transport, the concentrative capacity of the transporter and the sensitivity of the transport process to changes in the ion gradients all depend on it. As explained above, the transporter mediates a reversible process and the direction of transport (efflux or influx) depends on the driving forces. It is not known whether the exact mechanism of transport is the same in all cells expressing sodium dependent high affinity glutamate transport. In view of the existence of several different glutamate transporters (see below), studies done on different preparations with different techniques may not be directly comparable as different glutamate transporter subtypes may have been detected.

2.2. Substrate Selectivity The transport system does not have an absolute specificity for glutamate, as other compounds can be transported and competitively inhibit glutamate transport. Generally, interacting compounds share the common feature (Bridges et al., 1993) of being ct-amino-dicarboxylic acids where the acid groups are separated by two to three methylene groups. However, fl-glutamate (amino group at carbon-3) is also accepted (Balcar and Johnston, 1972). The distal COOH can be derivatized to hydroxamate or replaced by a sulphonate group as in cysteate (Roberts and Watkins, 1975). A hydroxyl group attached to carbon-3 in aspartate is accepted (Balcar et al., 1977), but 3-hydroxyglutamate is not (Balcar and Johnston,

*(R)-Cysteate is the same as L-cysteate. All L-amino acids occurring in proteins are referred to as (S)-amino acids in the (R,S) nomenclature system except L-cysteinewhich is referred to as (R)-cysteine.

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1972). The amino group may be part of a cyclic structure, but no other modifications of this are tolerated. The proximal carboxyl group cannot be altered in any way. It is interesting to note that (R)-glutamate is a poor substrate for the transport system while (S)-glutamate, (S)-aspartate, (R)-aspartate and (R)-cysteate* are taken up with roughly the same high affinity. Kainic acid (Johnston et al., 1979) and derivatives (dihydrokainate, acromelic acid and domoic acid) are not transported, but competitively inhibit glutamate uptake. Some sulphur-containing amino acids [(R)-cysteine sulphinate, (R)-cysteate, (S)-homocysteine sulphinate, (S)-homocysteate and (S)-sulpho-(R)-cysteine] may also have transmitter function (Griffiths, 1993; Cu6nod et al., 1993). These amino acids are probably taken up by the glutamate transporter (Wilson and Pastuszko, 1986; Griffiths, 1993). (R)-Aspartate has been used as a metabolically inert substrate (Davies and Johnston, 1976; Takagaki, 1978), which has proved useful particularly for tracing putative glutamatergic neurons by axonal transport of (R)-[3H]aspartate (e.g. Streit, 1980; Baughman and Gilbert, 1980; Storm-Mathisen and Wold, 1981; Fischer et al., 1986; Kisvfirday et al., 1989; Behzadi et al., 1990). Glutamate (2-aminopentanedioic acid) is a flexible molecule and there are several possible conformations that are only minimally less favourable energetically at room temperature than the lowest energy conformation. Bridges et al. (1991) have synthesized several conformationally restricted glutamate analogues and found one that bind with high affinity to the transporter without affecting glutamate receptors. This compound, (S)-trans-pyrrolidine-2,4-dicarboxylic acid, closely resembles one conformation of glutamate. Imagine the structure of the molecule in the following way: take (S)-glutamate and link the or-amino nitrogen via an extra carbon to carbon-4 in the glutamate molecule. A five-membered ring structure is obtained consisting of the three middle carbon atoms in glutamate, the amino nitrogen and the added carbon atom. Directly attached to the ring are the two carboxylic groups. The distal carboxylic group can be oriented in two directions, either on the same side of the ring as the proximal group or on the opposite side. The latter is the compound interacting with the transporter, i.e. the enantiomer in which the carboxylic groups are as far away from each other as possible, corresponding to the extended conformation of (S)-glutamate. Another series of conformationally restricted (S)-glutamate analogues have been synthesized (Ohfune et al., 1993). These are (S)-2-(carboxycyclopropyl)glycines in which the cyclopropyl group fixes the glutamate chain in either an extended or a folded form. Of the four compounds, one is a metabotropic receptor agonist (CCG-I), one is a N M D A receptor agonist (CCG-IV) and the two others (CCG-II/III) are uptake inhibitors (Ohfune et al., 1993; Robinson et al., 1993). A third series of conformationally restricted (S)-glutamate analogues are the 1-aminocyclobutanedicarboxylic acids (Allan et al., 1990; Fletcher and Johnston, 1991).

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All the glutamate analogues that bind to the transporter are either themselves transported by both glial cells and neurons (e.g. aspartate, hydroxyaspartate, pyrrolidine dicarboxylate) or have a much lower affinity for the transporter than glutamate (dihydrokainate). The concentration of glutamate in the synaptic cleft after synaptic release may be in the order of 1 mM (Clements et al,, 1992). To efficiently inhibit glutamate uptake in the presence of 1 mM glutamate by competitive inhibitors, one would need an inhibitor with a higher affinity for the transporter than glutamate. Otherwise exceedingly high concentrations would have to be used. Thus, in tissue slices it may not be possible to bring any currently available analogue through the narrow intercellular spaces between glial cells and neurons, into the synaptic cleft, in sufficient concentrations to block the uptake from the cleft. A non-transported analogue that binds with high affinity is, therefore, badly needed. The word "blocker" may be misleading when referring to inhibitors that can be transported as they do not prevent the molecular transport mechanism from working and may induce release of glutamate and aspartate by heteroexchange. 2.4. Purification of a Glutamate Transporter A glutamate transporter has been purified in an active form from rat brain by employing reconstitution of transport as the assay (Danbolt et al., 1990). The purification is based on solubilization of brain membranes with a detergent (CHAPS: Hjelmeland, 1980) and fractionation by affinity chromatography on a lectin (wheat germ agglutinin), followed by chromatography on hydroxylapatite and DEAE-cellulose. A 30-fold increase in specific activity was obtained. Due to a considerable inactivation, the purification ratio was roughly estimated to be about 100-fold. SDS-polyacrylamide electrophoresis gels of the most pure fraction showed one dominant broad band at around 73 kDa. This purification method has also been used to purify a glutamate transporter from pig brain (L6pezCorcuera et al., 1993). It is feasible to assume that the purified protein, which appears to be the same as GLT-1 (Pines et al., 1992; Levy et al., 1993a), is quite pure for two reasons. Firstly, the antibodies raised against the purified protein have been successfully used to clone a glutamate transporter (see section 2.6. below). Secondly, these antibodies give the same labelling of immunoblots and of tissue sections as a monoclonal antibody (Levy et al., 1993a) and antibodies against synthetic peptides corresponding to different parts of this cloned transporter (Lehre et al., 1993, 1995). The immunoblot labelling pattern closely resembles that of the silver stained electrophoresis gels of the purified protein (Danbolt et al., 1992). Consequently, the width of the band on the electrophoresis gels is due to the molecular properties of this class of transporters, rather than to incomplete purification. A number of proteins (created for instance by variable splicing of mRNA) sharing some epitopes would explain the broad electrophorectic bands (see Section 2.6 below).

If the protein is pure, this implies that this particular protein corresponds to about 1% of all brain membrane proteins. It is legitimate to ask whether this number is too high. However, in view of the high brain content of glutamate, the high toxicity of glutamate and the high glutamate transport activity, one would expect glutamate transporter proteins to be abundant. With specific antibodies, it will now be possible to determine if the above estimate is correct. The GLAST-1 protein (Storck et al., 1992) is separated (Lehre et al., 1995) from the GLT-1 protein during the second step of the purification procedure (Danbolt et al., 1990). Antibodies raised against a peptide corresponding to amino acid residues 522-541 in the transporter GLAST-1 (Storck et al., 1992) also label a broad band just like GLT-1, but with about 5-10kDa (cerebellum-cerebrum) lower apparent molecular mass (Lehre et al., 1993). 2.5. Pharmacological Demonstration of Glutamate Transporter Heterogeneity Heterogeneity of glutamate uptake has been expected from pharmacological studies (Schousboe and Divac, 1979; Ferkany and Coyle, 1986; Robinson et al., 1991, 1993; Fletcher and Johnston, 1991; Balcar and Li, 1992; Rauen et al., 1992). The uptake in cerebellum is different from that in the rest of the brain. It is insensitive to dihydrokainate and more sensitive to (S)-a-aminoadipate than the uptake in forebrain (Robinson et al., 1991; striatum (Ferkany and Coyle, 1986) and cerebral cortex and hippocampus (Fletcher and Johnston, 1991). Inhibition of the cerebellar glutamate uptake by kainate is best fit by a two site model (Ferkany and Coyle, 1986; Robinson et al., 1993). Furthermore, the uptake of glutamate in forebrain is also pharmacologically heterogeneous (Fletcher and Johnston, 1991; Robinson et al., 1993). Heterogeneity of uptake is also demonstrated immunocytochemically: antibodies against a glutamate transporter labels only gial cells (Danbolt et al., 1992; Hees et al., 1992; Levy et al., 1993a; Lehre et al., 1993, 1995) although nerve terminals also have glutamate uptake (Gundersen et al., 1993). 2.6. Cloning of Three Glutamate Transporters Three different groups have, simultaneously, but independently of each other cloned three different eukaryotic glutamate transporters: GLAST-I (Storck et al., 1992), GLT-1 (Pines et al., 1992) and EAAC-1 (Kainai and Hedinger, 1992). (The protein cloned by Tanaka (1993) is identical to GLAST-1 with the exception of amino acid residue 302.) The three proteins have about 55% amino acid identity with each other and no significant primary structure homology with any other known eukaryotic protein, but about 25-30% amino acid identity with a bacterial dicarboxylic acid transporter from Rhizobium meliloti (Engelke et al., 1989) and proton coupled glutamate transporters from E. coli (Wallace et al., 1990; Tolner et al., 1992), Bacillus stearotherrnophilus and Bacillus caldotenax. Thus, these proteins belong to a "new" protein family (Amara, 1992; Kanner, 1993; Taylor, 1993). There is no significant sequence identity with the N a + + C1--coupled transporters (see Section 2.8)

Excitatory Amino Acids or with the Na+-coupled glutamate transporter from E. coli (Deguchi et al., 1990). Recently, a human sodium dependent neutral amino acid transporter (for alanine, serine and cysteine) with structural similarity to the glutamate transporters have been cloned (Arriza et al., 1993; Shafqat et al., 1993). These transporters show 34-39% identity to the above eukaryotic glutamate transporters. It is noteworthy that a seven amino acid sequence A-A-I(V/L)-F-I-A-Q is conserved between the three eukaryotic and the two bacterial glutamate transporters. In view of the properties of the transporters, it may be speculated that this sequence could be involved in dicarboxylate handling. However, the lack of charged groups to attract the dicarboxylic acids speaks against a direct role in binding. The human equivalents of GLAST-1, GLT-1 and EAAC-1 display, when expressed in transfected COS-7 ceils, Kin-values for glutamate of 57, 101 and 70/tM. All three subtypes were specifically inhibited by (S)-trans-pyrrolidine-2,4-dicarboxylic acid and 3-hydroxyaspartate (Fairman et al., 1993). A variant of GLT consisting of 453 amino acids has been described in the rat (Roginski et al., 1993). 2.6.1. G L A S T - I Storck et aL (1992) were purifying a glactosyltransferase from rat brain and observed that a 66 kDa hydrophobic glycoprotein copurified with this protein. The purified protein was subjected to limited proteolysis, some amino acid sequences were obtained and used for synthesizing degenerate oligonucleotide probes for screening of a rat brain eDNA library. A 3 kb clone was isolated and sequenced. The sequence had considerable amino acid identity with several bacterial glutamate and dicarboxylic amino acid transporters. When GLAST-1 (Storck et al., 1992) is expressed in Xenopus laevis oocytes, electrogenic sodium dependent uptake is observed (Klrckner et al., 1993) suggesting a stoichiometry of 3 Na ÷, 1 glutamate and 1 K ÷ ions. The transport was not changed when the external pH was reduced from 7.4 to 6.0. This finding argues against a glutamate/proton cotransport. The /(ms for glutamate and Na ÷ were 11 and 41/~M, respectively. 2.6.2. G L T - I Pines et al. (1992) used the antibodies (Danbolt et al., 1992) raised against a purified glutamate transporter (Danbolt et al., 1990) to isolate eDNA clones by immunoscreening of a rat brain expression library constructed in the Lamda Zap vector. DNA sequencing of the isolated clones identified several that contained open reading frames with considerable homology to a proton coupled glutamate transporter from E. coli 0,Vailace et al., 1990; Tolner et al., 1992) and a dicarboxylic acid transporter from Rhizobium meliloti (Engelke et aL, 1989). Lysates of E. coli transformed by Bluescript plasmid derivatives of the identified Lamda Zap clones were immunoblotted. The antibodies identified a 25 kDa protein, which is significantly shorter than the deglycosylated purified

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transporter (Danbolt et al., 1992). The 25 kDa protein proved to be a fusion protein of LacZ coupled to the carboxy-terminal part of the glutamate transporter. The first part of the coding region of this immunopositive clone was used as a probe to screen another library with longer inserts. Some new clones were isolated and one of these directed the expression of sodium and potassium coupled glutamate transport activity in HeLa cells infected with a recombinant vaccinia virus containing the bacteriophage T7 RNA polymerase (Fuerst et al., 1986, 1987; Blakely et al., 1991b). This clone was designated GLT-1 and sequenced. The eDNA sequence predicts a protein of 573 amino acids and a molecular mass of 64 kDa which is consistent with the finding (Danbolt et al., 1992) that deglycosylation of the purified 73 kDa protein reduces its molecular mass by about 10 kDa. Two sequencing errors have been discovered after the publication and have resulted in a correction of the sequence of amino acid residue 260--289 (Kanner, 1993). It is assumed that the first ATG of the open reading frame is the translation initiation site because of similarity of the upstream sequence to the consensus sequence of Kozak (1987). Antibodies raised against a synthetic peptide corresponding to amino acid residue 2-11 recognize the GLT-1 transporter. This confirms the assumption that the translation starts at the first ATG (Lehre et al., 1995). The gene encoding GLT has been mapped to the short arm of chromosome 11 (Krishnan et al., 1993). It is interesting that the m R N A encoding GLT is as long as 11 kb (Pines et al., 1992; Roginski et al., 1993). 2.6.3. E A A C - I Kanai and Hediger (1992) isolated a eDNA clone encoding a glutamate transporter from a rabbit jejunum by Xenopus oocyte expression cloning using a technique that had led to the expression cloning of a Na+-dependent glucose transporter (Hediger et al., 1987). The eDNA sequence contains an open reading frame coding for a protein of 524 amino acids and a relative molecular mass of 57 kDa. The rat brain equivalent is 89.9% identical and 523 amino acids long (Kanai et al., 1993a). 2.7. Topology of the Cloned Glutamate Transporters Hydrophobicity analysis based on the primary structure of GLT-1 predicts eight or possibly nine transmembrane or-helices, whereas six (Storck et al., 1992) and 10 (Kanai and Hediger, 1992) transmembrane segments are predicted for GLAST-1 and EAAC-1, respectively. Twelve membrane-spanning domains are predicted for the two bacterial transporters GLTP (Tolner et al., 1992) and DCTA (Engelke et al., 1989). However, comparison of the hydropathy plots of these proteins reveals a pattern of hydrophobicity that is common to all of them. There may be some transmembrane ~t-helices at very similar positions at the amino-terminal part of the transporters, while there is more ambiguity at the carboxy-terminal side (Kanai et al., 1993b; Kanner, 1993). Structural analysis with other methods is needed to determine the folding of the protein.

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It has been shown by electron microscopy that the amino- and carboxy-terminal of both GLT-I and GLAST-1 are localized on the intracellular side of the plasma membrane indicating an even number of transmembrane domains (Danbolt et al., 1992; Levy et al., 1993a; Lehre et al., 1993, 1995). The finding that serine-ll3 in GLT-1 represents a physiological phosphorylation site (Casado et al., 1993) implies that also this part of the transporter has to be intracellular. 2.8. Neurotransmitter Transporter Families

2.8.1. Transporters in the Plasma Membrane Two distinct families of neurotransmitter transporters have now been identified: the sodium and potassium coupled (glutamate) transporter family (see above) and the sodium and chloride coupled (GABA) transporter family. All the transporters in the latter family are assumed to have 12 transmembrane domains. The molecular characterization of these proteins began with the purification (Radian and Kanner, 1985; Radian et al., 1986), amino acid sequencing and cloning of a GABA transporter from rat brain (Guastella et al., 1990). With the expression cloning of a human noradrenaline transporter (Pacholczyk et al., 1991), it was clear that a family of neurotransmitter carriers exists. Based on sequence homology with these two transporters several other sodium and chloride coupled transporters have been cloned. Several review articles have already appeared (Amara, 1992; Schloss et al., 1992; Uhl, 1992; Uhl and Hartig, 1992; Amara and Kuhar, 1993; Clark and Amara, 1993; Kanai et al., 1993b). While these transporters show great homology within the family, they show no primary structure homology with the glutamate transporter family. The cloned Na + + C1--coupled transporters include several transporters for GABA and for taurine and fl-alanine (Nelson et al., 1990; Borden et al., 1992; Clark et al., 1992; Liu et al., 1992a, 1993a; Lrpez-Corcuera et al., 1992; Smith et al., 1992b; Uchida et al., 1992; Yamauchi et al., 1992; Jhiang et al., 1993), glycine transporters (Guastella et al., 1992; Liu et al., 1992b, 1993b; Smith et al., 1992a; Borowsky et al., 1993), a proline transporters (Fremeau et al., 1992), a choline transporter (Mayser et al., 1992), several biogenic amine transporters (Blakely et al., 1991a, 1993; Giros et al., 1991; Hoffman et al., 1991 ; Kilty et al., 1991; Shimada et al., 1991; Usdin et al., 1991; Ramamoorthy et al., 1993; Lesch et al., 1993) and some molecules with unknown function resembling transporters (Mayser et al., 1991; Gingrich et al., 1992; Uhl et al., 1992; Liu et al., 1993c). 2.8.2. Transporters in Synaptic Vesicles In addition to the transporters located in the plasma membrane, there are also transporters in the synaptic vesicles. Five different transport systems have been described: one transporter for acetylcholine, one for glutamate (see Section 1.4 above), one for both GABA and glycine, one for ATP and one for biogenic amines (serotonin, catecholamines and histamine). Only the latter is cloned (Erickson et al., 1992; Liu et al., 1992c; Surratt et al., 1993) and it shows no significant primary

structure homology with the neurotransmitter transporters in the plasma membrane. 2.9. Anatomical Localization

2.9.1. Earlier Studies Glutamate uptake has been determined by uptake into synaptosomes prepared from various brain regions. The technique has proven very useful for biochemical studies, but offers tow anatomical resolution. Higher resolution has been obtained by autoradiographic detection of uptake of labelled amino acids in tissue slices (e.g. Storm-Mathisen and Iversen, 1979; Wilkin et al., 1982; Taxt and Storm-Mathisen, 1984; Garthwaite and Garthwaite, 1985) or after axonal transport of (R)-[3H]aspartate following injection of the tracer in vivo (Braughman and Gilbert, 1980; Streit, 1980; Storm-Mathisen and Wold, 1981; Fischer et al., 1986; Kisv~irday et al., 1989; Behzadi et al., 1990). [(R)-Aspartate is a metabolically almost inert probe for the excitatory amino acids uptake system (Davies and Johnston, 1976; Takagaki, 1978).] However, comparison of regions and quantification were difficult due to uneven penetration of the labelled substance into slices and at injection sites. To obtain comparable conditions for uptake among different locations it seemed essential to be able to work with thinner slices of CNS tissue. That is why sodium-dependent "binding" of glutamate to cryostat sections seemed to be an attractive method. Unfortunately, there are also problems with this method (see Section 3 below). It is still believed that nerve terminals releasing an amino acid as a transmitter would have re-uptake mechanisms for that particular transmitter. Glutamate uptake sites on nerve terminals have therefore been used as markers for glutamatergic neurons, and have proven useful for elucidating the connections of glutamatergic neurons (for review see: Fonnum, 1984; Ottersen and Storm-Mathisen, 1984). 2.9.2. Localization o f the Cloned Glutamate Transporters

The glutamate transporter GLT-I (Pines et al., 1992) has been identified immunocytochemically in the brain by polyclonal antibodies (Danbolt et al., 1992, 1993), a rnonoclonal antibody recognizing GLT-1 amino acid residues 518-525 (Levy et al., 1993a) and antibodies against peptides corresponding to GLT-1 amino acid residues 12-26 and 493-508 (Lehre et al., 1993, 1995). All the antibodies give the same labelling of both tissue sections and immunoblots. The GLT- 1 appears to be exclusively localized to glial cells and is preferentially expressed in the forebrain, the hippocampus, cerebral cortex and striatum in particular, but is present in lower amounts all over the brain (Lehre et al., 1993, 1995). The regional labelling pattern has been confirmed with in situ hydridization histochemistry except that GLT-I mRNA was detected in the hippocampal CA3 pyramidal cells (Torp et al., 1994). The reason for this disagreement is not known. Immunocytochemically these cells and their terminals in CA1 and CA3 are negative.

Excitatory Amino Acids The glutamate transporter GLAST-1 (Storck et al., 1992) has also been shown immunocytochemically (by an antibody raised against a peptide corresponding to GLAST-I amino acid residues 522-541) to be exclusively localized to glial cells. The protein is preferentially expressed in the molecular layer of the cerebellum, but this transporter is also present throughout the brain (Lehre et al., 1993, 1995). The same regional labelling pattern is also seen with in situ hybridization histochemistry (Storck et al., 1992; Torp et al., 1994). Studies with gold-labelled antibodies rule out any nerve terminal labelling and show that both GLT-1 and GLAST-I are localized to glial cell plasma membranes (Chaudhry et al., 1993). Both proteins appear to be present in the same cells, although in different proportions (Chaudhry et al., 1993; Lehre et al., 1993, 1995). It is not known whether the trans-porters exist as monomers or oligomers. A heterooligomeric structure, like in the case of glutamate receptors, could imply yet another level of heterogeneity. Little is known about the localization of the glutamate transporter EAAC-1. It has been localized by in situ hybridization histochemistry by probes corresponding to nucleotides 646-2783 (Kanai and Hediger, 1992). This region encompasses most of the coding sequence plus a large part of the 5'-end non-coding region. In view of the existence of a whole family of closely related proteins, the possibility exists that the labelling obtained represents the sum of the labelling of several different m R N A species. When one mRNA member of a large family is to be localized, one should preferentially use several different probes corresponding to different parts of the sequence and see if all of them give the same labelling. Labelling of pyramidal cells in hippocampus and granular cells in cerebellum suggest neuronal localization, but this conclusion needs to be confirmed. EAAC-1 has not been localized immunocytochemically.

2.10. Immunoprecipitation and Reconstitution Experiments The apparently exclusive glial localization was surprising especially in view of the amost complete immunoprecipitation of reconstitutable glutamate transport activity (Danbolt et al., 1992; L. M. Levy and N. C. Danbolt, unpublished observations). Either GLT-1 has to be quantitatively dominating in brain or the reconstitution assay preferentially detects the GLT-1 transporter. The latter interpretation is in line with the fact that no glutamate transport activity could be reconstituted with this method (Danbolt et al., 1990) from kidney brush border membranes (B. I. Kanner, unpublished observations). However, as both a glycine transporters (Lrpez-Corcuera et al., 1989, 1991) and a GABA transporter (Radian and Kanner, 1985) are reconstituted using virtually the same procedure, selective reconstitution of one glutamate transporter subtype would be surprising. A low density of glutamate transporters on nerve terminals seems unlikely. The electrogenic glutamate transport capacity of synaptosomes is so high that the application of 10-100/~i glutamate elicits a transporter mediated depolarization of the synaptosomes.

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The depolarization, which is glutamate receptor independent, is sufficient to elicit action potentials and calcium dependent exocytotic glutamate release (McMahon et al., 1989).

2.11. Regulation of Glutamate Uptake It is now clear that glutamate transporter proteins are regulated. Uptake of glutamate increases region-selectively in the brains of rats stressed by restraint in narrow cylinders (Gliad et al., 1990). In vivo electrical stimulation (for 10 min) of frontal cortex has been reported to increase the glutamate uptake in rat striatum by increasing the affinity of the transporter for glutamate (Nieoullon et al., 1983). The animals were killed for uptake measurements 20 min after cessation of electrical stimulation. The increase from basel level can be inhibited by dopaminergic activity (Kerkerian et al., 1987). The existence of putative phosphorylation sites (Pines et al., 1992) indicates that one glial glutamate transporter may be regulated by protein kinases and phosphatases. The finding that glutamate transport activity (V~x, but not Kin) is increased in cultured glial cells (but not neurons) after incubation of the cells with phorbol esters (protein kinase C activators) (Casado et al., 1991), suggested that the putative phosphorylation sites are physiolgically relevant. It has recently been shown that the activity of one glutamate transporter (GLT-1) is modulated by protein kinase C catalyzed phosphorylation of serine residue 113 (Casado et al., 1993). Furthermore, observations indicate that neurons release factors inducing glutamate transport activity (Drejer et al., 1983; Voisin et al., 1993) as well as ones inducing GABA transport activity (Nissen et al., 1992) in glial cells. This is in agreement with the recent finding that two astrocytic glutamate transporter proteins are downregulated in striatum after decortication (Levy et al., 1993b). Using an expression cloning technique, mRNA inducing GABA and glutamate transport activity in Xenopus laevis oocytes was demonstrated (Blakely et al., 1988), but this did not lead to the cloning of these transporters. Since Xenopus oocytes have endogenous glutamate transport activity (Steffgen et al., 1991), one may speculate that this mRNA actually encoded stimulatory factors, rather than the transporters.

2.12. Pathological Importance A substantial amount of evidence supports the idea that "excitotoxic" loss of nerve cells is a pathogenetic mechanism in many disorders of the nervous system (Olney, 1989, 1990; Choi, 1992; Meldrum, 1993; Whetsell and Shapira, 1993). It has been clearly demonstrated that excessive activation of glutamate receptors can cause neuronal death. The neurotoxicity appears to be more a consequence of intracellular accumulation of free Ca 2÷ than excitation per se (Choi and Hartley, 1993). Stimulation of the ionotrophic glutamate receptors has been shown to enhance rates of generation of reactive oxygen species (Bondy and Lee, 1993; Lafon-Cazel et al., 1993). One should not be surprised to find that excitotoxic mechanisms are important in a variety of diseases. As mentioned earlier, almost all neurons have glutamate

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receptors and the brain contains large amounts of excitatory amino acids. Any pathological process that causes, for any reason, an increased extracellular concentration of these amino acids would lead to excitotoxic damage. Thus, excitotoxicity could be a final pathway of neuronal death in several diseases. Some pathological conditions may be associated with a hypersensitivity to glutamate due to impaired cellular energy metabolism or possession of abnormal glutamate receptor subtypes (Albin and Greenamyre, 1992; Beal et al,, 1993). Simpson and Isacson (1993) report that mitochondrial impairment, induced by intraperitoneal administration of 3-nitropropionic acid, makes striatal neurons more vulnerable to NMDA-toxicity. The recent finding by Rosen et al. (1993) that a familial variant of amyotrophic lateral sclerosis (ALS) is linked to defects in the gene for cytosolic superoxide dismutase (SOD), is in line with this way of thinking. As depolarization of cells leads to formation of superoxide, via Ca 2÷ influx and activation of xanthine oxidase, reduced SOD activity renders the cells vulnerable to damage by physiological glutamate receptor stimulation (McNamara and Fridovich, 1993). Volterra's group report (1994) that glutamate uptake is inhibited by oxygen free radicals. This mechanism may represent another link between radical toxicity and excitotoxicity. 2.13. How Glutamate Transporters may be Involved in Pathology 2.13.1. Failure o f Transport If the glutamate transporters should, for some reason, stop working, there would be an extracellular build up of glutamate and aspartate causing neuronal excitation and leading to further increase in glutamate release. This would result in neuronal death. Reduced uptake of glutamate (as measured by uptake in synaptosomes) has been observed in autopsy material from patients with amyotrophic lateral sclerosis (Rothstein et al., 1992). Synaptosomal glutamate uptake declines progressively in the spinal cord of a mutant mouse with motor neuron disease (Battaglioli et al., 1993). Cerebrospinal fluid (CSF) from patients with ALS was toxic to neuronal cells in culture as compared to control CSF. The neurotoxic effect could be blocked by CNQX, and AMPA receptor antagonist (Couratier et al., 1993). Chronic inhibition of glutamate uptake by 3-hydroxyaspartate or (S)-transpyrrolidine-2,4-dicarboxylic acid in organotypic spinal cord cultures produces neurotoxicity over a period of a few weeks. Non-NMDA receptor antagonists (GYK1-52455 and CNQX) provide nearly complete protection (Rothstein et al., 1993). Reduced uptake as determined by so called sodium-dependent "binding" (see Section 3 for comments on the validity of the method) has also been reported in brains from patients with Alzheimer's disease (e.g. Cross et al., 1987; Cowburn et al., 1988; Simpson et al., 1988; Chalmers et al., 1990). However, *This compound is not excitatory in itself, but after reacting with HCO3- under physiological conditions it becomes exeitotoxic (Weiss and Choi, 1988; Allen et al., 1993).

reduced uptake in synaptosomes has been observed by some workers (Hardy et al., 1987), but not by others (Rothstein et al., 1992). Using bilateral intrahippocampal microdialysis in six patients with complex partial epilepsy, it has been shown (During and Spencer, 1993) that release of transmitter amino acids occurs in the conscious human brain during seizures. The glutamate concentration was higher in microdialysates from the epileptogenic hippocampus during seizures, while the opposite was true for GABA. Of particular interest is the finding that the glutamate concentration started to increase prior to the onset of seizures. One may speculate whether this rise in extracellular glutamate elicits the seizures. The increased concentration is either due to increased release or reduced uptake. The glutamate transporter is potently, but reversibly, inhibited by arachidonic acid (Rhoads et al., 1983; Barbour et al., 1989; Volterra et al., 1992). This could constitute a regulatory mechanism, but would be unselective as several sodium dependent uptake systems are inhibited (Rhoads et al., 1983) including the uptake system for GABA (Chan et al., 1983) and glycine (Zafra et al., 1990). Release of arachidonic acid may be one reason why the uptake systems fail during ischemia (Nicholls and Attwell, 1990). It is still unclear whether this effect is caused by a direct action on the transporter molecules, or indirectly, through an interaction with their lipidic environment. It has been reported (Brookes, 1988) that mercuric chloride inhibits glutamate uptake in cultured astrocytes reversibly and specifically at concentrations below 1 #M. Organotypic cultures derived from newborn rat cerebellum, tolerate 100 #u glutamate and 1/tra mercuric chloride when added separately, but not when added together (Matyja and Albrecht, 1993). It is concluded that mercury lowers the threshold for glutamate neurotoxicity presumably by inhibiting glutamate uptake. Several naturally occurring compounds can act as glutamate receptor agonists (Meldrum and Garthwaite, 1990; Krogsgaard-Larsen and Hansen, 1992; Meldrum, 1993). Some of these can enter the brain extracellular fluid after oral administration, but are not taken up by the glutamate transporter and thus bypass the brain's protective mechanisms. Several such compounds have been identified: (S)-(fl-N-oxalylamino)alanine (fl-ODAP, also abbreviated BOAA) from the chick pea, Lathyrus sativus, is a potent AMPA receptor agonist and thought to be the toxin responsible for neurolathyrism (Spencer et al., 1986). (S)-(fl-N-methylamino)Alanine* (BMAA) from the seeds of Cycas circinalis is an agonist at N M D A and metabotropic glutamate receptors and induces motor system dysfunction and parkinsonian features in monkeys (Spencer et al., 1987), although its role as a causative factor in Guamanian (Western Pacific) amyotrophic lateral sclerosis has been questioned (Appel, 1993; Stone, 1993). Domoic acid (a kainic acid analogue and a kainate receptor agonist) from mussels that feed on the algae Chondria armata or the plankton Nitzschia pungens can damage several brain regions including hippocampus and amygdala and cause memory impairment (Perl et al., 1990; Stewart et al., 1990; Teitelbaum et al., 1990). Acromelic acid (another kainic acid analogue) from poisonous

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mushrooms Clitocybe acromelalga (Shinozaki et aL, 1986) damages, in particular, interneurons in the spinal cord and hippocampus (Shin et al., 1992). It is possible that still unidentified environmental toxins of this kind exist.

3. COMMENTS ON THE SODIUM DEPENDENT "BINDING" OF EXCITATORY AMINO ACIDS

2.13.2. Reversal o f transport

The affinity of receptors for specific ligands have been utilized for studying receptor kinetics, pharmacology and distribution. The beauty of the method lies in its simplicity. The receptors can be studied while still unpurified in the native membranes (tissue sections or homogenates). The membranes are incubated with radioactively labelled ligands until equilibrium is obtained between free and receptor bound ligand. Subsequently, the membranes are rapidly washed to remove free ligand and the bound radioactivity is determined autoradiographically (e.g. Young and Kuhar, 1979) or by scintillation counting. Such binding studies have produced a substantial amount of important data on a large number of different receptors and have therefore been very important. Binding assays have also been used to monitor purification and expression cloning of receptors.

When the energy supply of the brain is insufficient (hypoxia, hypoglycaemia, ischaemia), toxic amounts of glutamate are released (Benveniste et al., 1984; Sandberg et al., 1986). This is thought to be due to reversal of the glutamate transporter (Kauppinen et al., 1988; S~nchez-Prieto and Gonz~tlez, 1988; Szatkowski et al., 1990). If the forces (ion gradients and membrane potential) driving the transporter are weakened, the high glutamate concentration intracellularly will drive the transporter backwards releasing glutamate (Kanner and Schuldiner, 1987; Nichols and Attwell, 1990). Thus, not surprisingly, ATP-depletion leads to reversal of the neuronal glutamate transporter (Madl and Burgesser, 1993). When one takes into account that neurons become more vulnerable to glutamate after energy deprivation (Novelli et al., 1988; Kohmura et al., 1990), this may be one of the main reasons why the brain is so vulnerable to energy failure. The glutamate uptake in glial cells is probably less sensitive to hypoxia than the uptake in neurons and is largely maintained during hypoxia provided glucose remains available (Swanson, 1992). Glial cells are not so strongly depolarized during ischaemia. This could be one reason why the uptake in glial cells is more resistant to energy deprivation. One might also suggest that glial cells require lower driving forces because of lower intracellular glutamate concentration. Most brain glutamate is stored in neuronal rather than glial compartments (Ottersen et al., 1992). These factors make neurons likely to be the most important source of glutamate efflux during energy failure. During ischaemia endogenous glutamate is reduced in neuronal perikarya, but increased in glia (Torp et al., 1991). 2.13.3. Excessive Transport

Increased transport activity could possibly result in glutamatergic hypofunction. It has been suggested that deficient stimulation of glutamate receptors may participate in the development of psychosis (Schizophrenia) (Carlsson and Cadsson, 1990). Whether this is a pathogenetic mechanism remains to be determined. It should also be kept in mind that hyperfunction of the transporter is not the only possible cause of glutamatergic hypofunction. Glutamatergic hypofunction may, for instance, also be the end stage after excitotoxic loss of cells with glutamate receptors (Olney, 1990). 2.13.4. Transport o f Toxic Substances Some substrates for the glutamate transporter [(S)-ct-aminoadipate, (S)-homocysteate, (S)-serine-Osulphate, (S)-2-amino-4-phosphonobutanoate and (S)-2-amino-3-phosphonopropanoate] are toxic to glial cells when taken up (Bridges et al., 1992).

3.1. Binding Studies Have Been Very Useful for Studying Receptors

3.2. Sodium Dependent "Binding" of Excitatory Amino Acids As transporter molecules must be able to bind the substrates in order to translocate them, several workers have proposed that techniques originally developed for studies of receptors, could also be used for studying binding of substrates to transporter molecules. However, as membrane fragments reseal to form tight vesicles, one must take care to exclude the possibility that the binding ligand may be translocated to the inside of such vesicles. If transport is occurring, this will affect the results in important ways (see Section 3.4. below). Binding to uptake sites, should, therefore, be studied with substrate analogues that the transporter of interest is unable to translocate. Unfortunately, no such analogues of glutamate (with sufficient affinity) are currently available. When brain tissue is homogenized, some of the endogenous excitatory amino acids are trapped inside resealing membrane fragments and can exchange with exogenously added radiolabelled amino acids through glutamate carrier proteins. Thus, the tight membrane vesicles can become radioactive in the absence of net transport. Both the so called "sodium dependent binding" of (S)-glutamate and (R)-aspartate (Danbolt and Storm-Mathisen, 1986a) and the Ca 2+ and Cl--dependent Na+-independent "binding" of glutamate (Pin et al., 1984; Kessler et al., 1986, 1987; Rrcasens et al., 1987; Zaczek et al., 1987) to brain membranes have been shown to represent sequestration ofligand into membrane bordered compartments, rather than binding to the active sites of transporter proteins. The "sodium dependent binding" of excitatory amino acids has a substrate selectivity similar to that of the sodium dependent uptake in synaptosomes and has, therefore, been assumed to represent "binding" to uptake sites (Roberts, 1974; Baudry and Lynch, 1979, 1981; Vincent and McGeer, 1980; Di Lavro et al.,

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1982; Parsons and Rainbow, 1983; Foster and Fagg, 1984; Ogita and Yoneda, 1986; Anderson and Sandier, 1993). It has been reported that this "binding" is inhibited by potassium (Danbolt and StormMathisen, 1984; Kramer and Baudry, 1984; Mena et al., 1985). The effect of potassium is not only due to inhibition of the interaction of the dicarboxylic amino acid with the transporter. Part of the effect of potassium is to allow net efflux of endogenous glutamate, thereby reducing the internal pool of amino acids against which the labelled amino acid exchanges (Danbolt and Storm-Mathisen, 1986b). This idea is consistent with the proposed model for glutamate uptake (Kanner and Bendahan, 1982; Kanner and Schuldiner, 1987; Bouvier et al., 1992). Rather than representing a regulatory feature, the potassium effect results from the nature of the mechanism of transport, revealing itself in an unusual way due to impaired electrochemical gradients (see Section 2.1). The sodium independent binding is very different from the "binding" seen in the presence of sodium, and has been thought to represent binding to receptors (Baudry and Lynch, 1981; Foster and Fagg, 1984).

3.3. Localization of Sodium Dependent "Binding" Sites Sodium dependent "binding" sites have been demonstrated autoradiographically in cryostat sections of fresh brain tissue thaw-mounted onto glass slides, dried and incubated with radioactive dicarboxylic amino acids in the presence of sodium (N.C. Danbolt, unpublished results; Greenamyre et al., 1990; Browne et al., 1991). The observed labelling is relatively uniform throughout the brain and is not reduced by lesioning of glutamatergic pathways, namely cortico-striatal, Schaffer-collateral, and perforant path fibres. The latter finding is in contrast to sodium dependent "binding" seen with membranes in suspension (Vincent and McGeer, 1980; McGeer et al., 1987; Slater et al., 1992c) indicating that the "binding" seen in the dried sections is different. Further, the finding is also in contrast to the results obtained from lesioning of glutamatergic pathways (Divac et al., 1977; Storm-Mathisen, 1977; Taxt and StormMathisen, 1984). The lesions caused a marked loss of the glutamate uptake in the target region of the lesioned fibres as determined in synaptosome-containing homogenates. Greenamyre and collaborators (1990) suggested that "sodium dependent binding" measures all the uptake sites (glial plus neuronal) whereas the synaptome assay preferentially detects neuronal uptake. If the majority of the uptake sites are located in glia rather than in nerve endings, a view held by several investigators (Schousboe, 1981), a reduction after lesioning would not be expected. However, this interpretation cannot account for the unexpected recent observations that glial glutamate transporters are also reduced after glutamatergic denervation (Levy et al., 1993b). Some investigators (Anderson et al., 1990) have tried to avoid the problem of sequestration of ligand by defatting the tissue in xylene in order to destroy all membrane encapsulated compartments in the preparation prior to incubation with labelled amino acid. Using this method (Anderson et al., 1991) an increase

of both Na+-dependent and Na+-independent CI-dependent "binding" of glutamate is observed in the hippocampus after entorhinal lesions. These apparent contradictions make interpretation of the binding experiments difficult. Several groups have now questioned the suitability of this method (McGeer et al., 1987; Greenamyre and Young, 1989; Greenamyre et al., 1990; Browne et al., 1991). The large number of glutamate binding proteins now identified (see above) may contribute to the difficulties in interpretation of the studies. Where possible, antibodies or oligonucleotide probes should now be the preferred tools for detecting the glutamate transporters.

3.4. The Importance of Distinguishing Uptake from Binding It is important to recognize whether the labelling of the membranes is due to exchange uptake or to binding. If not, the data will be collected and interpreted incorrectly: In binding experiments, the KD and Bmaxare determined at equilibrium, i.e. late in the time course when there is no longer any net increase in bound ligand. The KD represents the ligand concentration at which half of the binding sites are occupied, while the Bmax represents the total number of binding sites. In uptake studies, the Km and Vmax are determined at initial rate, i.e. early in the time course when the increase in labelling is still proportional to time. The Km represents the substrate concentration giving half maximal velocity (i.e. half saturation of transporter sites), while the Vm,x is the maximal velocity, i.e, the number of molecules taken up per time unit when the transporter sites are fully saturated with substrate. In studies on the sodium dependent "binding" of glutamate (e.g. Vincent and McGeer, 1980; Baudry and Lynch, 1981; Foster and Fagg, 1984; several studies on pathological material quoted below), the data are collected and treated as though they were binding data, i.e. collected at equilibrium. Since the process studied is predominantly an exchange diffusion uptake process, equilibrium is reached when the ratio between labelled and unlabelled amino acid is the same inside and outside of the membrane bounded compartments harbouring the transporter. The Bmaxwill be overestimated since the radioactivity retained will be the sum of that bound to the active site (at the time of termination of the reaction) and that taken up. If the exchange process is allowed to go on for long enough to reach equilibrium, the amount of retained radioactivity will be dependent on the amount of internal endogenous amino acid. In principle the retained radioactivity will be independent of the number of transporter molecules (as long as there is at least one in a given saccule). It will also be largely independent of the concentration of the exogenously added labelled amino acid (as long as the external volume can be considered infinite relative to that inside the saccules) and of its affÉnity for the transporter. However, these factors will determine the rate at which equilibrium is reached. Since retained radioactivity will not rise beyond the point at which all internal dicarboxylic amino acid has been exchanged with exogenous, when increasing the

Excitatory Amino Acids c o n c e n t r a t i o n o f labelled a m i n o acid, the retained radioactivity will quickly level off a n d the estimated affinity will be high, i.e. low a p p a r e n t ,~go,," T h e description a b o v e is o f a n idealized situation. F r o m the bell-shaped time course typical o f m e m b r a n e t r a n s p o r t processes (e.g. Fig. 2 in D a n b o l t a n d S t o r m - M a t h i s e n , 1986a), it is clear t h a t o t h e r factors (e.g. loss o f e n d o g e n o u s a m i n o acids) will affect the measurements, particularly at the long i n c u b a t i o n times needed to achieve equilibrium between labelled a n d unlabelled a m i n o acids at low external concentrations. Thus, the " b i n d i n g " will a p p e a r to be d e p e n d e n t o n the affinity, n u m b e r o f t r a n s p o r t e r molecules a n d c o n c e n t r a t i o n o f a d d e d exogenous a m i n o acid. T h e following three factors will affect the estimated "Bmax": the a m o u n t s o f e n d o g e n o u s a m i n o acids available for exchange, the percentage o f particles with t r a n s p o r t e r protein molecules a n d finally the ability o f m e m b r a n e s for form tight c o m p a r t m e n t s . One example: if the a m i n o acid content, e.g. o f a n Aizheimer b r a i n is reduced or some factor adversely affects the f o r m a t i o n o f tight vesicles, the estimated "Bmax" will be reduced a n d one would erroneously conclude t h a t the n u m b e r o f t r a n s p o r t e r molecules is reduced. A reduced n u m b e r o f t r a n s p o r t e r s (other things being equal) would n o t reveal itself by a reduced "Bmax" , b u t by a n increase in the time needed to reach equilibrium. ( L o n g i n c u b a t i o n time, however, would lead to a loss of g l u t a m a t e as the saccules are not completely tight.) These considerations cast d o u b t o n the i n t e r p r e t a t i o n o f several studies o n the sodium d e p e n d e n t g l u t a m a t e " b i n d i n g " (e.g. Cross a n d Slater, 1986; Cross et al., 1986a-c, 1987; Simpson et al., 1988, 1989; C h a l m e r s et al., 1990; Slater et al., 1992a, b).

4, C O N C L U D I N G R E M A R K S The g l u t a m a t e transporters are essential for n o r m a l b r a i n function a n d protection against excitatory a m i n o acid toxicity. T h e g l u t a m a t e u p t a k e system appears to be m o r e heterogeneous t h a n previously believed. Several g l u t a m a t e transporters are n o w cloned and, at least one o f them, G L T - I , is regulated by p h o s p h o r y l a t i o n . Regulated expression has been d e m o n s t r a t e d for GLT-1 a n d G L A S T - 1 . These two are also expressed in the same cells, but in different p r o p o r t i o n s in different parts o f the brain. Because there is such heterogeneity, it is reasonable to assume t h a t the rate a n d extent to which excitatory a m i n o acids are removed from the extracellular fluid is i m p o r t a n t for brain function. Thus, these transporters m a y take direct part in regulation o f n e u r o n a l excitability a n d synaptic transmission. M o r e knowledge o f these t r a n s p o r t e r s will p r o b a b l y be i m p o r t a n t for o u r u n d e r s t a n d i n g o f n o r m a l a n d pathological b r a i n functioning. Acknowledgements--I would like to thank L. M. Levy, O. P. Ottersen, J. Storm-Mathisen and B. Wharton for discussions and critical reading of the manuscript. JPNd414+~E

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