I. Introduction

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Intercellular communication in the central nervous system (CNS)² requires the precise control of the duration and the intensity of neurotransmitter action at specific molecular targets. After they have been released at the synapse, neurotransmitters activate pre- and/or postsynaptic receptors (Fig. 1). To terminate synaptic transmission, neurotransmitters are, in turn, inactivated by either enzymatic degradation or active transport in neuronal and/or glial cells by neurotransmitter transporters (Iversen, 1975). Once inside the neuronal cell, neurotransmitters can be further transported into synaptic vesicles by vesicular carriers (Fig. 1). These processes are responsible for the homeostasis of neurotransmitter pools within nerve endings (Fig. 1). Both at the plasma and the vesicular membranes, neurotransmitter influxes are directly coupled to transmembrane ion gradients which provide the energy for the retrotransport (Kanner and Schuldiner, 1987).

View larger version (69K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the main neurotransmission steps at a synapse. The neurotransmitter is synthesized in the presynaptic neuron, stored in synaptic vesicles (by the VNT), and released by exocytosis. The neurotransmitter then acts at metabotropic and/or ionotropic receptors (RNT, E: effector), and is removed from the synaptic cleft by uptake in presynaptic or postsynaptic neurons and/or glial cells. The uptake process is carried out by plasma membrane-bound neurotransmitter transporters (PNT).

Neurotransmitter transporters can be classified in superfamilies, families, and subfamilies according to their primary structures and site of action. In particular, the latter criterion allows the distinction of two superfamilies: 1) the plasma membrane transporters and 2) the vesicular membrane transporters. The superfamily of plasma membrane transporters can be further divided into two families depending on their ionic dependence: 1) the Na⁺/Cl-dependent transporters and 2) the Na⁺/K⁺-dependent transporters.

This review article describes the present status of the art about neurotransmitter transporters involved in the fine tuning of neuronal communication. Special attention is paid to their anatomical and cellular localization, pharmacological properties, and involvement in the physiology of the normal and pathological CNS.

	II. Plasma Membrane Neurotransmitter Transporters

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Plasma membrane neurotransmitter transporters are responsible for the high-affinity uptake of neurotransmitters by neurons and glial cells at the level of their plasma membrane. These membrane-bound proteins are all dependent on the Na⁺ intracellular/extracellular gradient for their activity; in addition they also may require either Cl or K⁺ (Kanner and Schuldiner, 1987; Kavanaugh et al., 1992; Zerangue and Kavanaugh, 1996). The advent of molecular cloning has allowed the pharmacological and structural characterization of a large family of related genes encoding Na⁺/Cl-dependent neurotransmitter transporters (SCDNTs; Fig. 2). The monoamine [dopamine (DA), norepinephrine and serotonin (5-HT)], amino acid [aa; -aminobutyric acid (GABA), glycine, proline, and taurine], and osmolite (betaine, creatine) transporters require Na⁺ and Cl and possess 12 hydrophobic structural motifs (Fig. 2). In contrast, excitatory aa (glutamate and aspartate) transporters are Na⁺/K⁺-dependent. They belong to another transporter family whose members possess 6 to 10 hydrophobic (transmembrane) domains, and share no sequence homology with the Na⁺/Cl-dependent carrier family (Amara, 1992).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Schematic structural organization of the different subfamilies of Na+/Cl-dependent transporters. A, classical Na+/Cl-dependent transporters. On the left side, the topology is derived from hydropathy plot analyses; on the right side, the representation is derived from the work of Bennett and Kanner (1997) and Olivares et al. (1997). B, orphan transporters with an atypical structure. All of these proteins have an enlargement of their fourth extracellular loop. The gray horizontal band represents the plasma membrane. , potentially N-glycosylated asparagine residue.

The molecular characterization of neurotransmitter transporters began with the purification, aa sequencing, and cloning of the rat GABA transporter (Radian et al., 1986; Radian and Kanner, 1986; Guastella et al., 1990). The cDNA encoding the GABA transporter (GAT) was expressed in Xenopus oocytes to establish the Na⁺/Cl dependence of the transport as well as its pharmacological characterization (Guastella et al., 1990). In parallel, the expression cloning of the human norepinephrine transporter (NET) was performed by Pacholczyk et al. (1991), and the high sequence homology between GAT and NET was unraveled. Consequently, these two clones were classified within the same gene family. Expression and homology cloning rapidly led to the enlargement of this family with the betaine, creatine, DA, glycine, proline, serotonin, and taurine transporters (Figs. 2 and 3). Moreover, subtypes and isoforms of GABA and glycine transporters have been characterized. All of the data concerning the SCDNT family are summarized in Table 1. Interestingly, these so called "high-affinity" neurotransmitter transporters have affinities for their respective substrates ranging from ~320 nM (for rat SERT/serotonin) to 930 µM (for human BGT/betaine).

View larger version (122K): [in this window] [in a new window]   Fig. 3.   Alignment of the aa sequences of Na+/Cl-dependent transporters. Classical members are rat DA [rDAT (Giros et al., 1991; Kilty et al., 1991; Shimada et al., 1991)], human norepinephrine [hNET (Pacholczyk et al., 1991)], rat serotonin [rSERT (Blakely et al., 1991; Hoffman et al., 1991], rat GABA [rGAT1 (Guastella et al., 1990)], rat proline [rPROT (Fremeau et al., 1992)], rat glycine [rGLYT1 (Guastella et al., 1992)], rat taurine [rTaurT (Smith et al., 1992b)], and rat creatine [rCREAT (Mayser et al., 1992)] transporters. Orphan members are Rxt1/NTT4 (Liu et al., 1993; El Mestikawy et al., 1994), V-7-3-2 (Uhl et al., 1992), rB21a (Smith et al., 1995), and ROSIT (Wasserman et al., 1994). The putative -helical membrane spanning domains (I-XII) are indicated by bars. Conserved residues are shaded.

View this table: [in this window] [in a new window]   TABLE 1 Na+/Cl-dependent transporters

Aside from these "classical" members, a new subfamily has emerged since 1993. The primary sequence and topology of the new members are clearly similar to those of prototypical Na⁺/Cl-dependent transporters (Uhl et al., 1992) (Figs. 2 and 3). However, some structural differences as compared to classical members have been reported. Moreover, because their transported substrates are, up to now, unknown, they have been named "orphan" transporters (Table 2).

1. Classical Members. The SCDNTs DA transporter (DAT), 5-HT transporter (SERT), GABA transporter [GAT(1-3)], norepinephrine transporter (NET), proline transporter (PROT), taurine transporter (Taurt or rB16a), glycine transporter GLYT(1a, -b, -c, and -2) share the same topology and are 40 to 60% homologous. The hydropathicity analysis of these clones revealed 12 stretches of 15 to 25 hydrophobic aas which have been interpreted as forming -helical transmembrane domains (TMs; Kyte and Doolittle, 1982). The N- and C-terminal regions are intracellular and the second large extracellular loop contains two to four potential N-glycosylation sites (Fig. 2). This initial theoretical topology has been recently challenged. Using the N-glycosylation scanning method, a somewhat different structural organization for GAT1 and GLYT1 could be established (Fig. 2) (Bennett and Kanner, 1997; Olivares et al., 1997). In this new model, the two thirds of the proteins on the C-terminal side (from TM4 to TM12) are organized as proposed in the initial theoretical topology. However, the introduction of N-glycosylation sites as reporters indicates that TM1 is not spanning the membrane. Bennett and Kanner (1997) suggest that this highly hydrophobic region might form a pore loop structure associated with the plasma membrane. Such a secondary structure has already been described for ion channels in which it can form a selectivity ion filter (MacKinnon, 1995). Consequently, 1) the former TM2 becomes the first transmembrane domain, 2) extracellular loop (EL) 1 is intracellular, and 3) an hydrophobic portion of EL2 becomes TM3 (see Fig. 2) (Bennett and Kanner, 1997; Olivares et al., 1997). Indeed this modified topology might well be relevant to all SCDNTs.

2. The Orphan Transporter Subfamily. Using the homology cloning strategy, four new members of the Na⁺/Cl-dependent transporter family have been isolated: Rxt1 (also named NTT4), V-7-3-2, ROSIT, and rB21a (Uhl et al., 1992; Liu et al., 1993; El Mestikawy et al., 1994; Wasserman et al., 1994; Smith et al., 1995) (Figs. 2 and 3). Up to now, the four new members are still to be established as actual transporters since their respective substrates have not been identified. Nevertheless, they are usually referred to as "orphan" transporters. These four proteins have all of the classical features of Na⁺/Cl-dependent transporters described above. However, they also exhibit original structural characteristics such as the enlargement of their fourth and sixth extracellular loops and the presence of an additional N-glycosylation site in the fourth extracellular loop (Fig. 2). The four transporter-like proteins have significant homology (~20-30%) with the "classical" members such as the DA, serotonin, GABA, or glycine transporters. However, the overall percentage of homology among Rxt1, V-7-3-2, ROSIT, and rB21a is higher, averaging 50 to 60%. Because they are more closely related to each other than to any other Na⁺/Cl-dependent transporter, they can be considered as forming a specific group within this family. In addition, their common structural features allow the hypothesis that these orphan transporters probably exhibit functional similarities. However, their transport activities have yet to be demonstrated.

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4. Cellular and Subcellular Localization. Thanks to the availability of their sequences, probes (cRNA and antibodies) have been produced for the determination of the detailed anatomical and cellular localization of SCDNTs. In particular, specific antibodies have been raised against DAT (Ciliax et al., 1995; Freed et al., 1995), GAT1-3 (Ikegaki et al., 1994; Minelli et al., 1995, 1996), GLYT1-2 (Jurski and Nelson, 1995; Zafra et al., 1995), NET (Bruss et al., 1995), PROT (Velaz-Faircloth et al., 1995), and SERT (Qian et al., 1995; Sur et al., 1996; Zhou et al., 1996). In summary, in situ hybridization and immunocytochemical data showed that DAT, NET, and PROT are present exclusively in neurons; GAT3 is found in glial cells; and GAT1, GLYT1-2, and SERT are synthesized both in neurons and astrocytes (Minelli et al., 1995; Zafra et al., 1995; Bel et al., 1997). On the other hand, GAT2 is expressed by arachnoid and ependymal cells (Ikegaki et al., 1994; Durkin et al., 1995).

5. Phosphorylation-Dependent Regulation of Transport. Before the advent of molecular cloning, the reuptake process appeared to be less regulated than other important steps of the neurotransmission cascade (such as the biosynthesis and the release of neurotransmitters or their binding to specific receptors). However, in a few cases, protein kinase-dependent modulation of neurotransmitter reuptake was described. For example, in primary cultures of astrocytes and neurons, GABA uptake could be modulated by Ca²⁺/calmodulin-dependent protein kinase, protein kinase C (PKC), or cAMP-dependent protein kinase A (PKA) (Gomeza et al., 1991, 1994; Corey et al., 1994). In addition, DA uptake in mouse striatum was significantly affected by a wide-spectrum inhibitor of protein kinases (Simon et al., 1997). On the other hand, the transport of serotonin in choriocarcinoma cells, as well as that of DA in hypothalamic neurons, was stimulated by cAMP-dependent phosphorylation (Kadowaki et al., 1990; Cool et al., 1991).

Large amounts of L-glutamate are found in the mammalian CNS where this aa acts as the major excitatory neurotransmitter (Fagg and Foster, 1983; Fonnum, 1984). The neurotransmitter pool of glutamate is highly concentrated in nerve terminals, and low levels of the aa (below 1 µM) are normally found in the extracellular space (Ottersen and Storm-Mathisen, 1984)_. It is now widely accepted that elevated levels of extracellular glutamate can induce severe damages to target neurons. Because glutamate is such a potent excitotoxin, its removal from the synaptic cleft is of key importance to maintain the integrity of neuronal tissues. The Na⁺-dependent transport of glutamate into neurons and glial cells represents the prime mechanism by which this aa is removed from the synaptic cleft (McBean and Roberts, 1985; Nicolls and Attwell, 1990). This high-affinity transport is in fact dependent on both Na⁺ and K⁺, but does not require Cl (Danbolt, 1994). Glutamate transporters were first studied and pharmacologically defined using brain synaptosomes. Then, in the early 1990s, molecular cloning of SKDGTs allowed the detailed characterization of the family of the Na⁺/K⁺-dependent glutamate transporters (Amara, 1992).

1. Molecular Cloning and Primary Structure. Three glutamate transporters, named GLAST1, GLT-1, and EAAC1 (Table 3), were cloned almost simultaneously but independently by different groups (Kanai and Hediger, 1992; Pines et al., 1992; Storck et al., 1992). GLAST1 was isolated using probes derived from the sequence of a protein copurified with UDP-galactose:ceramide galactosyl transferase (Storck et al., 1992). GLT-1 was first purified from the rat brain and then used to produce a specific antiserum (Danbolt et al., 1992) for immunoscreening of an expression library (Pines et al., 1992)_. On the other hand, an expression cloning strategy was successfully used in Xenopus oocytes to clone EAAC1 from the rabbit small intestine (Kanai and Hediger, 1992). The human homologs EAAT1 (=GLAST1), EAAT2 (=GLT1), and EAAT3 (=EAAC1) have all been found in the motor cortex (Arriza et al., 1994). Two new members of the SKDGT family, named EAAT4 and EAAT5 (see Fig. 5), were subsequently identified in the human cerebellum and retina, respectively (Fairman et al., 1995; Arriza et al., 1997). Interestingly, molecular cloning has also permitted the isolation of two neutral aa transporters, named ASCT-1 and -2 (Arriza et al., 1993; Shafqat et al., 1993; Utsunomiya-Tate et al., 1996), which belong to the same X_A,G system (McGivan and Pastor-Anglada, 1994) as the acidic aa transporters of the SKDGT family.

View this table: [in this window] [in a new window]   TABLE 3 Na+/Cl-dependent glutamate transporter family

View larger version (66K): [in this window] [in a new window]   Fig. 4.   Alignment of the aa sequences of SKDGTs. Glutamate transporters are rat EAAT-1/GLAST1 (Storck et al., 1992), rat EAAT-2/GLT1 (Pines et al., 1992), rat EAAT-3/EAAC1 (Bjoras et al., 1996), and rat EAAT-4 (Fairman et al., 1995). The putative -helical membrane spanning domains (I-VI) are indicated by bars. LHCS, large hydrophobic conserved domain. Conserved residues are shaded.

View larger version (37K): [in this window] [in a new window]   Fig. 5.   Hydropathy plots Kyte-Doolittle of the glutamate transporters. Putative -helical transmembrane domains of 20 to 25 aa are numbered (1-6) above each sequence. LHCS, large hydrophobic conserved domain.

View larger version (41K): [in this window] [in a new window]   Fig. 6.   Hypothetical topologies of glutamate transporters. The predictions of secondary structures of the identified glutamate transporters propose -helices for the first six transmembrane domains N-terminal domain. The secondary structure of the C-terminal domain is a matter of controversy. Some models predict 8 A or 10 B transmembrane domains (Kanai et al., 1993; Lesch et al., 1996; Slotboom et al., 1996; Gegelashvili and Schousboe, 1997), but other data are more in favor of the existence of 6 transmembrane domains only and 4 -sheets C (Wahle and Stoffel, 1996).

2. Ionic Dependence and Ligand-Gated Cl Channel Properties. Only a brief survey of this rapidly developing research area will be presented here. For more elaborate information, the reader can refer to the recent review of Palacin et al. (1998).

3. Cellular and Subcellular Localization. Northern blot studies showed that EAAT-1 and -2 are expressed exclusively in the brain (Pines et al., 1992; Storck et al., 1992; Nakayama et al., 1996). EAAT-3 mRNA is found in the intestine, kidney, heart, liver, and brain (Kanai and Hediger, 1992; Nakayama et al., 1996). EAAT-4 is present in the brain and placenta (Fairman et al., 1995), and the fifth SKDGT, EAAT-5, is expressed in the retina and, at lower levels, in the liver and brain (Arriza et al., 1997). Specific antibodies have been raised against the first four subtypes of glutamate transporters: EAAT-1 (Rothstein et al., 1994; Lehre et al., 1995; Wahle and Stoffel, 1996; Schmitt et al., 1997), EAAT-2 (Danbolt et al., 1992; Rothstein et al., 1994; Lehre et al., 1995), EAAT-3 (Rothstein et al., 1994; Shashidharan et al., 1997) and EAAT-4 (Yamada et al., 1996; Furuta et al., 1997; Nagao et al., 1997). Immunohistochemical labeling with anti-EAAT-1 and anti-EAAT-2 antibodies was observed throughout the brain, but with variable intensity from one region to another. Thus, EAAT-1 is especially abundant in the molecular layer of the cerebellar cortex (Chaudhry et al., 1995; Lehre et al., 1995; Shibata et al., 1996), whereas the areas containing the highest levels of EAAT-2 are the cerebral cortex, hippocampus, lateral septum, thalamus, striatum, nucleus accumbens, and cerebellum. In these regions, EAAT-1 and -2 are found primarily in the plasma membrane of astrocytes and Bergman glia (in the cerebellum) (Chaudhry et al., 1995; Lehre et al., 1995; Schmitt et al., 1996, 1997; Torp et al., 1997). In addition, EAAT-1 is also present in ependymal cells bordering ventricles (Schmitt et al., 1996, 1997; Torp et al., 1997), and EAAT-2 is expressed in subsets of hippocampal and cortical neurons (Schmitt et al., 1996; Torp et al., 1997).

4. Regulation of SKDGT Activity and Expression. The primary sequences of SKDGTs contain several PKA and PKC consensus phosphorylation sites (Kanai et al., 1993; Gegelashvili et al., 1997). For example, a conserved PKC site is present in the first intracellular loop of EAAT-1 to -5, and one PKA site is found just before the large conserved hydrophobic domain of EAAT-1-4 (Arriza et al., 1997). Indeed, numerous studies showed that glutamate transporters are regulated by protein kinases and phosphatases (Casado et al., 1991, 1993).

5. Pharmacological and Functional Aspects. Biochemical and pharmacological studies performed on brain preparations provided the first evidence in favor of the existence of several subtypes of SKDGTs (Ferkany and Coyle, 1986; Robinson et al., 1993). The pharmacological profile of each subtype could then be precisely determined using cell lines transfected with the corresponding cDNAs (Kanai and Hediger, 1992; Pines et al., 1992; Storck et al., 1992; Arriza et al., 1994, 1997; Tanaka, 1994; Fairman et al., 1995; Dowd et al., 1996). However, additional SKDGT subtypes are probably still to be discovered (Dowd et al., 1996). To date, only a few compounds are able to discriminate between the different subtypes. For example, kainate and cysteine inhibit preferentially EAAT-2 and EAAT-3, respectively (Vandenberg et al., 1997). All the drugs acting at the EAAT that are currently available are competitive inhibitors. However, many of them also act at glutamate receptors.