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Vol. 51, Issue 3, 439-464, September 1999
NeuroPsychoPharmacologie Moléculaire, Cellulaire et Fonctionnelle, Institut National de la Sante et de la Recherche Medicale U288, Faculté de Médecine Pitié-Salpêtrière, Paris, Cedex, France
I. Introduction
II. Plasma Membrane Neurotransmitter Transporters
A. Na+/Cl-Dependent Neurotransmitter Transporters (SCDNTs)
1. Classical Members.
2. The Orphan Transporter Subfamily.
3. Ionic Dependence and Electrogenic Properties.
4. Cellular and Subcellular Localization.
5. Phosphorylation-Dependent Regulation of Transport.
6. Pharmacological and Functional Aspects.
B. Na+/K+-Dependent Glutamate Transporters (SKDGTs)
1. Molecular Cloning and Primary Structure.
2. Ionic Dependence and Ligand-Gated Cl
3. Cellular and Subcellular Localization.
4. Regulation of SKDGT Activity and Expression.
5. Pharmacological and Functional Aspects.
III. Vesicular Neurotransmitter Transporters (VNTs)
A. Cloning of VNTs
1. The Vesicular Monoamine Transporter (VMAT)/Vesicular Acetylcholine Transporter (VAChT) Family.
2. Vesicular Inhibitory Amino Acid Transporter.
B. Regulation of Transport
C. Ionic Dependence and Electrogenic Properties
D. Pharmacological Properties
E. Cellular and Subcellular Localization
F. DysfunctioningModels for Neurodegenerative Disorders or Drug Abuse
IV. Conclusion
Acknowledgments
References
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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).
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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.
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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).
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A. Na+/Cl-Dependent Neurotransmitter
Transporters (SCDNTs)
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).
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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).
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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.
). It has been speculated that highly conserved regions
are pointing at structural elements important for the common functions
of these transporters (Giros et al., 1994). In particular, the highly
homologous N-terminal portion of these molecules (from TM1 to TM4) may
be involved in Na+/Cl
transport. On the other hand, the divergent C-terminal region (from TM7
to TM12) may be responsible for substrate recognition and inhibitor
binding (Zaleska and Erecinska, 1987; Kitayama et al., 1992; Buck and
Amara, 1994; Giros et al., 1994).
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.
3. Ionic Dependence and Electrogenic Properties.
All of the
SCDNTs are utilizing, as primary driving force, the
Na+ electrochemical gradient which is created and
maintained by the (Na+/K+)-ATPase across the
plasma membrane. They also required Cl to
transport their substrate against a concentration gradient from the
extra- to the intracellular compartment. However, under physiological
conditions, the energy derived from the Cl
electrochemical gradient is negligible when compared to that of
Na+.
).
Accordingly, SCDNTs would carry one or several positive charges for
each substrate molecule transported with a stoichiometry of 2 Na+/1 Cl/1 zwitterion.
However, because of the low turnovers of SCDNTs (0.5 to ~3
s-1), the predicted microscopic current
(1017-1019 A) is 5 to
7 orders of magnitude lower than the best resolution achieved with
patch-clamp recording. To really provide experimental support to this
inference, it was necessary to find cells with enough transporters
expressed on their surface for the recording of macroscopic
uptake-associated currents. These conditions were initially fulfilled
in some amphibian glial cells for the recording of the activity of
glutamate and GABA transporters (Brew and Attwell, 1987; Cammack and
Schwartz, 1993) and in invertebrate neurons expressing a serotonin
transporter (Bruns et al., 1993).
At the beginning of the 1990s, cloned SCDNTs could be successfully
expressed in Xenopus oocytes at a density higher than
103/µm2 (as established
from cryofracture experiments; Zampighi et al., 1995), allowing the
measurement of currents associated with the transport of
neurotransmitters. GAT1, the first cloned SCDNT, was also the first one
to be characterized electrophysiologically. In GAT1 expressing
Xenopus oocytes, application of GABA evoked a steady-state
inward current that could be recorded under voltage-clamp conditions
(Kavanaugh et al., 1992; Mager et al., 1993). The ionic coupling was
estimated at 1.29 charges per GABA molecule transported by determining
the ratio of [3H]GABA uptake over the integral
of the GABA-evoked current in the same transfected oocyte (Kavanaugh et
al., 1992); these data were in reasonable agreement with the predicted
stoichiometry of 2 Na+/1
Cl/1 GABA (Kanner and Schuldiner, 1987). The
uptake current was dependent on the presence of
Na+ and Cl ions and
blocked by specific GABA uptake inhibitors such as SKF-89976A [N-(4,4-diphenyl-3-butenyl)-3-piperidine carboxylic acid].
Interestingly, classical inhibitors of GABA or glycine transport, such
as nipecotic acid (for GAT1) or sarcosine (for GLYT1) were found to be
substrates. Like GABA, these compounds evoked a current when applied
alone (Kavanaugh et al., 1992; Supplisson and Bergman, 1997). Shortly after, the same type of measurements were extended to other SCDNTs such
as NET, SERT, DAT, GLYT1, and GLYT2 (Mager et al., 1994; Galli et al.,
1996a, 1997; Sonders et al., 1997; Lopez-Corcuera et al., 1998).
However, in the case of both SERT and NET, the measured ionic coupling
was in far excess when compared with the one predicted by the
stoichiometry (Lin et al., 1994; Mager et al., 1994; Galli et al.,
1996a,b, 1997). This discrepancy could not be explained by classical
models of cotransport with an alterned access at both sides of the
membrane. Rather, it suggested that some channel activity is associated
with the transporter cycle (for review, see Lester et al., 1994;
DeFelice and Blakely, 1996; Kavanaugh, 1998). This new feature of SCDNT
transport activity received more direct support from the recording of
single channel in cells expressing GAT1 or SERT (Cammack and Schwartz,
1993; Lin et al., 1996).
In addition to the electrogenic substrate translocation described
above, neurotransmitter transporters can also generate uncoupled currents that can be blocked by uptake inhibitors or the susbtrate itself. These uncoupled currents were first described for the transport
of glutamate in photoreceptor cells of the salamander (Sarantis et al.,
1988). With the advent of molecular cloning, this initial observation
has now been extended to other neurotransmitter transporters (for
review, see Sonders and Amara, 1996). For example, relatively large
current can be recorded by patch-clamping HEK293 cells that
express GAT1 (Cammack et al., 1994). This leakage current was
identified as resulting from the channel-like behavior of the
transporter (Cammack et al., 1994). However, it should be noted that
these observations were not reproduced with Xenopus oocytes
(Mager et al., 1993).
In addition, capacitive currents that can be suppressed by substrates
or inhibitors have been observed during voltage jumps for GAT1 (Cammack
and Schwartz, 1983; Mager et al., 1993, 1994, 1996) and TAUT (Loo et
al., 1996). Integration of the current and determination of the
saturating charges movement (Qmax) allowed an
estimate of the number of transporters expressed (Mager et al., 1993,
1998; Loo et al., 1996). In addition, the slope of the relationship
between the maximal uptake current (Imax) and the
Qmax has permitted the determination of
transporter turnover (for review, see Mager et al., 1998).
All of these data show that in addition to their known function as
neurotransmitter transporters, SCDNTs have complex and not yet
completely identified ion channel-like properties (Sonders and Amara,
1996).
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).
; Amara and Kuhar,
1993; Kanai et al., 1993; Borden, 1996). Nonetheless, this article will
concentrate on their distribution in the mammalian CNS.
DAT and NET are considered as specific markers of DAergic and
noradrenergic neurons in the CNS, respectively. Similarly, SERT can be
used as a marker of serotoninergic neurons because its synthesis in
astrocytes (Bel et al., 1997) seems to be hardly detected in the CNS of
adult intact (i.e., unlesioned) rats (F. C. Zhou, personal
communication). Apparently, all GABAergic neurons express GAT1 mRNA;
however, GAT1 is also synthesized in pyramidal glutamatergic cells in
the hippocampus and the cerebral cortex (Minelli et al., 1995; Yasumi
et al., 1997). GLYT1 is expressed in both glycinergic neurons in the
brainstem and in the spinal cord and glutamatergic neurons within the
forebrain (Zafra et al., 1995). Finally, the PROT is found in
glutamatergic neurons (Fremeau et al., 1992).
Some SCDNTs seem to be addressed to specific subcellular compartments,
whereas others are present all over the plasma membrane. For example,
DAT, NET, and SERT are present on dendrites, perikarya, axons, and
nerve endings of the corresponding monoaminergic neurons (Ciliax et
al., 1995; Freed et al., 1995; Qian et al., 1995; Nirenberg et
al., 1996, 1997b). Interestingly, previous results already demonstrated
the existence of dendritic [3H]DA uptake in the
substantia nigra (Gauchy et al., 1994) and somatodendritic
[3H]5-hydroxytryptamine (5-HT) uptake in the
dorsal raphe nucleus (Descarries et al., 1982). Accordingly, these
ultrastructural data formally establish a fact that has long been
suspected: SERT and DAT are not only present but are also functional
over the entire surface of serotoninergic and dopaminergic neurons,
respectively. In striatal dopaminergic terminals, which belong to
neurons located in the substantia nigra pars compacta, DAT is found on
the varicose and intervaricose plasma membrane but not in the active
synaptic zones (Hersch et al., 1997; Sesack et al., 1998).
Interestingly, in dopaminergic nerve endings of the rat prefrontal
cortex (which belong to neurons located in the ventral tegmental area),
very low levels of DAT immunoreactivity are observed, most of which being extrasynaptic (Sesack et al., 1998). These observations are in
line with the fact that extracellular concentrations of DA are higher
in the prefrontal cortex than in the striatum (Cass and Gerhardt,
1995).
In contrast, GAT1, GLYT2, and PROT seem to be restricted to axon
terminals (Ikegaki et al., 1994; Jurski and Nelson, 1995; Velaz-Faircloth et al., 1995; Riback et al., 1996). These data allow
the distinction of two groups of SCDNTs: those that are restricted to
nerve terminals and those that are not specifically addressed to this
cell compartment. Furthermore, it appears that a transporter such as
DAT may have different subcellular localization in different neurons.
The cellular mechanisms and molecular signals that are responsible for
the targeting of a given transporter are just beginning to be
investigated. For this purpose, transfection in polarized cells such as
the LLCPK-1 and MDCK-1 epithelial cell lines has proven to be a
valuable method. The plasma membrane of these cells can be divided in
two functionally and structurally different compartments. The
basolateral membrane of an epithelial cell seems to correspond to the
somatodendritic domain of a neuron, whereas the apical side is
apparently equivalent to nerve terminals (Dotti and Simons, 1990). In
line with their respective neuronal targeting (see above), DAT, NET,
and SERT are addressed to the basolateral (in LLCPK cells) and apical
(in MDCK cells) domains (Gu et al., 1996), whereas GAT1 is found only
at the apical domain of the plasma membrane of transfected epithelial
cells (Pietrini et al., 1994). Thus, it should be kept in mind, as
mentioned above for neurons (Sesack et al., 1998), that the subcellular
sorting of a transporter in heterologous systems is largely dependent
on the cell type that is used (Gu et al., 1996). However, despite this
important drawback, it should be feasible using such cellular models to
determine the molecular mechanisms responsible for the specific
targeting of a given transporter, using notably site-directed mutated
and/or chimeric transporters.
In addition to the above-mentioned SCDNsT, the orphan transporter
Rxt1/NTT4 has also been extensively studied with regard to its cellular
and subcellular localization (Liu et al., 1993; El Mestikawy et al.,
1994, 1997; Masson et al., 1995; Luque et al., 1996). In the rat CNS,
Rxt1/NTT4 is expressed both in glutamatergic cells and in subsets of
GABAergic neurons (such as reticular thalamic and Purkinje cells). At
the subcellular level, this transporter-like protein is found almost
exclusively in axon terminals. Surprisingly, Rxt1/NTT4 has recently
been demonstrated to be located on synaptic vesicles (Masson et al.,
1998), although it is clearly a member of the
Na+/Cl-dependent
transporter family. Furthermore, the proline transporter was
also found in small synaptic vesicles within subsets of presynaptic axons forming asymmetric excitatory synapses (Renick et al., 1999). Interestingly, in situ hybridization data support the idea that Rxt1/NTT4 and PROT are probably synthesized in the same subset of
glutamatergic neurons (Fremeau et al., 1992; El Mestikawy et al., 1994;
Velaz-Faircloth et al., 1995; Masson et al., 1997).
Although Rxt1/NTT4 and PROT are located in synaptic vesicular
membranes, both proteins share no sequence homology with the vesicular
proton-driven carrier. Moreover, as mentioned above, transporters of
the SCDNT family use the plasma membrane Na+
ionic gradient as energy source, whereas vesicular transport is coupled
to the H+ electrochemical gradient. Indeed, to
our knowledge, the existence of a Na+ gradient
between the lumen of the vesicle and the neuronal cytoplasm has never
been reported. Therefore, it can be hypothesized that in spite of their
Na+/Cl-dependent
transporter-like primary structure, Rxt1/NTT4 and PROT are able to use
the proton-generated energy to perform their presumed function in
vesicles. However, PROT is unable to drive L-proline vesicular uptake in HEK293-transfected cells (Miller et al., 1997). Thus, alternatively, vesicular Rxt1/NTT4 and PROT might represent pools
of "spare transporters" awaiting for a yet unidentified physiological signal to be addressed at the plasma membrane and to
become functional.
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).
; Huff et al., 1997).
Similarly, GLYT1b and SERT have been shown to be inhibited upon PKC
activation in transfected HEK293 cells (Sato et al., 1995; Sakai et
al., 1997; Ramamoorthy et al., 1998). In all cases, PKC-induced
inhibition was associated with a reduction in
Vmax with no modification in
Km (Kitayama et al., 1994; Sato et
al., 1995; Huff et al., 1997; Sakai et al., 1997), suggesting a
decrease in the cell density of functional transporters. Indeed, rapid internalization of SERT could be observed in transfected cells with
activated PKC (Blakely et al., 1998; Ramamoorthy et al., 1998).
However, the down-regulation of glycine and serotonin uptake is still
observed after site-directed mutagenesis of all five predicted PKC
consensus sites in GLYT1b as well as in SERT. At first it was
hypothesized that uptake inhibition was not due to a direct
PKC-dependent phosphorylation of the transporters (Sato et al., 1995;
Sakai et al., 1997). However, because a direct phosphorylation of SERT
has been recently demonstrated, it is more likely that PKC-phosphorylated sites are noncanonical (Ramamoorthy et al., 1998).
Corey et al. (1994) have demonstrated that PKC activation markedly
increased the activity of GAT1 expressed in Xenopus oocytes. Interestingly, this effect was associated with a shift in GAT1 subcellular localization from intracellular vesicles to the plasma membrane (Corey et al., 1994; Quick et al., 1997). Using site-directed mutagenesis and coinjection of various mRNAs, it was then found that
the redistribution of this GABA transporter was dependent on both the
presence of a leucine zipper motif in its second transmembrane domain
and the level of syntaxin expression (Corey et al., 1994; Quick et al.,
1997).
In summary, relevant studies clearly showed that the activity of SCDNTs
can be modulated by protein kinases. The resulting changes generally
concern the concentration of SCDNTs at the plasma membrane rather than
their intrinsic transport activity.
6. Pharmacological and Functional Aspects. Numerous psychiatric, neurological, and neurodegenerative disorders have been associated with alterations in the neurotransmission cascade. In this context, the complete elucidation of neurotransmitter transporter functions can be of strategic importance for the development of new therapies. Indeed, a wide range of pharmacological agents are known to interact with neurotransmitter transporters. Generally, these compounds act as transport inhibitors. This is particularly well illustrated with antidepressants and psychostimulants which act primarily as inhibitors of monoamine transporters (SERT, NET, and DAT).
The pharmacological properties of SCDNTs were first determined from uptake studies performed with brain synaptosomes. Subsequently, experiments were also performed using Xenopus oocytes injected with total mRNAs isolated from discrete rat brain regions (Sarthy, 1986; Blakely et al., 1988). However,
interpretation of the data obtained with such approaches could be
difficult because of the possible participation of more than one
transporter type in the uptake process. Furthermore, these approaches
are not especially appropriate for the study of human transporters.
Thanks to molecular cloning, the precise substrate specificity and
pharmacological profile of each monoamine transporter could be
determined in in vitro experiments performed on transfected mammalian
cells or Xenopus oocytes (Giros and Caron, 1993). One interesting finding of such studies is that the human DAT can transport
both DA and norepinephrine with Km
values of 2.5 and 20 µM, respectively (Giros et al., 1992, 1994).
Surprisingly, NET has a better affinity for DA
(Km = 0.67 µM) than DAT itself, on
one hand, and than for its proper substrate, norepinephrine (Km = 2.6 µM), on the other (Giros
et al., 1994). The capacity of SCDNTs to transport more than one
substrate is not unique to DAT and NET since the betaine transporter
can take up not only betaine but also GABA with high affinity (Yamauchi
et al., 1992; Borden et al., 1995a). Thus, the concept of "one
transporter/one substrate" can no longer be considered as a general rule.
In the human brain, DAT, NET, and SERT are the primary binding sites of
cocaine (Giros et al., 1992; Giros and Caron, 1993). Recent reports
have established that inhibition of DA reuptake may be the key event
leading to the rewarding action of cocaine and thus to addiction (Giros
et al., 1994, 1996). However, knockout mice that do not express DAT
(Giros et al., 1996) can still self-administer cocaine under certain
conditions (Rocha et al., 1998). Thanks to this model, it could be
unraveled that serotoninergic mechanisms, in addition to dopaminergic
systems, play an important role in the development of addiction to
cocaine (Rocha et al., 1998). However, using the place-preference
paradigm, Sora et al. (1998) recently found that the appetitive
properties of cocaine are lost neither in DAT nor in SERT knockout
strains. Thus, neither DAT nor SERT seems to be absolutely required for
the rewarding action of cocaine.
Among monoamine transporters, DAT seems to be implicated in the
etiology of various neurological or psychiatric syndromes. Thus, as
expected of the marked degeneration of dopaminergic neurons, a decrease
of DAT is regularly observed in Parkinson's disease (Boja et al.,
1994; Miller et al., 1997). In addition, the amounts of DAT in striatal
axon terminals are reduced in spinocerebellar ataxia of type 1 (Kish et
al., 1997). Aberrant dopaminergic neurotransmission is also associated
with disorders of the schizophrenic spectrum and Tourette's syndrome
(Pearce et al., 1989; Singer et al., 1991). However, no linkage was
found to date between DAT alleles and hereditary pathogenesis of
schizophrenia (Byerley et al., 1993a,b; Li et al., 1994; Persico et
al., 1995).
Monoamine transporters are also the primary sites of action for tri-
and heterocyclic antidepressant drugs (Blakely et al., 1994; Barker and
Blakely, 1995). Indeed, both the reduced serotonin transport in
platelets and, possibly, brain in depressed and suicidal patients
(Meltzer and Lowy, 1987), and the efficacy of selective SERT inhibitors
(fluoxetine, fluvoxamine, paroxetine, sertraline, citalopram, etc.) in
the treatment of depression (Anderson and Tomenson, 1994) are
compelling evidences in support of the involvement of this transporter
in the etiology of mood disorders. Decreased serotonin brain levels in
patients with disorders of the affective spectrum may reflect a
structural defect and/or dysregulation of SERT (Perry et al., 1983).
The gene coding for the human SERT has been cloned and localized on
chromosome 17q11.2 (Ramamoorthy et al., 1993). This gene spans over 35 kilobases and is organized in 14 introns. No allelic variation has been
observed in the coding region of the SERT gene in
patients with affective disorders (Altemus et al., 1996; Di Bella et
al., 1996). In contrast, multiple polymorphims are found in the
5'-flanking region and in the second intron (Lesch et al., 1994; Heils
et al., 1996). Interestingly, the two variants in the 5' region are
associated with different rates of SERT expression and the one leading
to the lowest transcription rate seems to be more frequent in subjects
with anxiety-related personality traits (Lesch et al., 1996a)
and in alcoholics with suicidal behavior (Gorwood et al., 1998).
Moreover, allelic forms at the second intron locus seem to be
associated with bipolar and unipolar disorders (Battersby et al., 1996;
Collier et al., 1996; Ogilvie et al., 1996; Bellivier et al., 1997).
Thus, during the last 2 years, numerous genetic studies have
strengthened the "serotonin hypothesis" of mood disorders. However,
molecular and genetic studies on neurotransmitter transporters in
relation with psychiatric diseases are just on the starting line, and
numerous investigations will have to be performed to get really
definitive and clear-cut data regarding the actual association of a
given polymorphism of the SERT gene and one or several of
these diseases.
GABA is the major inhibitory neurotransmitter in the mammalian brain.
Pharmacological compounds which modulate GABAergic neurotransmission, such as benzodiazepines and barbiturates, have proven to be efficient in the treatment of anxiety and epilepsy (During et al., 1995; Dalby
and Nielsen, 1997b). Pharmacological data have long been pointing at
the existence of two distinct GABA transporters in glial and neuronal
cells (Borden, 1996). Indeed, with the advent of molecular cloning,
five GABA transporter subtypes have been found: GAT1, GAT2, GAT3,
betaine/GABA transporter 1, and rB16a (Table 1). Specific inhibitors at
each subtype of GABA transporters are thus representing new potential
therapeutic agents for the treatment of epilepsy and anxiety.
Furthermore, reverse functioning of GABA transporters should allow the
inhibition of excessive neuronal firing due to excitatory aas during
stroke and seizures. Accordingly, pharmacological compounds able to
release GABA by reversing GAT's activity might represent a new class
of neuroprotective agents.
The precise pharmacological profiles of the various cloned GABA
transporters have been determined using stably transfected mammalian
cell lines (Borden et al., 1994, 1995b, 1996). In these studies, cis-3-aminocyclohexanecarboxylate, CI-966
(1-[2-[bis 4-(trifluoromethyl) phenyl]methoxy]
ethyl]-1,2,5,6-tetrahydro-3-pyridine carboxylic acid), nipecotic acid,
NNC 05-711 (1-[2-[[[diphenylmethylene]amino]oxy]ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid), SK&F 89976-A
(N-[4,4-diphenyl-3-butenyl]-3-piperidinecarboxylic acid),
and tiagabine [Gabitril;
(R)-1-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-piperidinecarboxylic acid] were shown to act selectively at GAT1 (Borden et al., 1994, 1995b). The rank order potency of these compounds at the human GAT1 is
as follows: NNC 05-711 > tiagabine > SK&F 89976-A > CI-966 (Borden, 1996). All of these compounds display anticonvulsive activity in relevant animal models (Dalby and Nielsen, 1997b). Tiagabine has even proven its efficiency in the treatment of complex and refractory myoclonic seizures (Dalby and Nielsen, 1997a). Other
compounds have been found to act selectively at GAT2 and GAT3:
-alanine, hypotaurine, NNC 05-2045 (1[3-[9H-carbazol-9-yl]-1-propyl]-4-[4-methoxyphenyl]-4-piperidinol) and NNC 05-2090 (1-[3-[9H-carbazol-9-yl)-1-propyl)-4-(2-methoxyphenyl]-4-piperidinol) (Clark and Amara, 1994; Dhar et al., 1994; Borden et al., 1995b). Interestingly, the latter drugs also display anticonvulsive properties (Borden et al., 1994, 1995b; Dalby et al., 1997). Thus, in the last few
years, the field of antiepileptic drugs has developed rapidly thanks to
the availability, for relevant pharmacological studies, of mammalian
cell lines expressing the various GABA transporters.
B. Na+/K+-Dependent Glutamate Transporters (SKDGTs)
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
XA,G system (McGivan and Pastor-Anglada,
1994) as the acidic aa transporters of the SKDGT family.
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;
Kanner, 1993; Taylor, 1993). As can be seen in Fig. 4, similarity is
particularly striking in the C-terminal moiety. Along with the neutral
aa carriers (transporting alanine, cysteine, and serine; Arriza et al.,
1993), the five subtypes of glutamate transporters define a new gene
family (Danbolt et al., 1992; Kanai et al., 1993). From a structural
point of view, this group of genes is completely distinct from the
SCDNT family described above. For the sake of clarity, the five
glutamate transporters are named EAAT-1 to -5 in the following sections
of this review (see Table 3).
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).
Hydropathy plots of the SKDGTs are rather homogeneous (Fig. 5) and show six clear membrane-spanning
-helices (Kanai et al., 1993) in the amino-terminal portion (Figs. 4
and 5). On the other hand, the carboxyl terminus (Fig. 5) is mostly
formed of a long hydrophobic and highly conserved domain (spanning over
~50 residues) (Kanai et al., 1993; Arriza et al., 1997). The motif
R-F-V-L-P-V-G-A-T-I/V-A-A-I/V-F-I-A-Q-X-N-X-X-L-G-Q-I, which is
found in the eighth transmembrane domain of the five EAAT cloned
so far, can be considered as a SKDGT signature. The prediction of
secondary structure as well as the consequences of deletions in this
region are more in favor of its organization as four short [8-9 aas
(aa)] -sheets instead of two to four long (20-25 aa) -helices
(Arriza et al., 1994, 1997; Slotboom et al., 1996; Wahle and Stoffel,
1996). Nonetheless, it has to be pointed out that the controversy is
not closed yet because a recent study is favoring the 10--helices
model (Slotboom et al., 1996). In the -sheet model, the C terminus
has been proposed to interact with the plasma membrane as depicted in
Fig. 6C. Accordingly, both the N and C
termini, which are rather poorly conserved in this family, are supposed
to be located in the cytoplasmic compartment. In addition, EAAT-1 to -5 have a long second extracellular loop, which is also poorly conserved,
bearing one to three consensus N-linked glycosylation
sites (N-X-S/T). As shown by their migration patterns in polyacrylamide
gel electrophoresis and deglycosylation experiments, EAAT-1, -2, -3, and -4 are actually glycosylated proteins (Rothstein et al., 1994;
Furuta et al., 1997; Kataoka et al., 1997).
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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).
;
Schousboe, 1981). The transporters involved have long been known to be
electrogenic (Brew and Attwell, 1987) and have thus been studied by
electrophysiological means (Arriza et al., 1994; Kanai et al., 1994;
Klochner et al., 1994; Wadiche et al., 1995a). An initial
stoichiometry was determined as follows: 2 Na+
and 1 glutamate cotransported in exchange of 1 K+ and 1 OH (Bouvier et
al., 1992; Kanai et al., 1995b).
During the postcloning era, SKDGTs were expressed in Xenopus
oocytes and transfected cell lines, which allowed the demonstration that EAAT-1 to -5 can transport L-glutamate as
well as L- and D-aspartate
with a high affinity (Table 3). In addition, using the reversal
potential of EAAT-3, Zerangue and Kavanaugh (1996) could establish a
more definitive stoichiometry for this transporter as follows: 3 Na+ and 1 H+ cotransported
with 1 glutamate for 1 K+
countertransported. This stoichiometry was recently extended to EAAT-2
(Levy et al., 1998).
In 1995, Fairman et al. demonstrated that EAAT-4 has an intrinsic
Cl conductance gated by glutamate, and
subsequent studies showed that this property is shared with the other
SKDGT, EAAT-1-3 (Wadiche et al., 1995; Billups et al., 1996). In fact,
EAAT-1 to -5 behave as true glutamate carriers, contaminated by a
Cl conductance (Wadiche et al., 1995; Otis and
Jahr, 1998). In other words, the uphill transport of glutamate by
EAAT1-5 is Cl-independent whereas
Cl permeation is linked to particular steps of
the glutamate transport cycle (Wadiche et al., 1995; Sonders and Amara,
1996; Otis and Jahr, 1998). Interestingly, EAAT-4 (in the
cerebellum) and EAAT-5 (in the retina) are characterized by a
relatively large glutamate-elicited Cl
conductance (33 Cl/glutamate transported)
(Arriza et al., 1997; Eliasof et al., 1998) when compared with the one
induced by EAAT-1, -2, and -3 (1-2
Cl/glutamate transported) (Arriza et al., 1994;
Wadiche et al., 1995; Sonders and Amara, 1996).
Consequently, EAAT-4 and -5 are bifunctional proteins, each acting as
both a transporter and an ion channel. However, the actual functional
significance of the channel activity is still unknown.
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).
; Rothstein et al., 1994; Kanai et al., 1995a; Velaz-Faircloth et al., 1996; Yamada et al., 1996; Furuta et al., 1997;
Nagao et al., 1997; J. Tanaka et al., 1997; Torp et al., 1997).
Interestingly, EAAT-3 is found in both glutamatergic (such as granule
cells in the dentate gyrus and pyramidal cells in the hippocampus and
cerebral cortex) and GABAergic (such as Purkinje cells in the
cerebellum and medium spiny neurons in the striatum) systems. At the
ultrastructural level, EAAT-3 immunoreactivity is observed in the
plasma membrane of the somas and dendrites of these neurons (Coco et
al., 1997). However, a different targeting was noted in the deep
cerebellar nuclei because EAAT-3 is locally associated with the axon
terminals of GABAergic Purkinje cells (Rothstein et al., 1994; Furuta
et al., 1997). In contrast, such a distribution has never been found in
case of glutamatergic neurons. Thus, axotomy of glutamatergic pathways
(i.e., the corticostriatal and the fimbria-fornix projections) does not
decrease EAAT-3 in projection areas (Ginsberg et al., 1995, 1996).
Indeed, EAAT-3 is a postsynaptic transporter at glutamatergic synapses.
EAAT-4, the other neuronal SKDGT, is almost entirely restricted to the
GABAergic Purkinje cells in the cerebellum. Thus, the levels of EAAT-4
are ~30-fold lower in the hippocampus than in the cerebellum
(Furuta et al., 1997). Interestingly, EAAT-4-immunoreactive material
exhibits a parasagittal compartmentation which is perpendicular to the
Purkinje cell layer in the cerebellum (Nagao et al., 1997). At the
electron microscopic level, EAAT-4 is never associated with axon
terminals or Bergman glia, but is exclusively confined to the plasma
membrane of Purkinje cell soma and dendritic spines (Yamada et al.,
1996; Furuta et al., 1997; Nagao et al., 1997). Indeed, both EAAT-3 and
-4 are colocalized in dendritic spines of Purkinje cells (Furuta et
al., 1997), whereas EAAT-1 and -2 are present in Bergman glia
surrounding these cells. For the Purkinje cells, the two neuronal
transporters EAAT-3 and EAAT-4 play a triple role: 1) by taking up
glutamate, they decrease the local extracellular concentration of this
excitatory aa; 2) their functioning produces Cl
influx (cf. I. Cellular and Subcellular Localization), which leads to local hyperpolarization, thereby preventing excessive excitation by extracellular glutamate (Fig. 7); and 3) by accumulating the latter aa into the cells, they provide the substrate for the neosynthesis of GABA (Furuta et al., 1997).
In summary, SKDGTs are strategically distributed to control the
extracellular levels of glutamate in brain. Emphasis has to be put on
the fact that although excitatory aa uptake by hippocampal glutamatergic terminals has been demonstrated (Gundersen et al., 1993,
1995), no "presynaptic" glutamate transporter has yet been isolated
to date.
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).
). This effect is associated with a 2.5-fold increase in its
Vmax. Furthermore, Davis et al. (1998)
have recently established by confocal microscopy that PMA triggers both
an increase in the number of EAAT-3 molecules and their clustering at
the cell surface. The rapid onset of PMA-induced stimulation of
glutamate uptake is compatible with a direct PKC-mediated
phosphorylation of EAAT-3. Indeed, this effect is abolished when Ser113
in the EAAT-3 sequence has been mutated into an asparagine residue
(Casado et al., 1993).
On the other hand, short-term PKC-dependent down-regulation of
EAAT-1/GLAST has been described in transfected HEK-293 cells and
Xenopus oocytes (Conradt and Stoffel, 1997).
Immunofluorescence experiments allowed the demonstration that the
resulting PKC-mediated inhibition of glutamate transport is not due to
an altered targeting of the transporter at the cell membrane. Although
its amplitude is proportional to the amount of
32P incorporated into EAAT-1 (Conradt and
Stoffel, 1997), PMA-induced inhibition of glutamate transport persists
when the three PKC consensus sites of EAAT-1 have all been removed by
site-directed mutagenesis (Conradt and Stoffel, 1997). It can therefore
be hypothesized that the decreased activity of EAAT-1 upon PKC
activation involves other phosphorylated protein(s) possibly
interacting with the transporter.
EAAT-1 is the only SKDGT expressed in undifferentiated astrocyte
monocultures (Swanson et al., 1997). However, when astrocytes are
cultured on a neuronal layer, their morphology changes and they express
EAAT-2 in addition to EAAT-1 (Swanson et al., 1997). Indeed, EAAT-2
mRNA and protein are found in astrocytes cultured in media conditioned
by cortical neurons (Gegelashvili et al., 1997). These data suggest
that soluble factors released by neurons are able to trigger the
transcription of the EAAT-2 gene in astrocytes (Gegelashvili
et al., 1997). Interestingly, long-term treatment of astrocytes with
dibutyryl-cAMP resulted in an increased expression of EAAT-1 and -2 as
well as an enhancement of
D-[3H]aspartate uptake
(Gegelashvili et al., 1996; Swanson et al., 1997). In addition,
glutamate receptor activation by glutamate itself or kainate, but
not -amino-3-hydroxy-5-methylisoxazole-4-propionic acid
or trans-(±)-1-amino-1,3-cyclopentane-dicarboxylic acid, also results in an up-regulation of both EAAT-1 expression and D-[3H]aspartate uptake
(Gegelashvili et al., 1996).
In summary, phosphorylation by PKC activates EAAT-3 (and also EAAT-2;
see Casado et al. (1991, 1993) but inhibits EAAT-1. On the other hand,
EAAT-1 and -2 can be up-regulated at the transcriptional level by
neuronal soluble factors including glutamate itself (possibly acting at
kainate receptors) and dibutyryl cAMP. However, the physiological
relevance of this complex pattern of short- and long-term regulation of
glutamate reuptake remains to be established.
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.
). The neurotoxicity of this aa is due to overstimulation of its ion channel
receptors leading to excessive intracellular level of Ca2+. Concentrations of glutamate in the
micromolar range are needed to stimulate excitatory receptors in the
CNS (Fonnum, 1984). Consequently, to prevent neurotoxicity, glutamate
levels have to be maintained below this range in the extracellular
space. It has often been claimed that the termination of glutamate
transmission occurs primarily via the action of SKDGTs. However, as
shown in Table 3, the Km values of the
human SKDGT range between 30 and 97 µM and are consequently at least
1.5 order of magnitude higher than the resting levels of glutamate in
the extracellular space (~1 µM). Thus, it can be surmised that,
because of their high Km values, SKDGTs must also have high Vmax values
to efficiently take up extracellular glutamate. Alternatively,
mechanisms other than reuptake might be in charge of the termination of
glutamatergic transmission. Indeed, ionotropic EAAT receptors, like all
ligand-gated ion channel receptors, desensitize very rapidly. If
receptor desensitization is the primary mechanism of glutamate
inactivation, then SKDGTs may play a critical role in the
resensitization of glutamate receptors.
A large body of evidence supports the idea that elevated concentrations
of glutamate in the extracellular space are implicated in the
pathophysiology of various neurodegenerative diseases, such as
amyotrophic lateral sclerosis (ALS), Huntington's chorea, and
Alzheimer's disease (Appel, 1993; Portera-Caillau et al., 1995; Lesch
et al., 1996; Kanai, 1997). Moreover, glutamate has been shown to
induce severe damage during trauma, stroke, ischemia, anoxia, and
status epilepticus (Benveniste et al., 1984; Sandberg et al., 1986;
Sherwin et al., 1988; Storm-Mathisen et al., 1992; During and Spencer,
1993; Attwell and Mobbs, 1994; Szatkowski and Attwell, 1994; Kanai et
al., 1995).
A loss of EAAT-2, but not of other SKDGTs, has been reported in
Huntington's chorea (Arzberger et al., 1997), Alzheimer's disease
(Scott et al., 1995; Masliah et al., 1996; Li et al., 1997), and ALS
(Rothstein et al., 1992, 1995; Glenn Lin et al., 1998). For example, in
postmortem brains of patients who died with Huntington's chorea, the
decrease in EAAT-2 mRNA was shown to be proportional to the severity of
the illness (Arzberger et al., 1997). In contrast, EAAT-2 mRNA levels
are not altered in postmortem brain and spinal cord of patients
suffering from Alzheimer's disease or ALS. Therefore, the 30 to 90%
reduction of the EAAT-2 protein (Rothstein et al., 1992, 1995; Li et
al., 1997) in the latter neurodegenerative diseases (as compared to
normal controls) probably results from multiple aberrant processing of
its mRNAs (Glenn Lin et al., 1998). On the other hand, a direct
inhibitory effect of the amyloid precursor protein on EAAT-2 also
probably contributes to the reduction of excitatory aa reuptake
in Alzheimer's disease (Keller et al., 1997; Li et al., 1997).
Glutamatergic neurotransmission has also been shown to be implicated in
the pathophysiology of epilepsy. Alteration of EAAT has notably been
reported in amygdala-kindled rats (Akbar et al., 1997; Prince Miller et
al., 1997). In this model, EAAT-1 amounts are decreased (
60%) in the
piriform cortex with no change in other limbic areas (Prince Miller et
al., 1997). In contrast, EAAT-2 mRNA and protein are unchanged in
limbic areas (amygdala, piriform cortex, and hippocampus). Finally, the
levels of the neuronal subtype EAAT-3 are augmented in the piriform
cortex (+40%) and hippocampus (+28%) of these kindled animals (Prince
Miller et al., 1997).
Interestingly, typical and atypical neuroleptics have recently been
shown to affect glutamatergic transmission (Meshul et al., 1996). In
particular, chronic treatment with clozapine or haloperidol was found
to reduce the striatal levels of EAAT-2 mRNA in rats (Schneider et al.,
1998). In line with these observations, a deficit in EAA reuptake has
been described in the basal ganglia of schizophrenic patients (Simpson
et al., 1992). Accordingly, not only neurological disorders but also
psychiatric diseases might well be associated with altered functioning
of SKDGTs, especially EAAT-2.
Transient down-regulation of EAAT-1, -2, and -3 has been achieved
through the chronic i.c.v. infusion of antisense oligonucleotides in
rats (Rothstein et al., 1996). Thus, the brain levels of EAAT-1, -2, and -3 could be reduced by 85, 60, and 78%, respectively (Rothstein et al., 1996). Interestingly, the down-regulation of both
astroglial transporters, EAAT-1 and -2, was found to be associated with
a dramatic increase in the extracellular concentration of glutamate, by
13- and 32-fold, respectively. In contrast, an almost complete loss of
EAAT-3 had no affect on the extracellular levels of glutamate
(Rothstein et al., 1996). That EAAT-2 plays a key role in glutamate
reuptake has been additionally evidenced by Tanaka et al. (1997) who
showed that the knockout of its encoding gene results in a 94%
decrease in this process. In contrast, no changes in glutamate levels
and uptake could be detected in knockout mice that do not express
EAAT-3 (Peghini et al., 1997). Altogether, these data led to the
following order of SKDGT efficiency for the clearance of glutamate in
brain: EAAT-2 
EAAT-1 EAAT-3. Neuronal degeneration and epilepsy
are observed in mice with deficits in the glial transporters, EAAT-1
and -2, but not in those with low levels of EAAT-3 (Rothstein et al.,
1996; Peghini et al., 1997; Tanaka et al., 1997). Nevertheless,
behavioral studies showed that the loss of EAAT-1, -2, and -3 results
in locomotor impairment (Rothstein et al., 1996; Peghini et al., 1997;
Tanaka et al., 1997).
In conclusion, EAAT-2 seems to play a pivotal role in the control of
glutamatergic transmission and in neurodegenerative processes. The
physiological relevance of EAAT-1 and -3 seems to be more subtle and
remains to be further evaluated. Because null-mutant mice have proven
to be valuable models for the assessment of the respective roles of
EAAT-2 and -3 in EAA neurotransmission, the knockout of EAAT-1 and -4 are also eagerly expected to gain clear-cut information on the
physiological implications of these transporters.
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III. Vesicular Neurotransmitter Transporters (VNTs) |
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Neurotransmitters are synthesized in neurons, where they are
concentrated in vesicles for their subsequent
Ca2+-dependent release (see Fig. 1). Vesicular
transport has been demonstrated for several classical neurotransmitters
including acetylcholine (Toll and Howard, 1980), monoamines (Njus et
al., 1986), glutamate (Disbrow et al., 1982; Shioi et al., 1989; Tabb et al., 1992), GABA (Fykse and Fonnum, 1988), glycine (Kish et al.,
1989; Burger et al., 1991), and ATP (Luqmani, 1981). The accumulation
of intraneuronal neurotransmitters into storage vesicles acts as an
amplification step for the overall process of
Na+-dependent uptake of these molecules from the
extracellular space, since it controls their concentration gradient
across the plasma membrane. Moreover, vesicular accumulation of
neurotransmitters protects these molecules from leakage and/or
intraneuronal metabolism. Finally, this storage process also prevents
the possible toxic effects of neurotransmitters that could occur when
their cytoplasmic concentration exceeds a critical level.
A. Cloning of VNTs
1. The Vesicular Monoamine Transporter (VMAT)/Vesicular
Acetylcholine Transporter (VAChT) Family.
In 1992, two groups
successfully isolated cDNAs encoding the vesicular monoamine
(serotonin, DA, norepinephrine, epinephrine, and histamine)
transporters (VMATs, see Fig.
8) in the rat. On the one hand, Erickson
et al. (1992) isolated a cDNA from mRNA of rat basophilic leukemia
cells. This cDNA was shown to promote 5-HT vesicular accumulation in
transfected and permeabilized CV-1 cells (Erickson et al., 1992). On
the other hand, Y. Liu et al. (1992) used an expression cloning
strategy based on the observation that PC12 cells (derived from
pheochromocytoma) are resistant to the neurotoxic agent
MPP+. Thus, CHO cells were transfected with PC12
cDNA library and MPP+-resistant clones were
isolated. In fact, resistance to MPP+ could be
attributed to the vesicular accumulation of this agent, notably because
reserpine, a potent inhibitor of vesicular transport, restored its
toxicity in transfected CHO cells (Y. Liu et al., 1992).
View larger version (21K):
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Fig. 7.
Schematic diagram illustrating the functional
roles of neuronal EAAT. Glutamate released by an excitatory bouton acts
at postsynaptic glutamatergic receptors (EAAR); in most cases, this
results in postsynaptic membrane depolarization. The removal of
glutamate from the synaptic cleft is carried out by the transporter
EAAT-4 in the plasma membrane of the postsynaptic neuron. In addition,
EAAT-4 also acts as a Cl channel, leading to local
hyperpolarization.
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), and SVAT or MAT for synaptic vesicle
amine transporter or monoamine transporter (Erickson et al., 1992; Y. Liu et al., 1992). Then, because SVAT expression appeared not to be
restricted to synaptic vesicles, a new nomenclature was adopted: CGAT
was renamed VMAT-1, and SVAT/MAT, VMAT-2 (Fig. 8; Table
4).
|
; Howell et al.,
1994) and humans (Erickson and Eiden, 1993; Lesch et al., 1993; Surratt
et al., 1993; Erickson et al., 1996) (see Table 4). VMAT-1 is primarily
present in endocrine and paracrine cells of peripheral organs; on the
other hand, VMAT-2 is the predominant monoamine vesicular transporter
in the CNS, and it is also found in histaminergic cells of the stomach,
adrenal medulla, and blood cells (Mahata et al., 1993; Peter et al.,
1995; Weihe et al., 1995; Erickson et al., 1996). Finally, it has to be
noted that VMAT-1 and -2 are mutually exclusive except in human and rat
chromaffin cells where they are coexpressed.
Hydropathic analysis of the primary sequences of all these clones
predicts 12 putative transmembrane segments (Fig.
9). In addition, a large hydrophilic loop
with three to five potential sites of N-linked glycosylation
probably exists between transmembrane domains 1 and 2. This loop has
been proposed to be inside the lumen of the vesicle, whereas both the N
and C termini should be in the cytoplasm. Major sequence divergences
between the different clones occur at this large luminal loop and at
the N- and C-terminal domains (Fig. 9). Interestingly, VMATs show some
sequence homology with several drug resistance H+
antiporters in bacteria (Schuldiner, 1994).
|
). However, characterization of a
VMAT-1/VMAT-2 chimera revealed that the domains located between TM6 and
TM11 could account for the better affinity of VMAT-2 than VMAT-1 for
histamine (Erickson, 1998). Furthermore, TM1 and TM11 to TM12 appeared
to contain residues critical for the specific binding of
tetrabenazine (Peter et al., 1996; Erickson, 1998). The same conclusion
was reached independently by microsequencing proteolytic peptides from
VMAT-2 covalently modified by photoactivable tetrabenazine or
ketanserin derivatives (Sagné et al., 1997b; Sievert and Ruoho,
1997).
Site-directed mutagenesis also contributed to the identification of
residues involved in VMAT functioning. Thus, Asp33 in TM1 and the
triplicate Ser180, 181, and 182 in TM3 are probably implicated in
substrate recognition (Merickel et al., 1995). Asp404 in TM10 and
Asp431 in TM11 probably bind protons (Steiner-Mordoch et al., 1996),
and His419 in the fifth cytosolic loop appears to participate in the
energetic coupling of the antiport (Shirvan et al., 1994). Recently,
Finn and Edwards (1997) identified several residues between TM9 and
TM12 which are required for the high-affinity interactions of VMAT-2
with multiple ligands and also account for the preferential recognition
of serotonin over DA by this transporter.
Successful cloning of the vesicular transporter for acetylcholine,
VAChT, initially derived from studies in invertebrates. In a first
step, homology screening with a probe from unc-17, a gene
responsible for the integrity of neuromuscular function in the nematode
Caenorhabditis elegans (Alfonso et al., 1993), allowed the
isolation of DNA clones from Torpedo marmorata and Torpedo ocellata (Varoqui et al., 1994). The homology of
these clones with VMAT-1 and VMAT-2 (43% identity) led to their
classification in the same gene family (Fig. 8). Indeed, the
corresponding proteins expressed in fibroblasts were able to bind
vesamicol and to transport acetylcholine (Varoqui et al., 1994).
Mammalian VAChTs (human and rat) were subsequently isolated (Erickson
et al., 1994; Varoqui and Erickson, 1996) (see Table 4).
2. Vesicular Inhibitory Amino Acid Transporter.
Several genes
have been identified in the nematode C. elegans, the
mutations of which cause defects in GABAergic transmission (McIntire et
al., 1993). In particular, the defect observed in unc-47
mutant suggested that it was due to a loss of GABA transport into
synaptic vesicles. Subsequent studies based on this hypothesis actually
led to the isolation of the vesicular GABA (and glycine) transporter
(McIntire et al., 1997; Sagné et al., 1997a).
successfully rescued the unc-47
mutant phenotype with injection of cosmids having the unc-47
gene. The rescuing activity was precisely localized, and a cDNA
encoding the vesicular GABA uptake could be isolated. The rat cDNA
homolog, designated VGAT, was then used for the transfection of PC12
cells. Uptake measurement using vesicles isolated from these cells
demonstrated that VGAT is endowed with the capacity for accumulating
GABA (McIntire et al., 1997).
On the other hand, Sagné et al. (1997a) used another strategy
based on the screening of genome databases. They mapped the region
spanning the unc-47 gene and selected a cDNA possibly
encoding the C. elegans vesicular GABA/glycine transporter.
Indeed, the selected sequence corresponded to a protein with the
expected secondary structure (i.e., more than six putative
transmembrane domains) and size. This candidate was used for homology
screening in mouse and human genome expressed sequence tag databases,
and the putative mouse vesicular GABA/glycine transporter could then be
isolated from corresponding expressed sequence tag clones. Indeed, in
situ hybridization and expression experiments subsequently demonstrated
that this transporter actually corresponds to the vesicular
GABA/glycine transporter in the mouse brain (Sagné et al.,
1997a).
Comparison of the respective sequences of vesicular GABA transporter
(VGAT) and vesicular inhibitory aa transporter (VIAAT) that were cloned
independently by McIntire et al. (1997) and Sagné et al. (1997a),
respectively, clearly showed that they correspond to the same protein.
Further comparison of VGAT/VIAAT with VMAT and VAChT allowed the
conclusion that they did not belong to the same gene family (McIntire
et al., 1997; Sagné et al., 1997a). Indeed, VGAT/VIAAT is the
first member of a new neurotransmitter transporter family. Its putative
structure predicts 10 TMs, a long NH2- and a
short COOH-intracytoplasmic termini, and a large intraluminal loop
between TM1 and TM2 (Fig. 9B).
B. Regulation of Transport
VNT expression and activity can be controlled by both long- and
short-term regulatory processes, as shown notably in studies on VMAT-2
in chromaffin cells which endogenously express this transporter (Desnos
et al., 1992, 1995; Krejci et al., 1993; Mahata et al., 1993; Laslop et
al., 1994). Thus, chronic stimulation (for several days) of these cells
by a high extracellular concentration of K+ was
shown to produce a marked increase in VMAT-2 synthesis, as assessed by
measurement of [3H]tetrabenazine-specific
binding (Desnos et al., 1992, 1995). This suggests that a functional
link exists among cell stimulation, catecholamine secretion, and the
synthesis of VMAT-2 in chromaffin cells (Krejci et al., 1993; Desnos et
al., 1995).
Numerous consensus sites for protein kinases (PKA and PKC) are present
in intracytoplasmic portions of VMAT-1 and -2, suggesting that second
messengers are able to play a role in post-translational regulation of
VMAT activity. Relevant investigations performed on PC12 cells have
shown that activation of the cAMP pathway leads to a decrease in
vesicular monoamine uptake (Nakanishi et al., 1995). Additonal studies
on transfected cells (CHO, PC12, and COS) demonstrated that VMAT-2 is
constitutively phosphorylated at two consensus sites (Ser512 and 514)
for casein kinase II (Krantz et al., 1997). On the other hand, no
phosphorylation of VMAT-1 could be detected, suggesting the existence
of different regulatory mechanisms for the two subtypes (Krantz et al.,
1997). Finally, it has to be emphasized that directed mutagenesis of
Thr154 in rat VMAT (rVMAT)-2, a potential site for phosphorylation by
PKC, does not affect monoamine transport function (Merickel et al., 1995). Indeed, whether PKA- and/or PKC-mediated modulations of VMATs
transport function result from the phosphorylation of the transporters
themselves or of other interacting protein(s) is still an unsolved question.
The VAChT gene is contained within the first intron of the
gene encoding choline acetyltransferase (ChAT, the key enzyme for acetylcholine synthesis). Moreover, these two genes are transcribed in
the same orientation (Bejanin et al., 1994; Erickson et al., 1994).
Several factors (nerve growth factor, retinoic acid, trophic factors, cholinergic differentiation factor, cAMP) were initially reported to affect the levels of ChAT mRNA (Usdin et al., 1995), and it
was of interest to examine whether they could also influence VAChT
transcription. Indeed, coregulation of both ChAT and
VAChT gene transcription by retinoic acid and
differentiation factor/leukemia inhibitory factor was actually
demonstrated in cultured sympathetic neurons and septal cells (Berrard
et al., 1995; Berse and Blusztajn, 1995).
Thus, the VNTs appear to be regulated at the transcriptional, translational, and post-translational levels in the various cell types where they are expressed.
C. Ionic Dependence and Electrogenic Properties
The vesicular transport system consists of two components: an
ATP-driven H+ pump that, on the one hand,
acidifies the organelle lumen (
pH) and, on the other hand, generates
a potential gradient (
), and a transporter that exchanges
internal H+ ions with a given substrate
(Schuldiner et al., 1978; Johnson and Scarpa, 1979). Studies on the
electrochemical components of the vesicular transport allowed
stoichiometric calculations and showed that the steady-state monoamine
concentration gradient depends both on and on 2 × pH
(Njus et al., 1986; Rottenberg, 1986; Johnson, 1988; Nguyen et al.,
1998). In particular, these investigations clearly established that the
monoamine and the acetylcholine vesicular transporters exchange two
protons with one molecule of respective substrate. Interestingly,
nigericin, which exchanges K+ for
H+, reduced VMAT-2 activity by 65% but rVGAT
activity by only 40% (McIntire et al., 1997). This observation
supports the idea that the transport activity of the three VNTs is
differentially sensitive to the pH and/or the . In particular,
VMAT-2 appears to be more dependent than VGAT on pH (McIntire et
al., 1997).
D. Pharmacological Properties
It is well established that VMAT-1 and -2 recognize each monoamine
(5-HT, adrenaline, DA, histamine, noradrenaline) with high affinity
(Johnson, 1988; Erickson et al., 1992, 1996; Peter et al., 1994;
Merickel and Edwards, 1995). However, VMAT-2 has a higher affinity than
VMAT-1 for some monoamine substrates, notably histamine (Merickel and
Edwards, 1995). In addition, VMAT-2 is approximately 10-fold more
sensitive to the inhibitor tetrabenazine than VMAT-1. Metamphetamine
also inhibits preferentially VMAT-2, apparently by competing at the
site of amine recognition (Peter et al., 1994; Erickson et al., 1996).
However, reserpine has a similar affinity for both VMATs (Peter et al.,
1994; Erickson et al., 1996).
Numerous acetylcholine (ACh) derivatives are recognized by VAChT
(Parsons et al., 1993), some of which being actively transported, just
as well as ACh itself (Clarkson et al., 1992). Vesamicol has been
identified as a selective high-affinity inhibitor of the vesicular
accumulation of ACh (Erickson et al., 1994; Varoqui and Erickson,
1996).
Experiments using vesicular fractions from PC12 cells stably
transfected with the rVGAT-encoding sequence showed that -vinyl-GABA inhibits the vesicular GABA transport but with low potency
(Km = 5 mM) (McIntire et al., 1997).
Thus, high-affinity-selective VGAT inhibitors are eagerly expected.
Studies with native brain synaptic vesicles suggested that a common
transporter is responsible for the vesicular accumulation of both GABA
and glycine (Christensen and Fonnum, 1991). Direct demonstration of
this hypothesis was recently provided by Sagné et al. (1997a),
who showed that COS-7 cells transfected by the VIAAT-encoding sequence
actively accumulated both aas, and that GABA and glycine interacted
competitively with the same transport mechanism. These findings
accounted for the name chosen for this vesicular transporter: VIAAT.
E. Cellular and Subcellular Localization
VMATs and VAChTs are selective markers of monoaminergic and
cholinergic neurons, respectively (Schäfer et al., 1994; McIntire et al., 1997; Nirenberg et al., 1997; Sagné et al., 1997a). Thus, Efange et al. (1997) proposed the use of selective VAChT ligands as
probes for assessing the loss of cholinergic projections in Alzheimer-type dementias.
The localization of these transporters on the secretory vesicles in
axon terminals determines their prime role, i.e., to accumulate the
neurotransmitter for subsequent quantal release at the synapse. VAChT
is localized in small, clear synaptic vesicles of axon terminals that
make symmetric contacts with dendrites (Gilmor et al., 1996). VMAT-2 is
also expressed in small synaptic vesicles, but its main location in
axon terminals is on large dense core vesicles (Nirenberg et al.,
1997). This peculiar subcellular targeting of VMAT-2 addresses the
question of the role of large dense core vesicles in the storage and
release processes for classical neurotransmitters such as monoamines.
Somatic and dendritic expressions have also been observed for both
VAChT and VMAT-2. The somatic localization probably represents newly
synthesized proteins associated with the Golgi complex and the
endoplasmic reticulum (Nirenberg et al., 1995; Gilmor et al., 1996). At
the level of dendrites, expression of VMAT-2 and VAChT supports the
proposal that DA and ACh can be stored in and released from these
neurites (Arvidsson et al., 1997; Nirenberg et al., 1997).
In situ hybridization experiments showed that VIAAT/VGAT is synthesized
in GABAergic neurons (McIntire et al., 1997; Sagné et al.,
1997a). Furthermore, Sagné et al. (1997a) provided evidence that,
in some cells, VIAAT can be coexpressed with GLYT2, a specific marker
of glycinergic neurons. These data further support the idea that
VIAAT/VGAT is a neuronal vesicular transporter for both GABA and
glycine. Indeed, the protein has been observed in GABAergic as well as
glycinergic terminal boutons (Chaudhry et al., 1998; Dumoulin et al.,
1999). At the ultrastructural level, this VIAAT/VGAT is restricted to
small synaptic vesicles. Because this transporter: 1) can accumulate
glycine and GABA in synaptic vesicles and 2) is present in both types
of nerve endings, the name VIAAT should be adopted. Interestingly, some
GABAergic as well as glycinergic terminals are devoid of immunoreactive
VIAAT/VGAT (Chaudhry et al., 1998; Dumoulin et al., 1999). This
suggests that other member(s) of this vesicular transporter subfamily
are still to be cloned.
F. Dysfunctioning
Models for Neurodegenerative Disorders or Drug
Abuse
Vesicular sequestering seems to be crucial for protecting neurons from potential toxic effects of neurotransmitters and/or intraneuronal metabolites. In this context, possible implications of vesicular transporters in pathogenic mechanisms have been the subject of numerous studies.
Reserpine, an inhibitor of vesicular amine transport, has been used in
the treatment of hypertension because it potently reduces blood
pressure. However, high dosages frequently produce a disabling effect
which resembles that observed in depressed patients (Frize, 1954).
Accordingly, VMATs may also be implicated in psychiatric disorders.
Indeed, the hVMAT-2 gene is localized in the vicinity of
chromosomal breaks that have been identified in some mentally ill
patients with cutis verticis gyrata (Surratt et al., 1993).
MPP+ is a substrate for both the plasmic DA
transporter and VMATs. This neurotoxin has been used in numerous
studies aimed at generating relevant animal models of parkinsonism (Y. Liu et al., 1992, Liu et al., 1994; Stern-Bach et al., 1992; Edwards, 1993). In light of the neuroprotective effect resulting from the sequestration of MPP+ in synaptic vesicles, it
can be hypothesized that alterations in the activity of VMAT-2 may
contribute to the peculiar vulnerability of nigrostriatal dopaminergic
neurons in case of drug-induced and/or idiopathic Parkinson's disease
(Tanner and Langston, 1990). Attempts to enhance the vesicular
accumulation of DA and/or neurotoxic compounds may well be of
therapeutic interest for (early-stage) parkinsonian patients.
Mutant mice lacking VMAT-2 have recently been of great help to further
assess the physiological importance of this protein (Fon et al., 1997;
Takahashi et al., 1997; Wang et al., 1997). Indeed, VMAT-2 (/)
animals exhibit marked deficits in locomotor activity and feeding
behavior, and die shortly after birth. However, their brains present no
obvious morphological changes, and monoaminergic pathways do not seem
to be altered by the mutation (Fon et al., 1997). Nevertheless,
biochemical measurements showed marked reductions in the levels of
monoamines in hetero- and homozygous mutants (Fon et al., 1997;
Takahashi et al., 1997; Wang et al., 1997). In addition, the catalytic
activity of tyrosine and tryptophan hydroxylases is augmented in
homozygous animals (Wang et al., 1997), possibly as a compensatory
adaptation to the accelerated degradation of monoamines. Interestingly,
behavioral studies showed that the increased locomotor activity evoked
by acute treatment with amphetamine, cocaine, and apomorphine was
significantly higher in heterozygous VMAT-2(±) mice than in wild-type
animals (Fon et al., 1997; Takahashi et al., 1997; Wang et al., 1997).
A larger release of DA through both the blockade of vesicular monoamine transport and the reverse functioning of the plasma membrane
transporter (DAT) by amphetamine probably accounted for the differences
in the behavioral response to this drug in the mutant versus the wild-type mice. In line with this interpretation, Fon et al. (1997) reported that amphetamine was more efficient to trigger DA release in
primary cultures of midbrain dopaminergic neurones from knockout mice
than from wild-type animals. On the other hand, the increased behavioral response to apomorphine is in favor of the presence of
supersensitive DA receptors in heterozygous VMAT-2(±) mice. However,
direct investigations of D1- and D2-dopaminergic receptors, as well as
of 1A- and -adrenergic receptors and
5-HT1A and 5-HT2 serotoninergic receptors revealed no changes in the mutant mice as
compared to wild-type animals (Takahashi et al., 1997).
With regard to cholinergic neurons, the coordinate regulation of the
genes encoding VAChT and ChAT (Berrard et al., 1995) may play a key
role, especially during development in the central and peripheral
nervous systems. In addition, functional alterations of cholinergic
neurotransmission in Alzheimer-type dementias might also concern both
of these genes and their regulation.
| |
IV. Conclusion |
|---|
|
TopPreviousNextReferences
|
|---|
Recent progress in molecular cloning and the elucidation of
carriers' structures allowed the identification of several gene families encoding neurotransmitter transporters. In particular, two
families of plasma, as well as two families of vesicular, membrane
neurotransmitter transporter genes were isolated. These proteins are
Na+/Cl-,
Na+/K+-, or
H+-dependent. Their pharmacological profiles,
regulatory properties, regional and cellular localizations, and
implications in neuropathologies begin to be elucidated but a lot of
questions still have to be addressed. Clearly, the knowledge of the
precise ultrastructural localization of these transporters should
contribute to the understanding of their implications in the
physiological and pathophysiological functioning of specific synapses.
On the other hand, addition, alteration, or deletion of genes encoding
the transporters allow the generation of animal models that are of
considerable interest for assessing the actual role of these proteins,
notably in relevant psychiatric and neurological diseases.
Exploration of neurotransmitter transporters as cell-specific markers should also be a key for progressing in the knowledge of brain functioning. At present, antibodies specific of neurotransmitter synthesizing enzymes, such as tyrosine hydroxylase for catecholaminergic neurons and glutamic acid decarboxylase for GABAergic neurons, are generally used as markers of specific neuronal populations. However, neurons using excitatory aas as neurotransmitters cannot be specifically labeled by such probes. Antibodies raised against transporter(s) selectively expressed in these neurons should constitute alternative tools for their specific labeling and visualization.
| |
Acknowledgments |
|---|
|
TopPreviousNextReferences
|
|---|
We thank C. Sais for excellent secretarial assistance. We are also extremely grateful to Dr. S Supplisson for expert scientific advice and support.
| |
Footnotes |
|---|
1 Address for correspondence: S. El Mestikawy, INSERM U288, Faculté de Médecine Pitié-Salpêtrière, 91 Boulevard de l'Hôpital, 75634 Paris Cedex 13, France. E mail: elmesti{at}ext.jussieu.fr
| |
Abbreviations |
|---|
CNS, central nervous
system;
GABA, -aminobutyric acid;
SCDNT, Na+/Cl-dependent neurotransmitter
transporter;
GAT, GABA transporter;
ALS, amyotrophic lateral sclerosis;
aa, amino acid;
5-HT, 5-hydroxytryptamine, serotonin;
CHO, Chinese
hamster ovary;
ChAT, choline acetyltransferase;
ACh, acetylcholine;
PKC, protein kinase C;
PKA, protein kinase A;
DAT, dopamine
transporter;
DA, dopamine;
EAAT, excitatory amino acid transporter;
GLYT, glycine transporter;
PMA, phorbol 12-myristate 13-acetate;
SERT, serotonin transporter;
TM, -helical transmembrane domain;
PROT, proline transporter;
SKDGT, Na+/K+-dependent
glutamate transporter;
VAChT, vesicular acetylcholine transporter;
VGAT, vesicular GABA transporter;
VIAAT, vesicular inhibitory amino
acid transporter;
VMAT, vesicular monoamine transporter;
VNT, vesicular
neurotransmitter transporter;
rVMAT, rat VMAT;
MPP+, 1-methyl-4-phenylpyridinium;
NET, norepinephrine transporter.
| |
References |
|---|
|
TopPrevious |
|---|
A putative vesicular acetylcholine transporter.
Science (Wash DC)
261:
617-619)-coupled gamma-aminobutyric acid transporter from rat brain.
J Biol Chem
272:
1203-1210-aminobutyric acid (GABA) transport system.
J Biol Chem
267:
21098-21104Storage and transport characteristics.
Neuron
7:
287-293[Medline].-alanine-sensitive neuronal GABA transporter.
Neuron
9:
337-348[Medline].-dependent transporter family: Chromosomal localization and distribution in GABAergic and glutamatergic neurons in the rat brain.
J Neurochem
62:
445-455[Medline].-dependent transporter, in the central nervous system of rats and mice.
Neuroscience
77:
319-333[Medline].-aminobutyric acid by a synaptic vesicle fraction isolated from rat brain.
J Neurochem
50:
1237-1242[Medline].-aminobutyric acid uptake in primary astrocyte cultures by phorbol esters and phospholipase C.
Biochem J
275:
435-439.-aminobutyric acid transporter.
J Biol Chem
267:
17491-17493-dependent neurotransmitter transporter family in the rat CNS.
Eur J Neurosci
8:
127-137[Medline].-dependent "orphan" transporter Rxt1 in the rat central nervous system.
J Neurosci Res
42:
423-432[Medline].-dependent transporters Rxt1 and V-7-3-2 have an overlapping expression pattern in the rat central nervous system.
Recept Channels
4:
227-242.-dependent transporter Rxt1 on synaptic vesicles in the rat central nervous system.
Eur J Neurosci
11:
1349-1361.-aminobutyric acid transporter GAT1 is sorted to the apical membranes of polarized epithelial cells.
J Biol Chem
269:
4668-4674-aminobutyric acid transport glycoprotein from rat brain.
J Biol Chem
261:
15437-15441-aminobutyric acid transport glycoprotein from rat brain.
J Biol Chem
260:
11859-11865-Aminobutyric acid (GABA) uptake by Xenopus oocytes injected with rat brain mRNA.
Mol Brain Res
1:
97-100.Roles of membrane potential, pH gradient and intravesicular pH.
J Biol Chem
267:
15412-15418Homology with the putative vesicular acetylcholine transporter unc-17 from Caenorhabditis elegans.
FEBS Lett
342:
97-102[Medline].-organic solute cotransporter.
Am J Physiol
267:
F688-F694-dependent transporter that is regulated by hypertonicity.
J Biol Chem
267:
649-652
0031-6997/99/5103-0439$03.00/0
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 I. Velasco, S. Tenreiro, I. L. Calderon, and B. Andre Saccharomyces cerevisiae Aqr1 Is an Internal-Membrane Transporter Involved in Excretion of Amino Acids Eukaryot. Cell, December 1, 2004; 3(6): 1492 - 1503. [Abstract] [Full Text] [PDF]
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D. Soulet, B. Gagnon, S. Rivest, M. Audette, and R. Poulin A Fluorescent Probe of Polyamine Transport Accumulates into Intracellular Acidic Vesicles via a Two-step Mechanism J. Biol. Chem., November 19, 2004; 279(47): 49355 - 49366. [Abstract] [Full Text] [PDF]
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 M Camilleri Is there a SERT-ain association with IBS? Gut, October 1, 2004; 53(10): 1396 - 1399. [Full Text] [PDF]
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 A. Soragna, S. A. Mari, R. Pisani, A. Peres, M. Castagna, V. F. Sacchi, and E. Bossi Structural domains involved in substrate selectivity in two neutral amino acid transporters Am J Physiol Cell Physiol, September 1, 2004; 287(3): C754 - C761. [Abstract] [Full Text] [PDF]
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 D. Fegley, S. Kathuria, R. Mercier, C. Li, A. Goutopoulos, A. Makriyannis, and D. Piomelli From the Cover: Anandamide transport is independent of fatty-acid amide hydrolase activity and is blocked by the hydrolysis-resistant inhibitor AM1172 PNAS, June 8, 2004; 101(23): 8756 - 8761. [Abstract] [Full Text] [PDF]
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T. Harkany, C. Holmgren, W. Hartig, T. Qureshi, F. A. Chaudhry, J. Storm-Mathisen, M. B. Dobszay, P. Berghuis, G. Schulte, K. M. Sousa, et al. Endocannabinoid-Independent Retrograde Signaling at Inhibitory Synapses in Layer 2/3 of Neocortex: Involvement of Vesicular Glutamate Transporter 3 J. Neurosci., May 26, 2004; 24(21): 4978 - 4988. [Abstract] [Full Text] [PDF]
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 X. Mu, P. D. Beremand, S. Zhao, R. Pershad, H. Sun, A. Scarpa, S. Liang, T. L. Thomas, and W. H. Klein Discrete gene sets depend on POU domain transcription factor Brn3b/Brn-3.2/POU4f2 for their expression in the mouse embryonic retina Development, March 15, 2004; 131(6): 1197 - 1210. [Abstract] [Full Text] [PDF]
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N. Axmacher, M. Stemmler, D. Engel, A. Draguhn, and R. Ritz Transmitter Metabolism as a Mechanism of Synaptic Plasticity: A Modeling Study J Neurophysiol, January 1, 2004; 91(1): 25 - 39. [Abstract] [Full Text]
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J.-Y. Chatton, L. Pellerin, and P. J. Magistretti GABA uptake into astrocytes is not associated with significant metabolic cost: Implications for brain imaging of inhibitory transmission PNAS, October 14, 2003; 100(21): 12456 - 12461. [Abstract] [Full Text] [PDF]
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J. W. Schwartz, R. D. Blakely, and L. J. DeFelice Binding and Transport in Norepinephrine Transporters. REAL-TIME, SPATIALLY RESOLVED ANALYSIS IN SINGLE CELLS USING A FLUORESCENT SUBSTRATE J. Biol. Chem., March 7, 2003; 278(11): 9768 - 9777. [Abstract] [Full Text] [PDF]
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M. K.-H. Schafer, H. Varoqui, N. Defamie, E. Weihe, and J. D. Erickson Molecular Cloning and Functional Identification of Mouse Vesicular Glutamate Transporter 3 and Its Expression in Subsets of Novel Excitatory Neurons J. Biol. Chem., December 20, 2002; 277(52): 50734 - 50748. [Abstract] [Full Text] [PDF]
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S. Cavallaro, V. D'Agata, P. Manickam, F. Dufour, and D. L. Alkon Memory-specific temporal profiles of gene expression in the hippocampus PNAS, December 10, 2002; 99(25): 16279 - 16284. [Abstract] [Full Text] [PDF]
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A. M. Carneiro, S. L. Ingram, J.-M. Beaulieu, A. Sweeney, S. G. Amara, S. M. Thomas, M. G. Caron, and G. E. Torres The Multiple LIM Domain-Containing Adaptor Protein Hic-5 Synaptically Colocalizes and Interacts with the Dopamine Transporter J. Neurosci., August 15, 2002; 22(16): 7045 - 7054. [Abstract] [Full Text] [PDF]
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H. Varoqui, M. K.-H. Schafer, H. Zhu, E. Weihe, and J. D. Erickson Identification of the Differentiation-Associated Na+/PI Transporter as a Novel Vesicular Glutamate Transporter Expressed in a Distinct Set of Glutamatergic Synapses J. Neurosci., January 1, 2002; 22(1): 142 - 155. [Abstract] [Full Text] [PDF]
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R. D. Blakely Physiological Genomics of Antidepressant Targets: Keeping the Periphery in Mind J. Neurosci., November 1, 2001; 21(21): 8319 - 8323. [Abstract] [Full Text] [PDF]
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 G. Forlani, E. Bossi, R. Ghirardelli, S. Giovannardi, F. Binda, L. Bonadiman, L. Ielmini, and A. Peres Mutation K448E in the external loop 5 of rat GABA transporter rGAT1 induces pH sensitivity and alters substrate interactions J. Physiol., October 15, 2001; 536(2): 479 - 494. [Abstract] [Full Text] [PDF]
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C. M. l. Cour, C. Boni, N. Hanoun, K.-P. Lesch, M. Hamon, and L. Lanfumey Functional Consequences of 5-HT Transporter Gene Disruption on 5-HT1A Receptor-Mediated Regulation of Dorsal Raphe and Hippocampal Cell Activity J. Neurosci., March 15, 2001; 21(6): 2178 - 2185. [Abstract] [Full Text] [PDF]
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S. Eskandari, M. Kreman, M. P. Kavanaugh, E. M. Wright, and G. A. Zampighi Pentameric assembly of a neuronal glutamate transporter PNAS, July 18, 2000; 97(15): 8641 - 8646. [Abstract] [Full Text] [PDF]
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 A. Tahara, M. Nishibori, A. Ohtsuka, K. Sawada, J. Sakiyama, and K. Saeki Immunohistochemical Localization of Histamine N-Methyltransferase in Guinea Pig Tissues J. Histochem. Cytochem., July 1, 2000; 48(7): 943 - 954. [Abstract] [Full Text]
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S. Takamori, D. Riedel, and R. Jahn Immunoisolation of GABA-Specific Synaptic Vesicles Defines a Functionally Distinct Subset of Synaptic Vesicles J. Neurosci., July 1, 2000; 20(13): 4904 - 4911. [Abstract] [Full Text] [PDF]
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R. Russnak, D. Konczal, and S. L. McIntire A Family of Yeast Proteins Mediating Bidirectional Vacuolar Amino Acid Transport J. Biol. Chem., June 22, 2001; 276(26): 23849 - 23857. [Abstract] [Full Text] [PDF]
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L. Bai, H. Xu, J. F. Collins, and F. K. Ghishan Molecular and Functional Analysis of a Novel Neuronal Vesicular Glutamate Transporter J. Biol. Chem., September 21, 2001; 276(39): 36764 - 36769. [Abstract] [Full Text] [PDF]
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S. Takamori, J. S. Rhee, C. Rosenmund, and R. Jahn Identification of Differentiation-Associated Brain-Specific Phosphate Transporter as a Second Vesicular Glutamate Transporter (VGLUT2) J. Neurosci., November 15, 2001; 21(22): RC182 - RC182. [Abstract] [Full Text] [PDF]
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, potentially
N-glycosylated asparagine residue.
