Tepper, Abercrombie & Bolam (Eds.

)
Progress in Brain Research, Vol. 160
ISSN 0079-6123
Copyright r 2007 Elsevier B.V. All rights reserved

CHAPTER 2

GABA: Homeostatic and pharmacological aspects

Arne Schousboe and Helle S. Waagepetersen

Department of Pharmacology and Pharmacotherapy, The Faculty of Pharmaceutical Sciences, University of Copenhagen,
DK-2100 Copenhagen, Denmark

Abstract: The central nervous system (CNS) operates by a fine-tuned balance between excitatory and
inhibitory signalling. In this context, the inhibitory neurotransmission may be of particular interest as it has
been suggested that such neuronal pathways may constitute ‘command pathways’ and the principle of ‘dis-
inhibition’ leading ultimately to excitation may play a fundamental role (Roberts, E. (1974). Adv. Neurol.,
5: 127–143). The neurotransmitter responsible for this signalling is g-aminobutyrate (GABA) which was
first discovered in the CNS as a curious amino acid (Roberts, E., Frankel, S. (1950). J. Biol. Chem., 187:
55–63) and later proposed as an inhibitory neurotransmitter (Curtis, D.R., Watkins, J.C. (1960). J. Neuro-
chem., 6: 117–141; Krnjevic, K., Schwartz, S. (1967). Exp. Brain Res., 3: 320–336). The present review will
describe aspects of GABAergic neurotransmission related to homeostatic mechanisms such as biosynthesis,
metabolism, release and inactivation. Additionally, pharmacological and therapeutic aspects of this will be
discussed.

Keywords: GAD; GABA-T; transport; inhibitors; epilepsy

GABA metabolism GAD67 reflecting their molecular weights of 65

Biosynthesis
In the first report (Roberts and Frankel, 1950) des-
cribing GABA as an interesting amino acid
present in relatively large amounts in the brain it
was demonstrated that the biosynthetic pathway
consisted of a decarboxylation of L-glutamate cat-
alysed by the enzyme L-glutamate decarboxylase
(GAD). Subsequent studies led to a detailed char-
acterization and purification of this enzyme
(Roberts and Simonsen, 1963; Wu et al., 1973;
Wu and Roberts, 1974). Further studies including
cloning have revealed the presence of two distinct
isoforms of GAD referred to as GAD65 and
Corresponding author. Tel.: +45 3530 6330;
Fax: +45 3530 6021; E-mail: as@dfuni.dk
and 67 kDa, respectively (Martin and Rimval,
1993; Soghomonian and Martin, 1998). The two
isoforms of GAD, which are encoded for by
different genes, exhibit distinct differences with
regard to regulation and subcellular localization in
neurons. It appears that GAD65 mainly exists as a
dormant apoenzyme, which may be rapidly acti-
vated by binding of the coenzyme, pyridoxal phos-
phate. This together with the demonstration that it
is preferentially associated with nerve endings in
GABAergic pathways makes it highly interesting
in relation to biosynthesis of neurotransmitter
GABA (Martin and Rimval, 1993; Waagepetersen
et al., 2001). GAD67, on the other hand, is present
in the cytosol throughout the GABAergic neurons
and it appears to exist mainly as the catalytically
active holoenzyme having the coenzyme tightly
bound (Martin and Rimval, 1993). Both isoforms

DOI: 10.1016/S0079-6123(06)60002-2 9
10
are regulated by protein kinase-mediated phos-
phorylation, GAD65 being activated and GAD67
being inhibited (Martin and Rimval, 1993). Since
the first immunocytochemical demonstrations of a
selective localization of GAD in GABAergic neu-
rons (Saito et al., 1974; McLaughlin et al., 1974;
Ribak et al., 1976) it has been the general notion
that GAD is only expressed in such neurons and
hence, it has served as a marker for GABAergic
neurons. Recent studies of the distribution of
GAD particularly in hippocampal structures have,
however, presented evidence that GAD is present
also in certain glutamtergic neurons (Sloviter et al.,
1996; Gutierrez, 2003). The functional implica-
tions of this are not well understood but it may be
speculated that it could be related to the neurotro-
phic actions of GABA during early neuronal de-
velopment as demonstrated in a number of studies
(Belhage et al., 1998; Waagepetersen et al., 1999;
Fiszman and Schousboe, 2004). In this context it
may be of interest that it has recently been dem-
onstrated that glutamatergic cerebellar granule
neurons in culture are able to transport and con-
centrate GABA which is synthesized via GAD in
and subsequently released by a small population
of GABAergic neurons present in these cultures
of dissociated cerebellum from 7-day-old mice
(Sonnewald et al., 2004, 2006).
Although glutamate is the immediate precursor
for GABA biosynthesis it has been demon-
strated in different brain tissue preparations that
GLN GLU
GABA
Fig. 1. An illustration of two pathways for GABA synthesis from glutamine. The bold arrow illustrates the predominant pathway
where the carbon skeleton of glutamine is metabolized via the TCA cycle prior to synthesis of GABA. The thin arrow illustrates the
direct synthesis of GABA from glutamine without involvement of the TCA cycle.
glutamine is a more efficient exogenously supplied
substrate (Reubi et al., 1978; Westergaard et al.,
1995). This is in keeping with the concept that
replenishment of GABA in vivo is accomplished
by a combination of its reuptake in the presy-
naptic nerve ending (see below) and supply of
glutamine from surrounding astrocytes by the
operation of the GABA–glutamine–glutamate
cycle (Waagepetersen et al., 2003). The synthesis
of GABA from glutamine via glutamate requires
the presence of phosphate-activated glutaminase
(PAG) in GABAergic neurons, a prerequisite
fulfilled by the demonstration of considerable
PAG activity in such neurons (Larsson et al.,
1985). It may, however, be noted that the two
enzymes have different subcellular localizations,
PAG being mitochondrial and GAD cytosolic
(Balazs et al., 1966). Due to this mitochondria
could play a functional role in GABA biosynthe-
sis. Although for decades this was not investigated
seriously, it has recently been demonstrated using
13
C-labelled glutamine as a precursor for GABA
synthesis in GABAergic neurons that a large
fraction (460%) of newly synthesized vesicular
GABA originates from glutamate which has been
produced in a fashion involving mitochondria and
the tricarboxylic acid (TCA) cycle as illustrated in
Fig. 1 (Waagepetersen et al., 2001, 2003). This may
add regulatory mechanisms to the biosynthetic
machinery involved in replenishment of vesicular
GABA.
Acetyl CoA
TCA
CYCLE
α-KG
GLU

Degradation
The carbon skeleton of GABA is channelled into
the TCA cycle as succinate by the concerted action
of GABA-transaminase (GABA-T) and succinic
acid semialdehyde dehydrogenase (Schousboe
and Waagepetersen, 2007). Together with GAD
this constitutes the GABA-shunt (Fig. 2), which
represents an alternative route for converting
a-ketoglutarate to succinate in the TCA cycle
circumventing the succinyl-CoA step (Machiyama
et al., 1970). This shunt is considered to account
for about 10% of the flux through the TCA cycle
in the brain (Balazs et al., 1970; Machiyama et al.,
1970). It should be noted that this version of the
TCA cycle yields slightly less ATP than the nor-
mally functioning TCA cycle due to the lack of
succinyl-CoA production. The key enzyme of the
Fig. 2. Schematic representation of the TCA cycle and the
GABA-shunt, which provides an alternative pathway for
conversion of aKG to succinate. Abbreviations: aKG: a-keto-
glutarate; SSADH: succinate-semialdehyde; GABA-T: GABA-
transaminase; GABA: g-aminobutyrate; GAD: glutamate
decarboxylase; Glu: glutamate.
11
degradative part of the shunt is GABA-T, which is
present in both neurons and astrocytes and in
many other organs than the brain (Schousboe
et al., 1977a; Wu et al., 1978; Larsson et al., 1985;
Schousboe and Waagepetersen, 2007). This en-
zyme has been purified to homogeneity by several
investigators and characterized with regard to Km
values for the substrates GABA and a-ketogluta-
rate (Schousboe et al., 1973, 1974; Cash et al.,
1974; Bloch-Tardy et al., 1974; Maitre et al., 1975;
John and Fowler, 1976). A high metabolic rate
of conversion of GABA to CO2 in situ is compa-
tible with the low Km values for both substrates
(Machiyama et al., 1970; Yu and Hertz, 1983)
and the fact that GABA-T is a mitochondrial en-
zyme associated with matrix (Schousboe et al.,
1977b). It should be noted, however, that CO2
production per se does not represent a net deg-
radation of the C-4 unit derived from GABA
(Fig. 2). Complete oxidation of the carbon skele-
ton of GABA requires pyruvate recycling, i.e.,
conversion of malate to pyruvate via malic en-
zyme and subsequently conversion of pyruvate to
acetyl CoA and CO2 by pyruvate dehydrogenase
(Waagepetersen et al., 2003). This process, how-
ever, is only marginally present in neurons but
does take place in astrocytes, making the latter cell
type likely to be the major site for complete
oxidative metabolism of GABA (Schousboe and
Waagepetersen, 2006). This has some fundamental
consequences for maintenance of optimal GABA-
ergic activity in the CNS as discussed in detail re-
cently (Schousboe et al., 2004a,b,c).
In vivo and ex vivo monitoring of GABA metabolism
using NMR technology
GABA metabolism has been monitored using
proton and 13C NMR spectroscopy and diffe-
rent labelled substrates such as [1-13C]glucose,
[1,6-13C]glucose and [2-13C]acetate or [1-13C]glu-
cose in combination with [1,2-13C]acetate in
both normal animals and in various disease
models (Cerdan et al., 1990; Haberg et al., 2001;
Sonnewald and Kondziella, 2003; Patel et al.,
2005). 13C-labelled GABA was detected from
[1-13C]glucose in an in vivo 13C NMR study in
12
humans suggesting substantial GABA turnover
(Gruetter et al., 1998). Glucose is metabolized in
both neurons and astrocytes while acetate is a
marker of astrocytic metabolism due to selective
uptake into astrocytes (Waniewski and Martin,
1998). Thus, employing these substrates informa-
tion regarding compartmentalized metabolism
at the level of astrocytes and neurons can be ob-
tained. Simultaneous injection of [1-13C]glucose
and [1,2-13C]acetate into the same animal, glial
metabolism is resolved from [4,5-13C]glutamate
and glutamine and [1,2-13C]GABA is formed from
[4,5-13C]glutamine via transfer from the astrocytes
to the GABAergic neurons and thus the
GABA–glutamate–glutamine cycle activity can be
estimated. On the other hand, [2-13C]GABA
derived from [2-13C]acetyl CoA (formed from
[1-13C]glucose) reflects metabolism in the neuro-
nal TCA-cycle (Haberg et al., 2001).
In an ex vivo study using [1-13C]glucose or
[2-13C]acetate as substrates it was suggested that
3-NPA, which inhibits succinate dehydrogenase,
somewhat selectively inhibits the TCA cycle of
GABAergic neurons (Hassel and Sonnewald,
1995). Also vigabatrin which inhibits GABA-T in
the neuronal compartment due to selective neu-
ronal uptake (Schousboe et al., 1986; Pow et al.,
1996) has been used in the monitoring of GABA
metabolism in both in vivo and ex vivo experi-
ments (Hassel et al., 1998; de Graaf et al., 2006;
Patel et al., 2005, 2006). Evidence has been pro-
vided supporting the notion that the non-inhibited
fraction of GABA-T, primarily localized in the
astrocytes, is up-regulated due to a rise in the cel-
lular GABA content upon acute vigabatrin treat-
ment. Such explanation requires that GABA-T is
not saturated in the astrocytic compartment. These
considerations would explain an observed un-
changed total GABA level, rate of GABA synthe-
sis from glutamine and total GABA-T activity in
vigabatrin treated animals (de Graaf et al., 2006).
The rate of the GAD-mediated GABA synthesis
has been estimated in the range of 0.2–0.3 mmol/g
per min (Hassel et al., 1998; Patel et al., 2005). It
should be mentioned that in vivo NMR studies are
performed under conditions of anaesthesia and the
choice of drug has a significant impact on the re-
sults. For instance using a-chloralose the rate of
GAD was almost two-fold higher than that ob-
served in rats exposed to halothane (Mason et al.,
2001; Patel et al., 2005). GABA-shunt activity was
estimated to be approximately 17% of total cere-
bral TCA cycle activity using [1-13C]glucose in an
ex vivo NMR spectroscopy study in mice (Hassel
et al., 1998), a value being significantly higher than
those discussed above.
The fraction of GABA which is taken up by the
presynaptic neuron compared to that taken up by
surrounding astrocytes has only been estimated
from indirect measurements. From NMR studies
it has been estimated that the GABA–gluta-
mate–glutamine cycle accounts for approximately
23% of the total cycling of glutamine from as-
trocytes to neurons (Patel et al., 2005). The rate of
the GABA–glutamate–glutamine cycle and the
glucose oxidation rate of GABAergic neurons in-
crease with cortical activity in line, although to a
smaller extent, with that observed for gluta-
matergic neurons and the glutamate-glutamine
cycle (Patel et al., 2005). It has previously been
suggested that particularly GAD65 and not GAD67
may be associated with synthesis of neurotrans-
mitter GABA during neuronal activity, mainly due
to its localization in the nerve terminal (Kaufman
et al., 1991). The importance of GAD65 for cat-
alysing GABA synthesis during seizure was inves-
tigated in an experimental design using in vivo 1H
NMR spectroscopy in vigabatrin treated rats, a
treatment which exhibits a selective inhibitory
effect on GAD67 expression (Sheikh and Martin,
1998; Mason et al., 2001) and GAD65 was shown
to be responsible for the majority of GABA syn-
thesis during seizures (Patel et al., 2006). It was
furthermore suggested that an increase in Pi, a
known activator of apoGAD65, during seizures
might transform the large amount of dormant
apoGAD65 into the active holoform (de Graaf
et al., 2006).
GABA release and uptake
Vesicular and non-vesicular release
In keeping with its role as a neurotrans-
mitter, GABA is released from nervous tissue

upon depolarisation in a Ca2+-dependent manner
(Curtis and Johnston, 1974; Schousboe et al.,
1976; Otsuka, 1996). This represents vesicular re-
lease (Otsuka, 1996) which is the combined result
of vesicular filling by transport of cytosolic GABA
into vesicles via vesicular GABA transporters and
subsequent depolarisation-coupled, Ca2+-depen-
dent fusion of the vesicular and plasma mem-
branes (Otsuka, 1996).
It has been demonstrated in a number of
neuronal preparations that depending upon the
nature of the depolarising signal release of GABA
may occur not only from vesicles but also by re-
versal of the GABA transporters (see below), i.e.,
release of GABA originating from the cytoplasmic
pool of GABA (Pin and Bockaert, 1989; Bernath,
1992; Belhage et al., 1993). An important feature
of this non-vesicular release is that it can be
blocked by non-transportable GABA transport
inhibitors such as diphenyl-butenyl-nipecotic acid
and tiagabine di(methyl-thienyl)-butenyl-nipecotic
acid (Belhage et al., 1993; Waagepetersen et al.,
2001). An analogous release of GABA may also
occur from glial cells (Minchin and Iversen,
1974). Altogether, such non-vesicular release of
GABA occurring preferentially at extrasynaptic
sites could be of considerable functional im-
portance considering the recent demonstration
that extrasynaptic GABA receptors may medi-
ate tonic GABAergic inhibition (Mody, 2001;
Krogsgaard-Larsen et al., 2004; Schousboe et al.,
2004b).
Neuronal and glial transport
GABA uptake into inhibitory nerve terminals as
well as glial elements was first reported using au-
toradiographic analysis of [3H]GABA uptake into
brain slices (Hokfelt and Ljungdahl, 1970; Bloom
and Iversen, 1971; Iversen and Bloom, 1972; Hösli
and Hösli, 1976). Kinetic studies of GABA trans-
port in a number of nervous tissue preparations
including slices, homogenates, synaptosomes and
bulk-prepared glial cells demonstrated high-
affinity GABA transport systems (Iversen and
Johnston, 1971; Beart and Johnston, 1973; Balcar
and Johnston, 1973; Henn and Hamberger, 1971;
13
Levi and Raiteri, 1973). Subsequent kinetic studies
in primary cultures of astrocytes and GABAergic
neurons clearly confirmed that both of these cell
types express high-affinity GABA transporters
with Km values around 10–20 mM (Schousboe
et al., 1977a; Larsson et al., 1981). Numerous
recent immunohistochemical studies using anti-
bodies directed against the cloned GABA trans-
porters (see below) have likewise demonstrated
that both neurons (synapticly and extrasynapticly)
as well as astroglial cells express a variety of
GABA transporters (for references see Conti et al.,
2004; Madsen et al., 2007).
Cloned transporters
The advent of the purification of a sodium and
chloride dependent GABA transporting 80 kDa
glycoprotein from rat brain synaptosomes (Radian
et al., 1986) was instrumental in the subsequent
first cloning of a rat brain GABA transporter
(GAT1) having a Km for GABA of 7 mM and
exhibiting a Na+ and Cl dependence (Guastella
et al., 1990). Subsequently, two other GABA
transporters with Km values of 8 and 12 mM were
cloned from rat brain (Borden et al., 1992) and
referred to as GAT2 and GAT3. Four GABA
transporters were almost simultaneously cloned
from mouse brain and these were given the names
GAT1, GAT2, GAT3 and GAT4 (Liu et al., 1992,
1993). One of these (GAT2) is homologous to
the betaine transporter cloned from rat brain
(BGT-1) by Yamauchi et al. (1992) and hence the
nomenclature of the GABA transporters is some-
what confusing since rat GAT2 and rat GAT3
corresponds to mouse GAT3 and GAT4, respec-
tively (Schousboe and Kanner, 2002). In the fol-
lowing, the mouse nomenclature will be used and
therefore GAT2 is synonymous with BGT-1
(Schousboe and Kanner, 2002). It should be noted
that the Km for GABA of GAT2 is somewhat
higher than that for the other three transporters
(Bolvig et al., 1999). Nevertheless, as will be dis-
cussed below, this transporter may well be of con-
siderable interest in relation to fine-tuning of
GABAergic neurotransmission (Schousboe et al.,
2004b,c).
14
Pharmacology of metabolism and uptake
Enzyme inhibitors
Inhibitors of the GABA synthesizing enzyme
GAD mostly acting through the formation of
covalent attachment to the carbonyl group of the
coenzyme pyridoxal phosphate (e.g., formation of
hydrazones) are always acting as convulsants since
ultimately such action results in decreased GABA
levels (Tapia, 1975). Since, so far no strategy has
been found by which GAD activity can be en-
hanced, pharmacological intervention with GABA
synthesis has not been a promising avenue to
facilitate GABA neurotransmission. It should,
however, be kept in mind that the GABA cata-
bolic enzyme GABA-T, like GAD, is a pyridoxal-
phosphate requiring enzyme (Schousboe et al.,
1973). Therefore, inhibitors of GAD are generally
also inhibitors of GABA-T (Tapia, 1975). In con-
trast to the problem with inhibitors of GAD acting
as chemical convulsants (Tapia, 1975), inhibitors
of GABA-T should theoretically increase GABA
levels thereby acting as anti-convulsants (Tapia,
1975). Interestingly, aminooxyacetic acid has been
shown to inhibit both GAD and GABA-T being
10-fold more potent as a GABA-T inhibitor (Wu
and Roberts, 1974; Schousboe et al., 1974). Thus,
the compound can act either as a convulsant or as
an anti-convulsant drug depending on concentra-
tion (Tapia, 1975). It is therefore clear that if one
could develop a carbonyl trapping agent which
specifically would inhibit GABA-T leaving GAD
activity intact, it might be possible to develop an
anti-convulsant drug. Since GABA is a substrate
for GABA-T with no affinity for GAD, it was used
as a lead structure to develop an active site di-
rected inhibitor of GABA-T by introducing an al-
kylating entity into the GABA molecule (Lippert
et al., 1977). This led to development of g-vinyl
GABA and GABAculline, two highly specific
suicide inhibitors of GABA-T (Lippert et al.,
1977). These compounds when administered to
animals or added to cultured GABAergic neurons
are able to increase the synaptic GABA level sig-
nificantly (Iadarola and Gale, 1980; Wood et al.,
1981; Gram et al., 1988) which is likely to explain
the anti-convulsive efficiency (Schechter et al.,
1977). Gamma-vinyl GABA was subsequently de-
veloped into a clinically active drug (vigabatrin)
used to treat certain types of epilepsy (Krämer,
2004).
Transport inhibitors
Inactivation of GABA as a neurotransmitter oc-
curs exclusively by diffusion in the synaptic cleft
and active transport into presynaptic nerve end-
ings and astroglial cells ensheathing the synapse
(for references see Schousboe and Waagepetersen,
2007). It is therefore clear that inhibition of such
transport could lead to increased GABA levels in
the synapse and thus to enhanced efficacy of in-
hibitory neurotransmission (e.g., Schousboe et al.,
1983). As proposed by Schousboe et al. (1983) it
would appear most attractive to selectively inhibit
astroglial GABA transport as this would allow
presynaptic transport to facilitate replenishment of
vesicular GABA levels. A recent study using a se-
ries of astroglial GABA transport inhibitors as
anti-convulsants in audiogenic seizure prone
Fring’s mice has demonstrated that this principle
may well be correct emphasizing the important
role of astroglial GABA transport in the regula-
tion of GABAergic efficacy (White et al., 2002;
Schousboe et al., 2004a).
Development of GABA transport inhibitors has
been the topic of pharmacological characterization
of the GABAergic system for more than 30 years.
Hence, early studies led to the notion that
b-alanine and diaminobutyric acid would act as
selective inhibitors of glial and neuronal GABA
transport, respectively (see Iversen and Kelly,
1975). However, particularly the concept of
b-alanine being specific for glial GABA transport
has been questioned partly because it acts as a
substrate for the taurine carrier, which is predom-
inantly expressed in glia (Larsson et al., 1986).
Subsequent studies using the cloned GABA trans-
porters have actually provided evidence that
b-alanine preferentially inhibits GAT3 and
GAT4 both of which are mainly expressed in glial
cells (Borden, 1996).
An extensive characterization of the pharmacol-
ogy of neuronal and glial GABA transport was
 15

initiated by the advent of the demonstration of Clinical applications

THPO (4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-
3-ol) and its isostere nipecotic acid being active as
GABA transport inhibitors with no activity on
GABA receptors (Krogsgaard-Larsen and Johnston,
1975). Thus, in a series of studies using cultured as-
trocytes and neurons as well as the cloned trans-
porters (GAT1–4) expressed in human embryonic
kidney cells (HEK cells) a large number of GABA
analogues of restricted conformation using nipecotic
acid, THPO and exo-THPO (3-hydroxy-4-amino-
4,5,6,7-tetrahydro-1,2-benzisoxazol) as the GABA
mimetic entity have been characterized leading to
the identification of drug candidates (for over-
view see Schousboe et al., 2004a; Clausen et al.,
2006a,b; Madsen et al., 2007). Among these EF 1502
(N-[4,4-bis (3-methyl-2-thienyl)-3-butenyl]-4-(methyl-
amino)-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol) first
described by Clausen et al. (2005) may be of parti-
cular interest as will be discussed below. As seen in
Table 1, it represents an interesting GABA analogue
among the series of compounds developed using the
bicyclic isoxazole moity of THPO as a GABA
molecular scaffold. It is the only compound of this
very large library of GABA analogues (Falch et al.,
1999; Clausen et al., 2005, 2006a,b), which potently
inhibits both GAT1 and GAT2. All other com-
pounds are GAT1 selective. The importance of this
in relation to development of a second-generation
clinically active anti-epileptic drugs is discussed in
the following section.
The development of the clinically active anti-epi-
leptic drug tiagabine the action of which specifically
relates to its inhibition of GABA transport (Suzdak
and Jansen, 1995) has provided proof of principle
that GABA transporters are indeed relevant drug
targets for development of anti-epileptic drugs.
Likewise, the application of vigabatrin in treatment
of epilepsy shows that GABA-T is a suitable drug
target. Recent attempts to develop new GABA
analogues aimed at inhibiting GABA-T (Choi and
Silverman, 2002) have, however, not led to deve-
lopment of clinically active drug candidates.
The demonstration that the glial selective
GABA transport inhibitor N-methyl-exoTHPO,
being more than 100-fold less potent than tiaga-
bine with regard to inhibition of GABA transport
(Table 1) but essentially equipotent with tiagabine
as an anti-convulsant (Table 1 and White et al.,
2002) has provided evidence that glial GABA up-
take may be an interesting drug target. Moreover,
the finding that EF1502 which inhibits GAT2 and
GAT1 equipotently not only is as potent as tiaga-
bine as an anti-convulsant (White et al., 2005) but
actually acts synergistically with tiagabine when
administered in combination with this drug (White
et al., 2005) provides new insights into the possi-
bility that non-GAT1 inhibitors may be of clinical
interest (Clausen et al., 2006a,b). This notion is
further substantiated by the demonstration that

Table 1. Inhibitory parameters and anti-convulsant activity of selected GABA analogues

Inhibition of uptake IC50/Km/Ki (mM)a Anti-convulsant activity

ED50 (nmol icv.) (mg/kg
Neurons Glia GAT1 GAT2 GAT3 GAT4 i.p.)

GABA 8 32 17 51 15 17 –
Nipecotic acid 12 16 24 41000 113 159 –
THPO 501 262 1300 3000 800 5000 –
Exo-THPO 780 250 1000 3000 43000 43000 136
N-methyl-exo- 405 48 450 43000 43000 43000 59
THPO
EF 1500 4 2 3 4130 4100 4100 –
EF 1502 2 2 7 26 4300 4300 4.4
Tiagabine 0.5 0.2 0.1 4100 4100 4100 22
a
Values for GABA uptake (Km) and its inhibition (Ki/IC50). Anti-convulsant activity of some of the analogues in Fring’s audiogenic
seizure susceptible mice.
Values cited from Larsson et al. (1981), Bolvig et al. (1999), White et al. (2002, 2005) and Sarup et al. (2003).
16
EF1502 is less toxic than tiagabine, i.e., it exhibits
a more favourable therapeutic index (White et al.,
2005).
Concluding remarks
Although the basic metabolic and homeostatic
mechanisms governing functional aspects of
GABA-mediated neurotransmission have been
worked out decades ago, modern analytical tech-
nology such as NMR spectroscopy as well as
cloning of enzymes, transporters and receptors
have provided a wealth of information allowing a
much more sophisticated and detailed knowledge
about these matters to be obtained. Among other
things it has become clear that the astrocytic entity
of synapses has a profound importance for the
regulation of GABAergic function. This has led to
development of new therapeutic strategies by
which neurological disorders involving distur-
bances of GABAergic activity may be treated.
Acknowledgements
The expert secretarial assistance of Ms. Hanne
Danø is highly appreciated. The experimental
work has been supported by grants from the
Lundbeck, Hørslev and Benzon Foundations as
well as the Danish Medical Research Council
(22-03-0250 and 22-04-0314).
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