Pharmacology & Therapeutics 125 (2010) 394–401

Contents lists available at ScienceDirect

Pharmacology & Therapeutics

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / p h a r m t h e r a

Associate editor: H. Bönisch

Neuronal and non-neuronal GABA transporters as targets for antiepileptic drugs

Karsten K. Madsen a, H. Steve White b, Arne Schousboe a,⁎
a
Department of Pharmacology and Pharmacotherapy, Faculty of Pharmaceutical Sciences, University of Copenhagen, Copenhagen, Denmark
b
Anticonvulsant Drug Development Program, Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah, United States

a r t i c l e i n f o a b s t r a c t

Keywords: Epileptic seizure activity is associated with an imbalance between excitatory and inhibitory synaptic
Tiagabine activities. The latter is mediated by GABA, and several currently used antiepileptic drugs target entities of the
SNAP-5114 GABAergic synapse such as the receptors or the inactivation mechanism consisting of transmembrane
EF 1502
transport and enzymatic degradation. The development of tiagabine selectively inhibiting the GABA
Extrasynaptic GABA receptors
transporter GAT1 constitutes a proof of concept that the GABA transporters are interesting drug targets in the
Betaine/GABA transporter-1
context of antiepileptic drugs. The review provides a detailed analysis of the role of such transporters
pointing in particular to an interesting role of the transporters located extrasynaptically. It is suggested that
the betaine–GABA transporter BGT1 should receive particular interest in this context as the GABA analogue
EF 1502 (N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-4-(methylamino)-4,5,6,7-tetrahydrobenzo[d]isoxazol-
3-ol) has been shown to possess a novel anticonvulsant profile in animal models of epilepsy, involving the
ability to inhibit GABA transport mediated by GAT1 and BGT1 at the same time.
© 2009 Elsevier Inc. All rights reserved.

Contents

1. Epilepsy: a heterogeneous neurological disorder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394

2. GABAergic neurotransmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
3. Characterization of neuronal and non-neuronal GABA transporters . . . . . . . . . . . . . . . . . . . . . . 395
4. The era of cloning: impact on GAT pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
5. Localization of the GATs in healthy tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396
6. Introducing lipophilic aromatic side chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396
7. GAT inhibitors and the control of neuronal excitability . . . . . . . . . . . . . . . . . . . . . . . . . . 398
8. Anticonvulsant activity of GAT inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
9. Therapeutic aspects of GAT inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399

1. Epilepsy: a heterogeneous neurological disorder
Epilepsy is a heterogeneous neurological disorder characterized by
the onset of spontaneous convulsive and non-convulsive seizures.
Worldwide over 50 million people are affected and it is estimated that
only 50% of the patients are adequately treated for their symptoms with
the currently available antiepileptic drugs (AEDs). Of the remaining
⁎ Corresponding author. Department of Pharmacology and Pharmacotherapy,
Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2,
2100 Copenhagen, Denmark. Tel.: +45 35 33 63 30.
E-mail address: as@farma.ku.dk (A. Schousboe).
patients about 20% are either inadequately treated or treated at the
expense of severe side effects leaving about 30% of the patients with
refractory epilepsy (White, 1999; Madsen et al., 2009). A common
feature in epilepsy seems to involve hyperexcitable neurons which
discharge in a highly synchronized manner to produce a seizure. It has
been suggested that seizure activity is a consequence of an imbalance
between the inhibitory and excitatory neurotransmission (Dalby &
Mody, 2001). The current available AEDs can be divided into three
groups based on their mechanism of action which includes: modulation
of voltage-dependent ion-channels, attenuation of excitatory neuro-
transmission, and enhancement of the inhibitory neurotransmission
(Kwan et al., 2001). Inhibiting GABA transaminase (GABA-T), the
catabolic enzyme involved in the degradation of GABA, has been shown

0163-7258/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.pharmthera.2009.11.007
 K.K. Madsen et al. / Pharmacology & Therapeutics 125 (2010) 394–401
to increase the GABA content in nerve terminals thereby facilitating the
inhibitory GABAergic neurotransmission which mediates its anticon-
vulsant activity (Gale & Iadarola, 1980). Moreover, tiagabine, an
inhibitor of GABA transport which increases the extracellular levels of
GABA has been shown to mediate seizure protection (Nielsen et al.,
1991). However, increasing the amount of extracellular GABA does not
alleviate all seizure types. Actually, tiagabine is mainly used as an add-on
antiepileptic drug for treatment of partial seizures and unfortunately
tiagabine is not without severe adverse side effects (Schousboe & White,
2009). In fact absence seizures commonly seen in children can be
exacerbated by non-selectively increasing GABA levels (Inaba et al.,
2009).
2. GABAergic neurotransmission
After the independent discovery of GABA in 1950 by Awapara et al.
(1950), Roberts and Frankel (1950) and Udenfriend (1950) much
attention was given to this small flexible molecule. What has come to
be widely accepted today is that GABA is the principal inhibitory
neurotransmitter in the mammalian brain; although this notion was
highly controversial at first (Krnjevic, 2004). The major route of GABA
biosynthesis involves decarboxylation of glutamate yielding GABA
and CO2 via the enzyme glutamate decarboxylase (GAD) (Roberts &
Kuriyama, 1968). GAD exists in two isoforms GAD65 and GAD67 both
activated by pyridoxal 5′-phosphate with the former isoform being in
a readily inducible state pending neuronal activity and the latter
isoform being almost fully activated. GAD67 is found ubiquitously in
GABAergic neurons, whereas GAD65 is preferentially located in the
nerve terminals. Based on these findings it has been suggested that
GAD65 is specialized to readily synthesize GABA under short-term
demand (Martin & Rimvall, 1993). In the nerve terminals GABA can
be released to the synaptic cleft by two different pathways; i.e., via a
Ca2+ dependent vesicular release or Ca2+ independent release via
transporter reversal (Belhage et al., 1993). Upon release to the
synaptic cleft GABA interacts with GABA receptors located pre and
postsynaptically. Presynaptic GABA receptors metabotropically me-
diate a negative feedback mechanism on GABA release whereas,
postsynaptic GABA receptors can be both metabotropic (GABAB) and
ionotropic (GABAA) and mediate usually hyperpolarization of the cell
(Watanabe et al., 2002). After dissociation from the receptor complex
GABA is transported back into the presynaptic nerve terminal or into
surrounding astrocytes via a high affinity GABA transport system
thereby terminating GABA's inhibitory action (Iversen & Neal, 1968).
It has been estimated that the neuronal GABA transport system is
three- to six-fold more efficient than the astrocytic GABA transport
mechanisms which could indicate a reutilization of GABA taken up in
the neuron (Hertz & Schousboe, 1987). The degradation of GABA is
brought about via the enzymes GABA transaminase (GABA-T) and
succinic semialdehyde dehydrogenase yielding succinate. GABA-T is
located in both neurons and astrocytes with the highest activity in the
latter cell type (Schousboe et al., 1980).
3. Characterization of neuronal and non-neuronal GABA transporters
Ten years before Iversen and Neal (1968) proved the existence of
high affinity GABA transporters (GATs) in neurons and astrocytes,
Elliott and van Gelder (1958), demonstrated that GABA in the
incubation medium could accumulate in slices of cerebral cortices.
The accumulation of GABA in neurons and astrocytes can be blocked
by the GABA analogue diaminobutyric acid (DABA) and β-alanine,
respectively, revealing a very important difference in the pharmacol-
ogy of neuronal and astrocytic GAT's (Iversen & Neal, 1968; Iversen &
Kelly, 1975). It has been hypothesized that selective inhibition of
astrocytic GABA transport could possess superior anticonvulsant
properties compared to inhibition of neuronal GABA transport, since
DABA has been shown to be proconvulsant and GABA taken up in
395
astrocytes is metabolized and lost thereby draining the presynaptic
neuron of inhibitory neurotransmitter (Schousboe, 1990, 2000; White
et al., 2002).
The initial characterization of GATs has to a large extent been made
possible due to a naturally occurring compound from the fly agaric
mushroom, muscimol (Fig. 1). Muscimol is a potent GABAA receptor
agonist with weak inhibitory effect on GAT and is a substrate for the
GABA-T. The pharmacological profile of muscimol was separated into
the rigid GABA analogue, THIP (Gaboxadol, (4,5,6,7-tetrahydroisox-
azolo[5,4-c]pyridin-3-ol)) which is a selective extrasynaptic GABAA
receptor agonist (Wafford & Ebert, 2006; Cremers & Ebert, 2007) and
the rigid β-alanine analogue THPO ((4,5,6,7-tetrahydroisoxazolo[4,5-
c]pyridin-3-ol)) which retained GAT activity (Krogsgaard-Larsen &
Johnston, 1975). As can be seen from Table 1, β-alanine and THPO
display a two-fold selectivity for astrocytic GAT compared to neuronal
GAT. However, the conversion of the isoxazolol group of THPO to the
carboxyl group resulted in the generation of two compounds, nipecotic
acid and guvacine which are very potent inhibitors but with a
concomitant loss of selectivity for astrocytic GAT (Schousboe et al.,
1979, 1981). Nipecotic acid and guvacine have subsequently been used
as lead structures in the efforts to synthesize potent GAT inhibitors.
The glial selectivity of THPO prompted the synthesis of a series of
compounds where the secondary amino group of THPO was moved to
an exocyclic position becoming a primary amino group (exo-THPO)
(Fig. 1). The pharmacological action of selected GAT inhibitors from
this series is summarized in Table 1. Of special interest is its mono-
methylated derivative (N-methyl-exo-THPO) which is the most
selective inhibitor for astrocytic versus neuronal GAT seen until now
(Falch et al., 1999).
4. The era of cloning: impact on GAT pharmacology
The existence of pharmacologically different GAT populations on
neurons and astrocytes was further elaborated as cloning revealed
four subtypes of high affinity GATs. In 1986 an 80 kiloDalton (kDa)
glycoprotein was isolated from the rat brain with an apparent Km for
GABA transport of 3 μM and it showed a dependence for Na+ and Cl−
for transport. In comparison, the apparent Km for GABA for neuronal
and astrocytic GAT is 8 and 32 μM, respectively. Antibodies raised
against GAT failed to cross-react with GABA receptors suggesting little
homology between these proteins (Radian et al., 1986). Four years
later Guastella et al. (1990) succeeded in cloning this transporter and
designated it GAT-1 which consists of 599 amino acids with a Km for
GABA of 7 μM. Inhibitors of neuronal and glial GATs suggested that
GAT-1 displayed a similar pharmacology as the neuronal GAT
(Guastella et al., 1990). That same year the human GAT-1 was cloned.
It was found to consist of 599 amino acids (Nelson et al., 1990). From
the mouse, four GATs have been cloned and named GAT1, GAT2,
GAT3, and GAT4 encoding proteins of 598, 614, 602, and 627 amino
acids, respectively with a Km for GABA of 7, 79, 18, and 0.7 μM,
respectively. GAT2 also shows affinity for betaine with a Km of 398 μM
(Liu et al., 1992, 1993). From canine, human and rat, a betaine–GABA
transporter (BGT-1) has been cloned which is homologous to mouse
GAT2, encoding proteins of 614 amino acids for the two former
species and 628 amino acids for the latter species, and like mouse
GAT2 they show a greater affinity for GABA than for betaine
(Yamauchi et al., 1992; Borden et al., 1995; Burnham et al., 1996).
The rat GAT-2 and GAT-3 clones are homologues of mouse GAT3 and
GAT4 encoding proteins of 602 and 627 amino acids, respectively with
a Km for GABA of 8 and 12 μM. Both were sensitive to β-alanine
suggesting a greater similarity to glial GAT (Borden et al., 1992;
Christiansen et al., 2007). The human GAT-2 and GAT-3 clones are also
homologous to mouse GAT3 and GAT4 and encode proteins of 602 and
632 amino acids (Borden et al., 1994; Christiansen et al., 2007).
Unfortunately, the cloning of the four GATs has resulted in a rather
confusing nomenclature. Table 2 summarizes the GAT nomenclature
396 K.K. Madsen et al. / Pharmacology & Therapeutics 125 (2010) 394–401
Fig. 1. Chemical structures of muscimol, THIP (Gaboxadol) and selected of GABA uptake inhibitors used for the characterization of neuronal and non-neuronal GABA transporters.
categorized in columns for species homology. Throughout the
remainder of this review the nomenclature as proposed by the
HUGO Gene Nomenclature Committee will be used. It seems that
GAT1 behaves as the neuronal GAT whereas GAT2 and GAT3 resemble
the astrocytic GAT when transport of GABA is inhibited with DABA
and β-alanine, respectively.
5. Localization of the GATs in healthy tissue
GAT1 and GAT3 have been found exclusively in the brain, whereas
GAT2 and BGT1 have been found in multiple other organs. GAT1 is
widely distributed throughout the entire brain (Durkin et al., 1995)
and its expression closely follows that of GABAergic pathways (Radian
et al., 1990). GAT1 is primarily found on presynaptic neurons in the
synapse and to a minor extent on distal astrocytic processes in close
proximity to axon terminals forming symmetric synapses (Borden,
1996; Conti et al., 1998). In polarized Madin Darby Canine Kidney
(MDCK) cells GAT1 was after transfection into the cells found
exclusively on the apical surface suggesting an axonal localization
(Pietrini et al., 1994). The localization of GAT3 is more restricted when
compared to GAT1 showing strong intensity in retina, olfactory bulb,
brainstem, diencephalon and low levels in hippocampus and cortex.
GAT3 is predominantly expressed on distal astrocytic processes in
direct contact with GABAergic neurons (Durkin et al., 1995; Minelli
et al., 1996). Expression in MDCK cells places GAT3 on the apical
surface (Ahn et al., 1996; Borden, 1996). The finding of GAT3 on
neurons in human tissue has been investigated further and it is
debated whether this localization is an artifact introduced after the
tissue has been removed in response to metabolic stress (Melone
et al., 2005). The expression of GAT2 in the brain is very limited and it
is found in the leptomeninges and the neonatal brain and maybe at
very low levels throughout the brain at extrasynaptic sites on both
neurons and astrocytes (Liu et al., 1993; Borden, 1996; Conti et al.,
1999). BGT1 has been found in the hippocampus and cortex,
surprisingly not located closely to GABAergic synapses but in an
extrasynaptic region involving to a large extent astrocytes (Borden
et al., 1995; Zhu & Ong, 2004) which could indicate a specialized
pharmacological action different from the other GATs. Both GAT2 and
BGT1 are found on the basolateral membrane in polarized MDCK cells
after transfection (Ahn et al., 1996) strikingly opposite to the
localization of GAT1 and GAT3. In the developing mouse cerebral
cortex, the onset of GAT1 expression in the axon terminals is closely
related to synapse formation. GAT3 expression follows a couple of
days later with astrocyte sealing of the synapse (Takayama & Inoue,
2005). Fig. 2 is a graphical representation of the primary localization
of GAT1, BGT1 and GAT3 summarized in one synapse.
6. Introducing lipophilic aromatic side chains
The subtype selectivity of THPO and the exocyclic derivatives
showed an interesting feature; i.e., they were all GAT1 selective but the
inhibitory effect was greater on astrocytic GAT rather than neuronal
GAT. This was somewhat unexpected given the localization of GAT1.
Direct intracerebroventricular injections of N-methyl-exo-THPO, N-
ethyl-exo-THPO, exo-THPO, and tiagabine were evaluated for anti-
convulsant activity in the audiogenic seizure (AGS) susceptible Frings
mouse, a reflex seizure model that is non-discriminatory with respect
to seizure type. The IC50 value of the drugs obtained in neurons and
 K.K. Madsen et al. / Pharmacology & Therapeutics 125 (2010) 394–401 397

Table 1
Inhibition of GABA uptake by selected GABA analogues.
Data summarized from Schousboe (1979), Larsson et al. (1981, 1983, 1986, 1988), Suzdak
et al. (1992), Borden (1996), Thomsen et al. (1997), Bolvig et al. (1999), Falch et al. (1999),
White et al. (2002) and Sarup et al. (2003).

Compound GABA uptake inhibition IC50 or ⁎Km/i (μM)

Neurons Astrocytes GAT1 GAT2 GAT3 GAT4

GABA 8a 32a 17a 51a 15a 17a

Nipecotic acid 12 16 24 >1000 113 159
Guvacine 32 29 39 >1000 228 378
DABA 1000 >5000 128 528c 300 710
ACHC 200 700 132 1070c >1000 >10,000
β-Alanine 1666b 843b 2920 1100c 66 110
THPO 501b 262b 1300 3000 800 5000
Exo-THPO 780 250 1000 3000 >3000 >3000
N-methyl-exo-THPO 405 48 450 >3000 >3000 >3000
N-ethyl-exo-THPO 390 301 320 >1000 >1000 >1000
N-2-hydroxyethyl- 300 200 >500 >500 >500 >500
exo-THPO
N-4-phenylbutyl- 100 15 7 >500 >1000 >1000
exo-THPO
N-acetyloxyethyl- 200 18 550 >1000 >1000 >1000 Fig. 2. A graphical representation of the primary subcellular localization of GAT1, BGT1, and
exo-THPO GAT3. GAT1 is primarily located presynaptically. GAT3 is primarily located on distal astrocytic
(R/S)-EF1502 2 2 7 26 >300 >300 processes in close proximity to the synapse. BGT1 is located in the extrasynaptic region.
(R)-EF1502 1.5 0.65 4 22 >150 >150
(S)-EF1502 >100 >100 120 34 >150 >150
N-DPB-THPO 38b 26 30 200 >300 >1000
Overcoming this obstacle to produce systemically active com-
N-DPB-Nipecotic acid 1.3b 2.0b 0.64 7210c 550 4390
N-DPB-guvacine 4.9b 4.2b – – – – pounds was initiated by introducing lipophilic aromatic side chains
N-DPB-exo-THPO 1.4 0.6 6 100 >100 >100 such as 4,4-diphenylbut-3-en-1-yl (DPB) to the parent compounds
N-DPB-N-methyl- 5 2 2 200 >100 >100 such as R-nipecotic acid and guvacine yielding SKF89976A and
exo-THPO SKF100330A, respectively. These SKF compounds were approximately
NNC 05-2090 – – 19b 1.4b 41b 15b
20 times more potent than the parent structures thereby producing
SNAP-5114c – – 388 140 21 5
NO-711 1.24 0.64 – – – – inhibitors with an IC50 in the mid nanomolar range. Moreover, the two
Tiagabine 0.45 0.18 0.11 >100 >100 800 compounds showed no inhibitory effect towards 3H-muscimol
DABA: diaminobutyric acid; ACHC: amino-cyclohexane-carboxylic acid. binding in the nanomolar range confirming GAT selectivity (Yunger
a
Km. et al., 1984; Ali et al., 1985).
b
Ki. Based on this discovery, guvacine, R- and S-nipecotic acid were
c
Human homologue of BGT1. used as the GABA mimetic moiety in the synthesis of more potent GAT
inhibitors in which the length and electro-negativity of the linker was
astrocytes correlated extremely well with their anticonvulsant activity altered along with the di-aromatic region at the end of the linker
in the AGS susceptible Frings mouse. Furthermore, the anticonvulsant (Fig. 3) (Braestrup et al., 1990; Andersen et al., 1993, 1994, 1999;
activity correlated (r2 = 0.988) nicely with the IC50 obtained in the Knutsen et al., 1999; Andersen et al., 2001a,b). One compound which
astrocytes contrary to the IC50 in neurons (r2 = 0.5336) suggesting that gained particular interest was tiagabine (Gabitril®), a derivative of R-
selective inhibition of astrocytic GAT has therapeutic potential (White nipecotic acid. Tiagabine is approved for the treatment of partial
et al., 2002). epileptic seizures and is the only GAT inhibitor currently available for
The compounds described so far possess IC50 values in the low the treatment of epilepsy. Recently, selected compounds from the
micromolar range (Table 1); however, the therapeutic potential of above mentioned series were reevaluated for inhibitory effect on the
these molecules is limited due to their inability to penetrate the BBB at four cloned GATs in order to supplement the inhibitory effect
physiological pH. This inability to penetrate the blood–brain barrier is previously measured on synaptosomes. Not surprisingly these
due to the zwitterion characteristic of the drugs which is governed by compounds were found to preferentially inhibit GAT1; however,
the ionized/unionized (I/U) ratio of the compounds (Krogsgaard- some effect was also seen on the other subtypes. It seems that by
Larsen et al., 2000). increasing the length of the carbohydrate linker of diphenylamine
derivates a greater inhibitory effect was obtained on the non-GAT1
subtypes along with an almost unchanged affinity for GAT1; i.e., they
Table 2 lost their GAT1 preference (Clausen et al., 2006).
GABA transporter nomenclature across species.
Very few compounds developed possess selectivity towards non-
Species Nomenclature GAT1 subtypes. SNAP-5114 (Fig. 3) was synthesized and tested on the
SLC6 gene SLC6a1 SLC6a12 SLC6a13 SLC6a11
human GAT clones and found to display an 80-fold selectivity towards
a b c
GAT3 over GAT1 (Borden et al., 1994). This selectivity is not as
Rat GAT-1 BGT-1 GAT-2 GAT-3c
pronounced in mice where the selectivity between mouse GAT3 and
Human GAT-1d BGT-1e GAT-2f GAT-3g
Mouse GAT1h GAT2h GAT3h GAT4h GAT1 is about 30-fold (Kragler et al., 2005). A series of compounds
HUGO GAT1 BGT1 GAT2 GAT3 have recently been synthesized using proline and pyrrolidine-2-acetic
a
Guastella et al. (1990). acid as the GABA mimetic moiety substituted with three lipophilic side
b
Burnham et al. (1996). chains DPB, the side chain from tiagabine (4,4-[di(3-methylthiophen-
c
Borden et al. (1992). 2-yl)]phenylbut-3-en-1-yl), and the side chain from SNAP-5114 (2-
d
Nelson et al. (1990). [tris(4-methoxyphenyl)methoxy]ethyl). The results obtained with
e
Borden et al. (1995).
f
Christiansen et al. (2007).
these molecules suggest that the SNAP-5114 side chain conveys
g
Borden et al. (1994). selectivity towards GAT3 which is independent of the affinity of the
h
Liu et al. (1993). GABA mimetic moiety towards GAT1 and GAT3 (Fulep et al., 2006).
398 K.K. Madsen et al. / Pharmacology & Therapeutics 125 (2010) 394–401
Fig. 3. General structure of GAT inhibitors. The vertical dashed lines cut the structure into the diaryl-region, the linker, and the amino acid moiety. DPB is presented as an example of
an aromatic side chain. Tiagabine, EF1502, SNAP-5114 and Lu-32-176B are the compounds used for anticonvulsant testing as described in this review.
Unfortunately, a drug with a better selectivity profile than SNAP-5114
has not been found in either study, thereby leaving SNAP-5114 as one
of the best commercially available pharmacological tools for evaluat-
ing the impact of GAT2/3 inhibition.
Recently, the development of EF1502 ((RS)-4-[N-[1,1-bis(3-methyl-
2-thienyl)but-1-en-4-yl]-N-methylamino]-4,5,6,7-tetrahydrobenzo[d]
isoxaxol-3-ol) in which N-methyl-exo-THPO serves as GABA mimetic
moiety substituted with the lipophilic side chain from tiagabine (Fig. 3)
was reported to be a GAT1/BGT1 selective inhibitor. Interestingly, GAT1
selectivity was confined to R-EF1502 whereas the BGT1 selectivity was
independent of the stereochemistry of the GABA mimetic moiety
(Table 1) (Clausen et al., 2005). In summary, tiagabine, SNAP-5114, and
EF1502 represent the three currently available drugs for investigation of
the therapeutic potential of the different GAT subtypes.
In addition to the subtype selectivity of the amino acid based
inhibitors like DABA and β-alanine and the lipophilic aromatic inhibitors
like tiagabine, EF1502, and SNAP-5114, these compounds also display
different kinetics regarding inhibition of GABA uptake and some act as
substrates for transport. While the simple amino acid related inhibitors
(Fig. 1) have proven to be competitive substrates for the transporter, the
lipophilic derivatives (Fig. 3) display multiple forms of kinetics and are
not substrates (Larsson et al., 1988). From the recently reevaluated
lipophilic compounds described above, the individual inhibitors showed
differences in kinetic profiles between the GAT subtypes (Clausen et al.,
2006). The therapeutic implication of different kinetics of GAT inhibitors
has not been investigated; however, theoretical considerations can be
made. Competitive inhibitors work at low concentrations of extracel-
lular GABA and loose effect due to competition as the concentration of
GABA increases. Non-competitive inhibitors, on the other hand, are
independent of the extracellular concentration of GABA if the occupancy
of the available transporters is nearly complete. However, if the
occupancy is low as may be the situation in-vivo, the non-competitive
inhibitor will behave as a competitive inhibitor, i.e. its effect will be
concentration dependent.
Why have we failed in producing non-GAT1 selective drugs? The
reason for this could be a consequence of the parent structures used to
synthesize new analogues and the fact that the in-vitro assays
employed express primarily GAT1, thereby missing lead structures for
non-GAT1 inhibitors.
7. GAT inhibitors and the control of neuronal excitability
Neuronal excitability is controlled by two different inhibitory
mechanisms mediated through synaptic and extrasynaptic GABA
receptors. Synaptically located GABAA receptors mediate a phasic
inhibition in response to high concentrations (low micromolar) of
intermittently released GABA from the presynaptic neuron. Extra-
synaptically located GABAA receptors respond to ambient levels of
GABA that have diffused away from the synapse and mediate a
continuous tonic inhibition and show no, or very little, desensitization
in contrast to their synaptic counterparts (Saxena & MacDonald, 1994;
Brown et al., 2002).
From adult rat hippocampal slices the tonic conductance in
granule cells of the dentate gyrus is about 4 times larger than
spontaneous inhibitory postsynaptic currents (IPSC) when activated
only by ambient GABA. Furthermore, by inhibiting GAT1 the tonic
inhibition was increased by 330% without affecting spontaneous IPSC
(sIPSC) and zolpidem enhanced the phasic conductance by 66%
without producing any effect on the tonic current (Nusser & Mody,
2002). From 3 to 4 week old guinea pig hippocampal slices a tonic
conductance was seen in interneurons but not in pyramidal cells
under basal conditions. The tonic current was pharmacologically
different from phasic currents and much larger. Inhibition of GAT1
produced a comparable tonic inhibition in pyramidal cells as that
observed in interneurons under basal conditions without affecting the
tonic conductance in interneurons. Reducing the tonic conductance in
interneurons increases the inhibitory drive to pyramidal cells
(Semyanov et al., 2003). Microdialysis and electrophysiological
studies on GAT1 knockout mice versus wildtype mice show that
KCl-evoked release of GABA was significantly increased along with a
tonic conductance in layer II/III pyramidal cells. Moreover, the decay
time of evoked IPSC (eIPSC) was prolonged significantly contrary to
spontaneous IPSC. Immunohistochemistry confirms that GAT2 or
GAT3 expression was not altered; however, an increase in GAD65/67
expression was observed. These results demonstrate that GAT1
regulates both phasic and tonic GABAA receptor mediated inhibition
in the cerebral cortex of mice (Bragina et al., 2008). In monkey globus
pallidus the inhibition of either GAT1 or GAT3 increased the ambient
GABA concentration resulting in a decreased neuronal firing (Galvan
et al., 2005). Recordings of GABA currents from rat neocortical slices
(P17–22) were evaluated in the presence of either NO711 (GAT1
selective inhibitor) or SNAP-5114 (GAT2/3 selective inhibitor) or a
combination of both. NO711 decreased the amplitude and increased
the decay time of eIPSC, whereas SNAP-5114 was without effect.
However, co-application synergistically decreased the amplitude and
increased the decay time. Neither NO711 nor SNAP-5114 alone
increased the tonic inhibitory current but co-application substantially
increased it. Taken together these results indicate that GAT1 regulates
 K.K. Madsen et al. / Pharmacology & Therapeutics 125 (2010) 394–401
the phasic inhibitory currents in the synapse whereas GAT1 and
GAT2/3 in combination can regulate the extrasynaptic tonic inhibitory
currents (Keros & Hablitz, 2005). SNAP-5114 has also been shown to
increase GABA levels in the thalamus to 247 ± 27% of basal conditions
but had no effect on GABA levels in the hippocampus, whereas
tiagabine was able to increase GABA levels both in the thalamus and
hippocampus (Dalby, 2000). Neuronal excitability can be fine tuned
by the use of GAT inhibitors. However, whether phasic or tonic
inhibitory currents are activated seems to depend on the neuronal
network being investigated, which undoubtedly is a consequence of
both GABA transporter and receptor density and subtype expression.
8. Anticonvulsant activity of GAT inhibitors
The therapeutic potential of GAT inhibition has been confirmed
with the successful development of the GAT1 selective drug tiagabine
(Nielsen et al., 1991), albeit tiagabine has limited efficacy in epilepsy
management and exhibits numerous side effects such as agitation,
sedation and psychotic-like episodes in patients predisposed to
psychiatric illness (Schousboe & White, 2009). Unfortunately, the
development of selective non-GAT1 inhibitors has been disappoint-
ing. However, the GAT1/BGT1 inhibitor EF1502 and the GAT2/GAT3
inhibitor SNAP-5114 do allow us to evaluate the therapeutic potential
of non-GAT1 inhibitors although the drugs are only semi-selective and
the results must be conservatively interpreted. An interesting
question is: to what extent does the individual GATs contribute to
the control of neuronal excitability?
The electrophysiological data presented above suggests different
mechanisms of action of GAT1 inhibitors depending on the circuitry
being investigated, and it is conceivable that the same fact holds for the
other GAT subtypes. In-vivo studies in AGS susceptible Frings mice have
attempted to address this question. From an isobologram study
conducted by White et al. (2005) we gained several important pieces
of information about the combination between GAT1 and BGT1
inhibitors. The two selective GAT1 inhibitors tiagabine and LU-32-
176B in combination resulted in an additive effect at three fixed dose
mixtures indicating that both drugs indirectly interacted with the same
GAT population. Interestingly, the combination of the GAT1/BGT1
selective inhibitor EF1502 with either tiagabine or LU-32-176B (GAT1
selective) produced a synergistic anticonvulsant interaction at all dose
mixtures, strongly indicating that the additional inhibition of BGT1 was a
requirement for the synergism. The combination between tiagabine and
EF1502 was also reevaluated in a model of partial epilepsy, the corneal
kindling model described by Matagne and Klitgaard (1998) and the
synergistic effect was seen at one dose mixture (Madsen et al., 2009). The
same study examined the effect of combining SNAP-5114, the GAT2/3
inhibitor with tiagabine. This resulted in an additive anticonvulsant
effect at all dose mixtures, whereas SNAP-5114 in combination with
EF1502 produced a synergistic interaction in one dose mixture and an
additive interaction in the two others (Madsen et al., 2009).
GAT inhibitors time dependently increase the extracellular level of
GABA which then interacts with different GABA receptor populations
thereby facilitating inhibitory neurotransmission. Thus, GAT inhibi-
tors only produce an indirect inhibition of neuronal excitability. It is
known that tiagabine, EF1502 and SNAP-5114 possess anticonvulsant
activity in several established models of epilepsy (Nielsen et al., 1991;
Dalby, 2000; White et al., 2005). The isobologram studies suggest that
in order to achieve synergism, BGT1 needs to be inhibited along with
either GAT1 or GAT3. A possible explanation for this can be found in
the localization of the GATs (see Fig. 2). As described previously, GAT1
is localized to the presynaptic neuron and perisynaptic distal
astrocytic processes (Borden, 1996), GAT3 is located exclusively on
distal astrocytic processes perisynaptically (Durkin et al., 1995;
Minelli et al., 1996), and BGT1 is found exclusively extrasynaptically
at asymmetric synapses (Zhu & Ong, 2004). It is very likely that the
synergism obtained using EF1502 arises because GABA levels rise
399
extrasynaptically as a result of inhibition of BGT1 which then interacts
with a GABA receptor population that is different from those GABA
receptors activated by inhibition of GAT1 and GAT3. Therefore it could
be argued that in order to achieve synergism one should use GAT
inhibitors that produce a different regional increase in GABA levels
and thus activate different GABA receptor populations; e.g., synaptic
and extrasynaptic. On this basis it may be argued that development of
inhibitors of extrasynaptically located GABA transporters such as
BGT1 may represent a novel avenue to obtain antiepileptic drugs with
a therapeutic potential different from that of tiagabine.
9. Therapeutic aspects of GAT inhibitors
Tight regulation of the synthesis, release, and removal of
synaptically released GABA is of fundamental importance for the
maintenance of brain function at all levels. In addition to their value in
controlling seizure activity in patients with epilepsy, it is highly likely
that drugs acting on GABA transporters may represent potential
therapeutic candidates for the treatment of other neurologic and
psychiatric conditions associated with dysfunction of the GABA
system; e.g., anxiety, sleep disorders, chronic pain, post-traumatic
stress disorder, migraine, and others. Actually, several clinically
effective drugs used for the treatment of these disorders do in fact
act through an interaction with GABA receptors; i.e., the benzodia-
zepines for the treatment of anxiety and sleep disorders. In this
context, inhibitors of GABA transporters exert their beneficial effects
by making more GABA available at synaptic and extrasynaptic GABA
receptors. Although the development of THIP (Gaboxadol) for the
treatment of sleep disorders has been halted, it certainly demon-
strates the therapeutic potential whereby increasing extrasynaptic
GABA levels could play an important role in the control of CNS
function (Krogsgaard-Larsen et al., 2004). In this context, it is
becoming increasingly clear that the anatomical distribution, regula-
tion and trafficking of GABA transporters may have important
implications for the treatment of a number of neurological disorders
and diseases. To this point, a greater understanding of the molecular
biology, distribution, and factors that regulate their function will be
critical for developing a new class of therapeutic agents that target
this important regulator of the CNS function.
Acknowledgment
The secretarial assistance of Ms. Hanne Danø is cordially
acknowledged. The experimental work has been supported by the
Lundbeck Foundation (R19-A2199).
References
Ahn, J., Mundigl, O., Muth, T. R., Rudnick, G., & Caplan, M. J. (1996). Polarized expression
of GABA transporters in Madin–Darby canine kidney cells and cultured hippocam-
pal neurons. J Biol Chem 271, 6917–6924.
Ali, F. E., Bondinell, W. E., Dandridge, P. A., Frazee, J. S., Garvey, E., Girard, G. R., et al.
(1985). Orally active and potent inhibitors of gamma-aminobutyric acid uptake.
J Med Chem 28, 653–660.
Andersen, K. E., Begtrup, M., Chorghade, M. S., Lee, E. C., Lau, J., Lundt, B. F., et al. (1994).
The synthesis of novel GABA uptake inhibitors. 2. Synthesis of 5-hydroxytiagabine,
a human metabolite of the GABA reuptake inhibitor tiagabine. Tetrahedron 50,
8699–8710.
Andersen, K. E., Braestrup, C., Grønwald, F. C., Jørgensen, A. S., Nielsen, E. B., Sonnewald,
U., et al. (1993). The synthesis of novel GABA uptake inhibitors. 1. Elucidation of the
structure–activity studies leading to the choice of (R)-1-[4,4-bis(3-methyl-2-
thienyl)-3-butenyl]-3-piperidinecarboxylic acid (tiagabine) as an anticonvulsant
drug candidate. J Med Chem 36, 1716–1725.
Andersen, K. E., Lau, J., Lundt, B. F., Petersen, H., Huusfeldt, P. O., Suzdak, P. D., et al.
(2001). Synthesis of novel GABA uptake inhibitors. Part 6: preparation and
evaluation of N-Omega asymmetrically substituted nipecotic acid derivatives.
Bioorg Med Chem 9, 2773–2785.
Andersen, K. E., Sørensen, J. L., Huusfeldt, P. O., Knutsen, L. J., Lau, J., Lundt, B. F., et al.
(1999). Synthesis of novel GABA uptake inhibitors. 4. Bioisosteric transformation
and successive optimization of known GABA uptake inhibitors leading to a series of
potent anticonvulsant drug candidates. J Med Chem 42, 4281–4291.
400 K.K. Madsen et al. / Pharmacology & Therapeutics 125 (2010) 394–401
Andersen, K. E., Sørensen, J. L., Lau, J., Lundt, B. F., Petersen, H., Huusfeldt, P. O., et al.
(2001). Synthesis of novel gamma-aminobutyric acid (GABA) uptake inhibitors.
5. (1) Preparation and structure–activity studies of tricyclic analogues of known
GABA uptake inhibitors. J Med Chem 44, 2152–2163.
Awapara, J., Landua, A. J., Fuerst, R., & Seale, B. (1950). Free gamma-aminobutyric acid in
brain. J Biol Chem 187, 35–39.
Belhage, B., Hansen, G. H., & Schousboe, A. (1993). Depolarization by K+ and glutamate
activates different neurotransmitter release mechanisms in GABAergic neurons:
vesicular versus non-vesicular release of GABA. Neuroscience 54, 1019–1034.
Bolvig, T., Larsson, O. M., Pickering, D. S., Nelson, N., Falch, E., Krogsgaard-Larsen, P., et al.
(1999). Action of bicyclic isoxazole GABA analogues on GABA transporters and its
relation to anticonvulsant activity. Eur J Pharmacol 375, 367–374.
Borden, L. A. (1996). GABA transporter heterogeneity: pharmacology and cellular
localization. Neurochem Int 29, 335–356.
Borden, L. A., Dhar, T. G., Smith, K. E., Branchek, T. A., Gluchowski, C., & Weinshank, R. L. (1994).
Cloning of the human homologue of the GABA transporter GAT-3 and identification of a
novel inhibitor with selectivity for this site. Receptors Channels 2, 207–213.
Borden, L. A., Smith, K. E., Gustafson, E. L., Branchek, T. A., & Weinshank, R. L. (1995).
Cloning and expression of a betaine/GABA transporter from human brain. J Neurochem
64, 977–984.
Borden, L. A., Smith, K. E., Hartig, P. R., Branchek, T. A., & Weinshank, R. L. (1992).
Molecular heterogeneity of the gamma-aminobutyric acid (GABA) transport
system. Cloning of two novel high affinity GABA transporters from rat brain. J Biol
Chem 267, 21098–21104.
Braestrup, C., Nielsen, E. B., Sonnewald, U., Knutsen, L. J., Andersen, K. E., Jansen, J. A., et al.
(1990). (R)-N-[4, 4-bis(3-methyl-2-thienyl)but-3-en-1-yl]nipecotic acid binds with
high affinity to the brain gamma-aminobutyric acid uptake carrier. J Neurochem 54,
639–647.
Bragina, L., Marchionni, I., Omrani, A., Cozzi, A., Pellegrini-Giampietro, D. E., Cherubini,
E., et al. (2008). GAT-1 regulates both tonic and phasic GABA(A) receptor-mediated
inhibition in the cerebral cortex. J Neurochem 105, 1781–1793.
Brown, N., Kerby, J., Bonnert, T. P., Whiting, P. J., & Wafford, K. A. (2002).
Pharmacological characterization of a novel cell line expressing human alpha(4)
beta(3)delta GABA(A) receptors. Br J Pharmacol 136, 965–974.
Burnham, C. E., Buerk, B., Schmidt, C., & Bucuvalas, J. C. (1996). A liver-specific isoform
of the betaine/GABA transporter in the rat: cDNA sequence and organ distribution.
Biochim Biophys Acta 1284, 4–8.
Christiansen, B., Meinild, A. K., Jensen, A. A., & Brauner-Osborne, H. (2007). Cloning and
characterization of a functional human gamma-aminobutyric acid (GABA)
transporter, human GAT-2. J Biol Chem 282, 19331–19341.
Clausen, R. P., Madsen, K., Larsson, O. M., Frolund, B., Krogsgaard-Larsen, P., & Schousboe, A.
(2006). Structure–activity relationship and pharmacology of gamma-aminobutyric
acid (GABA) transport inhibitors. Adv Pharmacol 54, 265–284.
Clausen, R. P., Moltzen, E. K., Perregaard, J., Lenz, S. M., Sanchez, C., Falch, E., et al. (2005).
Selective inhibitors of GABA uptake: synthesis and molecular pharmacology of 4-N-
methylamino-4, 5, 6, 7-tetrahydrobenzo[d]isoxazol-3-ol analogues. Bioorg Med
Chem 13, 895–908.
Conti, F., Melone, M., De Biasi, S., Minelli, A., Brecha, N. C., & Ducati, A. (1998). Neuronal
and glial localization of GAT-1, a high-affinity gamma-aminobutyric acid plasma
membrane transporter, in human cerebral cortex: with a note on its distribution in
monkey cortex. J Comp Neurol 396, 51–63.
Conti, F., Zuccarello, L. V., Barbaresi, P., Minelli, A., Brecha, N. C., & Melone, M. (1999).
Neuronal, glial, and epithelial localization of gamma-aminobutyric acid transporter
2, a high-affinity gamma-aminobutyric acid plasma membrane transporter, in the
cerebral cortex and neighboring structures. J Comp Neurol 409, 482–494.
Cremers, T., & Ebert, B. (2007). Plasma and CNS concentrations of gaboxadol in rats
following subcutaneous administration. Eur J Pharmacol 562, 47–52.
Dalby, N. O. (2000). GABA-level increasing and anticonvulsant effects of three different
GABA uptake inhibitors. Neuropharmacology 39, 2399–2407.
Dalby, N. O., & Mody, I. (2001). The process of epileptogenesis: a pathophysiological
approach. Curr Opin Neurol 14, 187–192.
Durkin, M. M., Smith, K. E., Borden, L. A., Weinshank, R. L., Branchek, T. A., & Gustafson, E. L.
(1995). Localization of messenger RNAs encoding three GABA transporters in rat
brain: an in situ hybridization study. Brain Res Mol Brain Res 33, 7–21.
Elliott, K. A., & van Gelder, N. M. (1958). Occlusion and metabolism of gamma-
aminobutyric acid by brain tissue. J Neurochem 3, 28–40.
Falch, E., Perregaard, J., Frølund, B., SLkilde, B., Buur, A., Hansen, L. M., et al. (1999).
Selective inhibitors of glial GABA uptake: synthesis, absolute stereochemistry, and
pharmacology of the enantiomers of 3-hydroxy-4-amino-4, 5, 6, 7-tetrahydro-1, 2-
benzisoxazole (exo-THPO) and analogues. J Med Chem 42, 5402–5414.
Fulep, G. H., Hoesl, C. E., Hofner, G., & Wanner, K. T. (2006). New highly potent GABA
uptake inhibitors selective for GAT-1 and GAT-3 derived from (R)- and (S)-proline
and homologous pyrrolidine-2-alkanoic acids. Eur J Med Chem 41, 809–824.
Gale, K., & Iadarola, M. J. (1980). Seizure protection and increased nerve-terminal
GABA: delayed effects of GABA transaminase inhibition. Science 208, 288–291.
Galvan, A., Villalba, R. M., West, S. M., Maidment, N. T., Ackerson, L. C., Smith, Y., et al.
(2005). GABAergic modulation of the activity of globus pallidus neurons in
primates: in vivo analysis of the functions of GABA receptors and GABA
transporters. J Neurophysiol 94, 990–1000.
Guastella, J., Nelson, N., Nelson, H., Czyzyk, L., Keynan, S., Miedel, M. C., et al. (1990).
Cloning and expression of a rat brain GABA transporter. Science 249, 1303–1306.
Hertz, L., & Schousboe, A. (1987). Primary cultures of GABAergic and glutamatergic
neurons as model systems to study neurotransmitter functions. I Differentiated
cells. In A. Vernadakis, A. Privat, J. M. Lauder, P. S. Timiras, & E. Giacobini (Eds.),
Model systems of development and aging of the nervous system (pp. 19–32). Boston:
Martinus Nijhoff Publishing.
Inaba, Y., D'Antuono, M., Bertazzoni, G., Biagini, G., & Avoli, M. (2009). Diminished
presynaptic GABA(B) receptor function in the neocortex of a genetic model of
absence epilepsy. Neurosignals 17, 121–131.
Iversen, L. L., & Kelly, J. S. (1975). Uptake and metabolism of gamma-aminobutyric acid
by neurones and glial cells. Biochem Pharmacol 24, 933–938.
Iversen, L. L., & Neal, M. J. (1968). The uptake of [3H]GABA by slices of rat cerebral
cortex. J Neurochem 15, 1141–1149.
Keros, S., & Hablitz, J. J. (2005). Subtype-specific GABA transporter antagonists
synergistically modulate phasic and tonic GABAA conductances in rat neocortex.
J Neurophysiol 94, 2073–2085.
Knutsen, L. J., Andersen, K. E., Lau, J., Lundt, B. F., Henry, R. F., Morton, H. E., et al. (1999).
Synthesis of novel GABA uptake inhibitors. 3. Diaryloxime and diarylvinyl ether
derivatives of nipecotic acid and guvacine as anticonvulsant agents. J Med Chem 42,
3447–3462.
Kragler, A., Hofner, G., & Wanner, K. T. (2005). Novel parent structures for inhibitors of
the murine GABA transporters mGAT3 and mGAT4. Eur J Pharmacol 519, 43–47.
Krnjevic, K. (2004). How does a little acronym become a big transmitter? Biochem
Pharmacol 68, 1549–1555.
Krogsgaard-Larsen, P., Frolund, B., & Frydenvang, K. (2000). GABA uptake inhibitors. Design,
molecular pharmacology and therapeutic aspects. Curr Pharm Des 6, 1193–1209.
Krogsgaard-Larsen, P., Frolund, B., Liljefors, T., & Ebert, B. (2004). GABA(A) agonists and
partial agonists: THIP (Gaboxadol) as a non-opioid analgesic and a novel type of
hypnotic. Biochem Pharmacol 68, 1573–1580.
Krogsgaard-Larsen, P., & Johnston, G. A. (1975). Inhibition of GABA uptake in rat brain
slices by nipecotic acid, various isoxazoles and related compounds. J Neurochem 25,
797–802.
Kwan, P., Sills, G. J., & Brodie, M. J. (2001). The mechanisms of action of commonly used
antiepileptic drugs. Pharmacol Ther 90, 21–34.
Larsson, O. M., Thorbek, P., Krogsgaard-Larsen, P., & Schousboe, A. (1981). Effect of
homo-∃-proline and other heterocyclic GABA analogues on GABA uptake in neurons
and astroglial cells and on GABA receptor binding. J Neurochem 37, 1509–1516.
Larsson, O. M., Johnston, G. A. R., & Schousboe, A. (1983). Differences in uptake kinetics
of cis-3-aminocyclohexane carboxylic acid into neurons and astrocytes in primary
cultures. Brain Res 260, 279–285.
Larsson, O. M., Griffiths, R., Allen, I. C., & Schousboe, A. (1986). Mutual inhibition kinetic
analysis of (γ-aminobutyric acid, taurine, taurine and β-alanine high affinity
transport into neurons and astrocytes: evidence for similarity between the taurine
and β-alanine carriers in both cell types. J Neurochem 47, 426–432.
Larsson, O. M., Falch, E., Krogsgaard-Larsen, P., & Schousboe, A. (1988). Kinetic
characterization of inhibition of gamma-aminobutyric acid uptake into cultured
neurons and astrocytes by 4, 4-diphenyl-3-butenyl derivatives of nipecotic acid
and guvacine. J Neurochem 50, 818–823.
Liu, Q. R., Lopez-Corcuera, B., Mandiyan, S., Nelson, H., & Nelson, N. (1993). Molecular
characterization of four pharmacologically distinct gamma-aminobutyric acid
transporters in mouse brain [corrected]. J Biol Chem 268, 2106–2112.
Liu, Q. R., Mandiyan, S., Nelson, H., & Nelson, N. (1992). A family of genes encoding
neurotransmitter transporters. Proc Natl Acad Sci U S A 89, 6639–6643.
Madsen, K. K., Clausen, R. P., Larsson, O. M., Krogsgaard-Larsen, P., Schousboe, A., &
White, H. S. (2009). Synaptic and extrasynaptic GABA transporters as targets for
anti-epileptic drugs. J Neurochem 109(Suppl 1), 139–144.
Martin, D. L., & Rimvall, K. (1993). Regulation of gamma-aminobutyric acid synthesis in
the brain. J Neurochem 60, 395–407.
Matagne, A., & Klitgaard, H. (1998). Validation of corneally kindled mice: a sensitive
screening model for partial epilepsy in man. Epilepsy Res 31, 59–71.
Melone, M., Barbaresi, P., Fattorini, G., & Conti, F. (2005). Neuronal localization of the
GABA transporter GAT-3 in human cerebral cortex: a procedural artifact? J Chem
Neuroanat 30, 45–54.
Minelli, A., DeBiasi, S., Brecha, N. C., Zuccarello, L. V., & Conti, F. (1996). GAT-3, a high-
affinity GABA plasma membrane transporter, is localized to astrocytic processes,
and it is not confined to the vicinity of GABAergic synapses in the cerebral cortex.
J Neurosci 16, 6255–6264.
Nelson, H., Mandiyan, S., & Nelson, N. (1990). Cloning of the human brain GABA
transporter. FEBS Lett 269, 181–184.
Nielsen, E. B., Suzdak, P. D., Andersen, K. E., Knutsen, L. J., Sonnewald, U., & Braestrup, C.
(1991). Characterization of tiagabine (NO-328), a new potent and selective GABA
uptake inhibitor. Eur J Pharmacol 196, 257–266.
Nusser, Z., & Mody, I. (2002). Selective modulation of tonic and phasic inhibitions in
dentate gyrus granule cells. J Neurophysiol 87, 2624–2628.
Pietrini, G., Suh, Y. J., Edelmann, L., Rudnick, G., & Caplan, M. J. (1994). The axonal
gamma-aminobutyric acid transporter GAT-1 is sorted to the apical membranes of
polarized epithelial cells. J Biol Chem 269, 4668–4674.
Radian, R., Bendahan, A., & Kanner, B. I. (1986). Purification and identification of the
functional sodium- and chloride-coupled gamma-aminobutyric acid transport
glycoprotein from rat brain. J Biol Chem 261, 15437–15441.
Radian, R., Ottersen, O. P., Storm-Mathisen, J., Castel, M., & Kanner, B. I. (1990).
Immunocytochemical localization of the GABA transporter in rat brain. J Neurosci
10, 1319–1330.
Roberts, E., & Frankel, S. (1950). γ-Aminobutyric acid in brain: its formation from
glutamic acid. J Biol Chem 187, 55–63.
Roberts, E., & Kuriyama, K. (1968). Biochemical-physiological correlations in studies of
the gamma-aminobutyric acid system. Brain Res 8, 1–35.
Sarup, A., Larsson, O. M., Bolvig, T., Frølund, B., Krogsgaard-Larsen, P., & Schousboe, A.
(2003). Effects of 3-hydroxy-4-amino-4, 5, 6, 7-tetrahydro-1, 2-benzisoxazol (exo-
THPO) and its N-substituted analogs on GABA transport in cultured neurons and
astrocytes and by the four cloned mouse GABA transporters. Neurochem Int 43,
445–451.
 K.K. Madsen et al. / Pharmacology & Therapeutics 125 (2010) 394–401
Saxena, N. C., & MacDonald, R. L. (1994). Assembly of GABAA receptor subunits: role of
the delta subunit. J Neurosci 14, 7077–7086.
Schousboe, A. (1979). Effects of GABA analogues on the high-affinity uptake of GABA in
astrocytes in primary cultures. In P. Mandel, & F. V. De Feudis (Eds.), GABA-
Biochemistry and CNS Function (pp. 219–237). New York: Plenum Publishing Corp.
Schousboe, A. (1990). Neurochemical alterations associated with epilepsy or seizure
activity. In M. Dam, & L. Gram (Eds.), Comprehensive epileptology (pp. 1–16). New
York: Raven Press, Ltd.
Schousboe, A. (2000). Pharmacological and functional characterization of astrocytic
GABA transport: a short review. Neurochem Res 25, 1241–1244.
Schousboe, A., & White, H. S. (2009). In P. Schwartzkroin (Ed.), Modulation of excitability
via glutamate and GABA transportersEncyclopedia of basic epilepsy research Vol. 1.
(pp. 397–401) Oxford, UK: Elsevier Ltd.
Schousboe, A., Larsson, O. M., Hertz, L., & Krogsgaard-Larsen, P. (1981). Heterocyclic
GABA analogues as new selective inhibitors of astroglial GABA transport. Drug Dev
Res 1, 115–127.
Schousboe, A., Saito, K., & Wu, J. Y. (1980). Characterization and cellular and subcellular
localization of GABA–transaminase. Brain Res Bull 5, 71–76.
Schousboe, A., Thorbek, P., Hertz, L., & Krogsgaard-Larsen, P. (1979). Effects of GABA
analogues of restricted conformation on GABA transport in astrocytes and brain
cortex slices and on GABA receptor binding. J Neurochem 33, 181–189.
Semyanov, A., Walker, M. C., & Kullmann, D. M. (2003). GABA uptake regulates cortical
excitability via cell type-specific tonic inhibition. Nat Neurosci 6, 484–490.
Suzdak, P. D., Frederiksen, K., Andersen, K. E., Sørensen, P. O., Knutsen, L. J., & Nielsen, E. B.
(1992). NNC-711, a novel potent and selective gamma-aminobutyric acid uptake
inhibitor: pharmacological characterization. Eur J Pharmacol 224, 189–198.
Takayama, C., & Inoue, Y. (2005). Developmental expression of GABA transporter-1 and
3 during formation of the GABAergic synapses in the mouse cerebellar cortex. Brain
Res Dev Brain Res 158, 41–49.
Thomsen, C., Sørensen, P. O., & Egebjerg, J. (1997). 1-(3-(9H-carbazol-9-yl)-1-propyl)-
4-(2-methoxyphenyl)-4-piperidinol, a novel subtype selective inhibitor of the
mouse type II GABA-transporter. Br J Pharmacol 120, 983–985.
401
Udenfriend, S. (1950). Identification of gamma-aminobutyric acid in brain by the
isotope derivative method. J Biol Chem 187, 65–69.
Wafford, K. A., & Ebert, B. (2006). Gaboxadol—a new awakening in sleep. Curr Opin
Pharmacol 6, 30–36.
Watanabe, M., Maemura, K., Kanbara, K., Tamayama, T., & Hayasaki, H. (2002). GABA
and GABA receptors in the central nervous system and other organs. In K. W. Jeon
(Ed.), A survey of cell biology (pp. 1–47). San Diego: Academic Press, Inc.
White, H. S. (1999). Comparative anticonvulsant and mechanistic profile of the
established and newer antiepileptic drugs. Epilepsia 40(Suppl 5), S2–S10.
White, H. S., Sarup, A., Bolvig, T., Kristensen, A. S., Petersen, G., Nelson, N., et al. (2002).
Correlation between anticonvulsant activity and inhibitory action on glial gamma-
aminobutyric acid uptake of the highly selective mouse gamma-aminobutyric acid
transporter 1 inhibitor 3-hydroxy-4-amino-4,5,6,7-tetrahydro-1,2-benzisoxazole
and its N-alkylated analogs. J Pharmacol Exp Ther 302, 636–644.
White, H. S., Watson, W. P., Hansen, S. L., Slough, S., Perregaard, J., Sarup, A., et al.
(2005). First demonstration of a functional role for central nervous system betaine/
{gamma}-aminobutyric acid transporter (mGAT2) based on synergistic anticon-
vulsant action among inhibitors of mGAT1 and mGAT2. J Pharmacol Exp Ther 312,
866–874.
Yamauchi, A., Uchida, S., Kwon, H. M., Preston, A. S., Robey, R. B., Garcia-Perez, A., et al.
(1992). Cloning of a Na(+)- and Cl(−)-dependent betaine transporter that is
regulated by hypertonicity. J Biol Chem 267, 649–652.
Yunger, L. M., Fowler, P. J., Zarevics, P., & Setler, P. E. (1984). Novel inhibitors of gamma-
aminobutyric acid (GABA) uptake: anticonvulsant actions in rats and mice. J Pharmacol
Exp Ther 228, 109–115.
Zhu, X. M., & Ong, W. Y. (2004). A light and electron microscopic study of betaine/GABA
transporter distribution in the monkey cerebral neocortex and hippocampus.
J Neurocytol 33, 233–240.