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GABA (A) receptor channel pharmacology.

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 Current Pharmaceutical Design, 2005, 11, 1867-1885 1867

GABAA Receptor Channel Pharmacology

Graham A.R. Johnston*

The Adrien Albert Laboratory of Medicinal Chemistry, Department of Pharmacology, The University of Sydney, NSW
2006, Australia

Abstract: GABA A receptor channels are ubiquitous in the mammalian central nervous system mediating fast inhibitory
neurotransmission by becoming permeant to chloride ions in response to GABA. The emphasis of this review is on the
rich chemical diversity of ligands that influence GABAA receptor function. Such diversity provides many avenues for the
design and development of new chemical entities acting on GABAA receptors. There is also a significant diversity of
GABAA receptor subtypes composed of different protein subunits. The discovery of subtype specific agents is a major
challenge in the continuing development of GABAA receptor pharmacology. Leads for the discovery of new chemical
entities that influence GABAA receptors come from using recombinant GABAA receptors of known subunit composition
as has been elegantly demonstrated by the refining of benzodiazepine actions with α1 subunit preferring agents showing
sedative properties but not anxiolytic properties. The most recent advances in the therapeutic use of agents acting on
GABAA receptors concern the promotion of sound sleep. Many herbal medicines are used to promote sleep and many of
their active ingredients include flavonoids and terpenoids known to modulate GABAA receptor function.

INTRODUCTION [4]. There is also diversity in GABAB metabotropic GABA

GABA (γ-aminobutyric acid), the major inhibitory
neurotransmitter in the brain, is essential for the overall
balance between neuronal excitation and inhibition that is
vital to normal brain function. Too much inhibition, or too
little excitation, can lead to coma, depression, low blood
pressure, sedation or sleep. Too much excitation, or too little
inhibition, can result in a range of conditions including
convulsions, anxiety, high blood pressure, restlessness and
insomnia. Either imbalance in the extreme can result in
death. The exact symptoms depend on what regions of the
brain are involved and exactly what nerve cells are out of
balance. Restoration of the balance between excitation and
inhibition is a major aim of therapies that target GABA-
mediated neuronal inhibition.
GABA produces neuronal inhibition by acting on an
amazing diversity of membrane-bound receptors. These
receptors can be divided into two major types: ionotropic
receptors that are ligand-gated ion channels (GABAA and
GABAC receptors), and metabotropic receptors that are G-
protein coupled receptors (GABAB receptors) that act via
second messengers [1, 2]. The ionotropic GABA receptors
belong to the nicotinicoid superfamily of ligand-gated ion
channels as described by Le Novere and Changeux [3] that
includes nicotinic acetylcholine, strychnine-sensitive glycine
and 5HT3 receptors. The family of ionotropic GABA
receptors is divided into two subfamilies, GABAA and
GABAC receptors, on the basis of their ability to form
endogenous heteromeric and homomeric receptors
respectively, and differences in their physiological and
pharmacological properties [1], although GABAC receptors
are sometimes classified as subtypes of GABAA receptors
*Address correspondence to this author at the The Adrien Albert Laboratory
of Medicinal Chemistry, Department of Pharmacology, The University of
Sydney, D06, Sydney, NSW 2006, Australia; Tel: 61 2 9351 6117; Fax: 61
2 9351 2891; E-mail: grahamj@mail.usyd.edu.au
receptors that are heteromeric dimers [5]. There is evidence
for the existence of functional GABA A, GABAB and GABAC
receptors, as well as strychnine-sensitive glycine receptors,
on a single population of retinal ganglion cells [6].
Like other members of the nicotinicoid superfamily of
ligand-gated ion channels, ionotropic GABA receptors are
considered to consist of 5 protein subunits arranged around a
central pore that constitutes the actual ion channel [1]. Each
subunit has a large extracellular N-terminal domain which
incorporates part of the agonist/antagonist binding site,
followed by three membrane spanning domains (M1-3), an
intracellular loop of variable length and a fourth membrane
spanning domain (M4), with the C-terminal end being
extracellular. Each subunit arranges itself such that the
second membrane-spanning domain (M2) forms the wall of
the channel pore and the overall charge of the domain
determines whether the channel conducts anions or cations.
Both GABAA and GABAC receptors are GABA-gated
chloride ion channels causing inhibition of neuronal firing,
with GABAA receptors being heteromeric, i.e. made up of
different subunits (e.g. α1, β2 and γ2 subunits) and GABAC
receptors being homomeric (e.g. made up exclusively of
either ρ1, ρ2 or ρ3 subunits; in addition ‘pseudoheteromeric’
GABAC receptors made up of ρ1 and ρ2 subunits have been
described). The cytoplasmic loop, between the third and
fourth transmembrane domains (M3 and M4), is believed to
be the target for protein kinases, required for subcellular
targeting and membrane clustering of the receptor. There are
16 different subunits comprising the GABAA receptor
family: α1-6, β1-3, γ1-3, δ, ε, π and θ [7]. In addition, there
are splice variants of many of these subunits. If all of these
subunits could co-assemble to form functional pentameric
receptors the total number of GABAA receptors would
be huge. Even if the combinations were restricted to
those containing two α, two β and one other subunit, then
more than 2000 different GABAA receptors could exist [8].

1381-6128/05 $50.00+.00 © 2005 Bentham Science Publishers Ltd.

1868 Current Pharmaceutical Design, 2005, Vol. 11, No 15
In fact, studies of native GABAA receptors suggest that there
may be less than 20 widely occurring GABAA receptor
subtype combinations, with the major combinations being
α1β2/3γ2, α3β3γ2 and α2β3γ2 [7, 9].
This review is directed at some recent highlights,
together with a re-evaluation of some older data, relevant to
GABAA receptors as therapeutic targets with an emphasis on
the chemical diversity of ligands that influence GABAA
receptor function. Such diversity provides many avenues for
the design and development of new chemical entities acting
on GABAA receptors. There are many reviews on aspects
GABAA receptors including methodological approaches to
the study of GABAA receptors [10, 11], GABAA receptor
subtypes [7, 12-15], neurosteroid modulation [16, 17], drug
interactions [18], specific agonists and partial agonists [19],
receptor recycling and regulation [20], novel modulators
[21], medicinal chemistry [1, 22], and analysis of GABAA
receptors through mouse genetics [23]. GABAC receptors as
therapeutic targets have been the subject of a recent review
[24].
THERAPEUTIC USES OF AGENTS ACTING ON
GABAA RECEPTORS
Agents acting on GABAA receptors have widespread
therapeutic use as anaesthetics, anticonvulsants, anxiolytics
and sedative-hypnotics. In the main, these agents act to
increase GABA-mediated synaptic inhibition either by
directly activating GABAA receptors or, more usually, by
enhancing the action of GABA on GABAA receptors. This
latter action is known as positive modulation [8] and is
considered to involve agents acting on allosteric sites on
GABAA receptors remote from the GABA recognition sites
(orthosteric sites). Such allosteric sites are regarded as good
targets for the development of subtype specific drugs since
there is generally greater diversity between receptor subtypes
in amino acid sequence at allosteric sites than at orthosteric
sites [25]. Agents that reduce the action of GABA on
GABAA receptors are known as negative allosteric modula-
tors (once known as ‘inverse agonists’); they have the
opposite actions to those of the classical benzodiazepines.
Agents that block the actions of both positive and negative
allosteric modulators are known as neutralising allosteric
modulators, e.g. the classical benzodiazepine ‘antagonist’
flumazenil [8]. Benzodiazepines and barbiturates are
examples of widely used therapeutic agents that act as
positive allosteric modulators at GABAA receptors.
A rich chemical diversity of agents acting GABAA recep-
tors is known [1]. The discovery of subtype specific GABAA
receptor agents is a major challenge in the continuing
development of therapeutic agents acting on specific wild
type and mutant GABAA receptors.
DISORDERS INVOLVING GABAA RECEPTORS
Not surprisingly, GABA as the major inhibitory
neurotransmitter is involved, directly or indirectly, in many
disorders of brain function. The major disorders for which
GABAA receptors represent important therapeutic targets
include anxiety disorders, cognitive disorders, epilepsies,
moods disorders, schizophrenia and sleep disorders.
Graham A.R. Johnston
Heritable mutations are known to occur across the
nicotinicoid superfamily of ligand-gated ion channels
including GABAA receptors [26]. Angelman syndrome, a
neurodevelopmental disorder characterised by severe mental
retardation, epilepsy and delayed motor development has
been associated with deletions of GABAA receptor β3
subunits [27]. GABAA receptor β3 knockout mice have
epilepsy and a phenotype with marked similarities to
Angelman syndrome [28, 29].
GABA systems have been implicated in the pathogenesis
of anxiety, depression and insomnia. These symptoms are
part of the core and comorbid psychiatric disturbances in
post-traumatic stress disorder (PTSD). In a study of PTSD
patients, heterozygosity of β3 GABAA receptor subunits was
associated with higher levels of anxiety, insomnia, social
dysfunction and depression than found in homozygosity
[30]. There is increasing evidence for GABA abnormalities
in mood disorders [31] and in alcohol dependence [32, 33].
Some of the neurological symptoms of guanidinoacetate
methyltransferase deficiency may be due to the partial
agonist action of increased levels of guanidinoacetate on
GABAA receptors [34].
The use of herbal medicines to treat depression may be
associated with actions on GABAA receptors [35]. GABAA
receptor ligands have a potential role in the treatment of
schizophrenia [36, 37], acute ischaemic stroke [38], tinnitus
[39] and impaired cognition [40].
Epilepsies
Studies on the genetics of human epilepsies show that
epilepsy syndromes that have monogenic inheritance are
associated with mutations that encode subunits of voltage
gated and ligand gated ion channels [41]. Heritable muta-
tions in GABAA receptor subunits are strongly implicated in
idiopathic generalised epilepsies [42]. Mutations in γ2
GABAA receptor subunits have been described in two
families with generalised epilepsy syndromes [43, 44]. One
mutation associated with febrile seizures and generalised
epilepsy is in the extracellular loop connecting the TM2 and
TM3 domains. Studies using recombinant receptors in frog
oocytes revealed that this mutant showed smaller GABA-
activated currents than did receptors lacking this mutation
[43]. The other mutation was in the distal part of the N
terminus thought to make up part of the benzodiazepine
binding pocket and was associated with childhood absence
epilepsy and febrile seizures. Studies in frog oocytes showed
that this mutation had diminished sensitivity to positive
allosteric modulation by benzodiazepines [44]. These
findings have been questioned recently as a result of patch
clamp studies using a mammalian expression system (HEK
cells) that showed that the TM2-TM3 loop γ2 GABAA
receptor mutation resulted in faster deactivation rates, while
the N terminus mutant reduced current amplitude without
altering benzodiazepine sensitivity [45]. Mutations in
intracellular regions of γ2 GABAA receptor subunits between
TM1 and TM2 and between TM3 and TM4 have also been
associated with epilepsies. It appears that all of these
mutations in γ2 GABAA receptor subunits may result in
diminished synaptic inhibition mediated by GABAA recep-
tors and epilepsy by diverse mechanisms [46]. In addition to
GABAA Receptor Channel Pharmacology
these mutations, a γ2 GABAA receptor subunit splice site
mutation has been associated with childhood absence
epilepsy and febrile convulsions, but it is not known how
this influences GABAA receptor properties [47]. An
association analysis has shown that polymorphism in the γ2
GABAA receptor gene is a susceptibility factor for febrile
seizures [48]. A mutation in the TM3 region of the α1
GABAA receptor subunit that influences both the efficacy
and affinity of GABA has been associated with juvenile
myoclonic epilepsy [49]. Studies on recombinant receptors
containing this TM3 α1 mutation show that it results in
reduced channel open time with no change in single channel
conductance [50].
Although heritable epilepsies represent only a small
fraction of epilepsies, the observed mutations associated with
heritable epilepsies provide clues as to possible problems
with GABAA receptors implicated in other epilepsies. They
also provide targets for pharmacogenomic therapies using
drugs acting on specific mutant GABAA receptors.
Sleep Disorders
GABA systems are known to play an important role in
sleep and positive allosteric modulators of GABAA receptors
are widely used to promote restful sleep [51]. Two
observations indicate the importance of β3 GABA A receptor
subunits in sleep. Oleamide, an endogenous sleep promoting
fatty acid, is inactive in β3 GABAA receptor subunit
knockout mice [52]. A mutation in β3 GABAA receptor
subunits has been described in a patient with chronic
insomnia. Functional characterisation of this mutant showed
a slower rate of desensitisation compared with normal
GABAA receptors [53].
The treatment of insomnia is regarded as a developing
market for agents acting on GABAA receptors. Drugs
currently used to treat insomnia include zolpidem
(Ambien), zaleplon (Sonata) and zopiclone (Imovane).
These drugs, Fig. (1), show some selectivity for α1 subunit
containing GABAA receptors, acting as positive allosteric
modulators. The structurally related indiplon, Fig. (1), which
is in phase III clinical trials for the treatment of insomnia,
acts in a similarly selective manner [54, 55]. Also in phase
III clinical trials is gaboxadol (THIP), a directly acting
GABAA receptor partial agonist, discussed in Section 5, Fig.
(3), that interacts with a GABAA receptor population that is
insensitive to benzodiazepines, zolpidem, zaleplon, zolpidem
and indiplon [56].
Many herbal preparations are used to promote sleep. For
example, chamomile tea contains the flavonoid apigenin (see
Section 7.1) which has been shown to enhance the positive
allosteric modulating effects of benzodiazepines on GABAA
receptors [24] and Valerian contains a variety of agents (see
Section 7.2) that act on GABAA receptors [57].
GABAA RECEPTOR SUBUNITS
The importance of drug interactions with receptor
subtypes made up of specific protein subunits has been
highlighted by a number of recent findings involving
genetically modified mice and ligands that show some
selectivity for particular receptor subtypes. The finding that
Current Pharmaceutical Design, 2005, Vol. 11, No.15 1869
different pharmacological actions of benzodiazepines result
from interactions with different GABAA receptor subtypes
was pivotal to these studies [14].
O
N N N
N Cl
N
N
O
O
O
N N
N
Zolpidem Zopiclone
CN O S
N
N
N
N
N
N
O
O
N
N
Indiplon
Zaleplon
Fig. (1). Structures of sedative/hypnotic substances that interact
preferentially with α1 subunit containing GABAA receptors.
It must be remembered, however, that it is not only the
nature of the protein subunits that influence the diversity of
function of GABAA receptor subtypes in vivo. Major
contributors to this diversity are: presynaptic factors
including release probability and number of release sites;
factors that determine synaptic GABA transients in the cleft,
including diffusion and the actions of GABA transporters;
and postsynaptic factors, including GABA receptor subtypes,
their location and number, their modulation by endogenous
and exogenous factors, and their interactions with postsynap-
tic-anchoring proteins [58]. The transport, clustering and
turnover of GABA A receptors are known to be influenced by
a variety of proteins}. [20]. The phosphorylation state of the
subunits is very important with GABAA receptors as with
many other ligand-gated ion channels [59]. For example, the
sensitivity of GABAA receptors to ethanol is dependent on
receptor phosphorylation; mice lacking protein kinase Cε
show increased sensitivity to ethanol while those lacking
protein kinase Cγ show decreased sensitivity [59]. Further-
more, there are many endogenous ligands that influence
GABAA receptor function in vivo including metal ions such
zinc [60], steroids [17], and chemicals derived from our diet
such as flavonoids [57].
Influence of α1 and α2 Subunits – Sedative and
Anxiolytic Actions of Benzodiazepines
Benzodiazepines are considered to act on GABAA
receptors at a binding pocket at the interface between the γ2
subunit and α subunits that contain a conserved histidine
residue in the benzodiazepine binding domain on the
1870 Current Pharmaceutical Design, 2005, Vol. 11, No. 15
extracellular N-terminus (α1, α2, α3 and α5 subunits).
Mutation of this histidine to an arginine results in GABAA
receptors that are insensitive to benzodiazepines in vitro
[61]. GABAA receptors containing α4 or α6 subunits are
relatively insensitive to benzodiazepines.
Knock-in point mutations in the genes that code for α1 or
α2 subunits produced mice that had different responses to
benzodiazepines. Mice with the mutant α1 subunits showed
the normal anxiolytic responses to benzodiazepines but not
the sedative effects [62, 63]. The reverse was true for mice
with the mutant α2 subunits showing sedative but not
anxiolytic effects in responses to benzodiazepines [64].
While there is general agreement on the importance of the
α1 GABAA receptor subunit in the sedative actions of
benzodiazepines, there has been some doubt on the relative
contributions of the α2 and α3 subunits to the anxiolytic
action due to confounding effects on locomotor activity that
influence the assessment of anxiety [65]. Furthermore, in
profiling the influence of a range of agents on
benzodiazepine binding differences in functional activity
need to be taken into account [66]. Ultimately, validation of
GABAA receptor subtype-selective drugs needs to be carried
out using genetically modified mice [12]. Drugs selective for
α2-containing GABAA receptors (found in about 15% of
diazepam-sensitive GABA A receptors) would be expected to
be anxiolytics with greatly reduced sedative effects
compared to the non-selective benzodiazepines currently in
clinical use [14]. The non-sedative anxiolytic L-838, 417,
Fig. (2), is a neutralising modulator at α1 containing GABAA
receptors but is a positive modulator at α2, α3 and α5
containing GABAA receptors [63]; this agent thus offers a
double pronged approach to anxiolysis without sedation by
enhancing the action of GABA at α2, α3 and α5 containing
GABAA receptors while diminishing the action of endo-
genous benzodiazepines on α1 containing GABAA receptors.
New chemical entities with functional selectivity for α2 over
N HO 2C
N H
N N
O N F N
N N
H2NO2 S
N N Cl
F
L-838,417 Furosemide
O
S(CH2) 2OH
O O
HN
OH
Cl N S
'Compound 4' 'Compound 43'
Fig. (2). Structures of substances showing selectivity for GABAA receptors containing specific subunits.
Graham A.R. Johnston
α1 subtypes of GABAA receptors are being developed, e.g.
3-heteroaryl-2-pyridones [67]. Recently a series of 3-phenyl-
6-(2-pyridyl)methoxy-1, 2, 4-triazolo[3, 4-a]phthalazines
have been developed with Compound 62, Fig. (2), showing
binding selectivity for α2, α3 and α5 over α1 subunits and
acting as an anxiolytic in the rat elevated plus maze [68]. It
showed a good pharmacokinetic profile making it a useful
tool to explore the effect of a GABAA α2/α3 selective
agonist in vivo. The sedative effects of the α1-selective agent
zolpidem are diminished in the α1 knock-in mouse,
consistent with sedation being mediated via α1-containing
GABAA receptors [69].
Quinolone antibiotics have long been known to interact
with receptors for GABA and other neurotransmitters [70].
Modification of norfloxacin has yielded molecules such as
Compound 4, Fig. (2), that positively modulates GABAA
receptors with α2 subunit selectivity and is a non-sedating
anxiolytic [71].
Importance of α5 Subunits in Spatial Memory
GABAA receptor α5 subunits account for less than 5% of
GABAA receptors in the brain. They are localised mainly to
the hippocampus where they may play a key role in
cognitive processes by controlling a component of synaptic
transmission in the CA1 [72]. Mice lacking the α5 gene
show improved performance in the Morris water maze model
of spatial learning, whereas the performance in non-
hippocampal-dependent learning and in anxiety tasks were
unaltered in comparison with wild-type controls [73].
Novel selective α5 negative allosteric modulators, e.g. 6,
6 - dimethyl - 3 - (2 - hydroxyethyl) thio - 1 - (thiazol - 2 - yl) - 6, 7 -
dihydro-2-benzothiophen-4(5H)-one (Compound 43, Fig.
(2)) have been developed that enhance spatial learning but
lack the convulsant or proconvulsant activity associated with
non-selective GABAA receptor negative allosteric modula-
tors [74].
O
HO
OH O
H
Salicylidene salicylhydrazide
N
S N
N
O N
N N
'Compound 62'
GABAA Receptor Channel Pharmacology
Furosemide and α6-Subunits
The loop diuretic furosemide, Fig (2), has been described
as ‘the most receptor-subtype specific’ allosteric modulator
acting on GABAA receptors [75]. Furosemide acts as a
negative allosteric modulator of α6 subunit containing
GABAA receptors. Such GABAA receptors are largely
restricted to the cerebellum and show low sensitivity to the
classic GABAA antagonist bicuculline and to positive
modulation by diazepam, but can be influenced by other
benzodiazepine receptor ligands [76, 77]. Furosemide
exhibits approximately 100-fold selectivity for α6 containing
receptors over α1 containing receptors. It also acts on α4
containing receptors. Mutation of a threonine to a isoleucine
in the TM1 region of α1 subunits increases furosemide
sensitivity by 20-fold [78]. Structure-activity studies show
that the diuretic properties of agents related to furosemide
are distinct from the α6 GABAA receptor negative
modulation [79]. The positive allosteric modulator (+)-
ROD188 shows selectivity for α6 GABAA receptors [80].
The GABAC receptor agonist cis-4-aminocrotonic acid also
acts as a bicuculline-sensitive agonist at recombinant
α6β2γ2S GABAA receptors that are insensitive to the
GABAC receptor antagonist TPMPA [81]. Niflumic acid, a
nonsteroidal anti-inflammatory drug, acts as an antagonist at
recombinant α6β2 receptors in a manner similar to that of
furosemide, but also acts as a positive modulator at α1β2γ2
receptors [82].
Gene knockout of the α6 subunit in mice resulted in an
associated inhibition of δ subunit expression without
influence on exploratory activity in the open field or learning
in a horizontal wire task [83]. Other studies showed the lack
of effect of α6 knockout on responses to ethanol,
pentobarbital and general anaesthetics [84]. In a rotating rod
test, however, α6 knockout mice were significantly more
impaired by diazepam than were wild-type mice [85]. This
diazepam-induced ataxia in α6 knockout mice could be
reversed by flumazenil, indicating the involvement of the
remaining α1β2/3γ2 GABAA receptors on the cerebellar
granule cells. This led to the conclusion that α6 subunit-
dependent actions in the cerebellar cortex could be
compensated by other receptor subtypes; but, if not for the
α6 subunit, patients on benzodiazepine medication would
suffer considerably from ataxic side-effects [85]. There is
electrophysiological evidence for the coexistence of α1 and
α6 subunits in a single functional GABAA receptor [86], for
furosemide-sensitive and furosemide-insensitive GABA-
mediated effects on cerebellar granule cells [87] and for a
tonic diazepam-sensitive GABA-mediated inhibition on
cultured rodent cerebellar granule cells [88].
Allelic variants in α6 GABAA subunits are associated
with abdominal obesity and cortisol secretion [89]. In a study
of 100 patients of the effects of midazolam, a point mutation
(Pro385Ser) in the α6 GABAA receptor subunit did not
affect baseline sedation, anxiety or memory, but significantly
attenuated the anxiolytic affect of low-dose midazolam [90].
Anaesthesia and Sedation Involving β2 and β3 Subunits
The intravenous general anaesthetic etomidate provides
another example of distinct actions involving different
GABAA receptor subtypes, in this case involving β2 and β3
Current Pharmaceutical Design, 2005, Vol. 11, No. 15 1871
subunits. Using genetically modified mice with etomidate-
insensitive β2 subunits, it was shown that in wild-type mice
etomidate produces sedation via the β2 subunit and
anaesthesia via the β3 subunit [91]. Furthermore, the
recovery of function in the genetically modified mice was
considerably improved after etomidate anaesthesia suggest-
ing that β3 selective agents could be used as anaesthetics
with significantly improved recovery profile [91]. The β2
subunit has been shown to mediate the hypothermic effect of
etomidate [92]. Loreclezole and mefenamic acid show
similar selectivity to etomidate with respect to β2 and β3
subunits [93]. Salicylidene salicylhydrazide, Fig. (2), has
been shown to be a selective inhibitor of GABAA receptors
that contain β1 subunits and thus may be a useful agent with
which to study β subunit selectivity [94].
Splice Variants of γ2 Subunits and Sedation
The γ2 GABA A receptor subunit is generally considered
to be vital to the classical actions of benzodiazepines on
GABAA receptors. Alternate splicing results in two splice
variants, a short (γ2S) and a long (γ2L) variant. Mice lacking
the γ2L variant are more sensitive to the sedative effects of
midazolam and zolpidem, while responses to etomidate and
barbiturates are unchanged [95]. It is suggested that the lack
of the γ2L variant may shift the state of α1-containing
GABAA receptors from a negative allosteric modulator
preferring conformation towards a positive allosteric
modulator preferring conformation.
Ethanol and δ Subunits
The importance of the δ GABAA receptor subunit has
been highlighted by the discovery that ethanol at low
concentrations known to affect humans enhances the action
of GABA on α4β3δ and α6β3δ receptor subtypes [96].
Reproducible ethanol enhancement of GABA responses
occurred at 3 mM, i.e. concentrations that are reached with
moderate ethanol consumption producing blood-ethanol
levels well below the legal level for driving in most
countries. Ethanol has been long known to influence the
functioning of a variety of receptors usually at concentra-
tions in excess of 50 mM. This had been true for recombi-
nant GABAA receptors [97] until the studies on δ subunit
containing receptors. The δ subunits appear to associate
almost exclusively with α4 and α6 subunits forming
functional receptors that are 50 fold more sensitive to GABA
and desensitise more slowly than receptor subtypes that do
not contain δ subunits [83, 98]. The δ subunit protein is
expressed in brain regions expressing α4 (high in thalamus,
dentate gyrus, striatum and outer cortical layers and low in
hippocampus) and α6 subunit proteins (cerebellum) and
appears to be associated with extrasynaptic rather than
synaptic GABA A receptors [99]. Knocking out the δ subunit
gene in mice reduces their sensitivity to neurosteroids [100]
and increases their susceptibility to seizures [101]. Knocking
out the α6 subunit gene in mice does not alter their
susceptibility to ethanol [84].
GABAA RECEPTOR AGONISTS
Muscimol and THIP, Fig. (3), are widely used as
selective GABA A receptor agonists [1]. However, they have
1872 Current Pharmaceutical Design, 2005, Vol. 11, No. 15
potent actions on GABAC receptors which mean that
interpretation of studies with these agents should be treated
with some caution. No “selective” GABAA receptor agonist
is known that does not have significant action on either
GABAB and/or GABA C receptors. Muscimol, a conforma-
tionally restricted analogue of GABA in which a hydroxy-
isoxazole moiety replaces the carboxyl group of GABA
[102], is more potent at GABAC receptors than at GABAA
receptors [103].
THIP and Ionotropic GABA Receptors
THIP (Gaboxadol, 4, 5, 6, 7-tetrahydroisoxazolo(5, 4-c)
pyridin-3-ol), Fig. (3), is a conformationally restricted anal-
ogue of muscimol [102].
It is a potent GABAA receptor partial agonist of high
efficacy [104] that has proved to be a moderately potent
GABAC receptor antagonist [103]. Unlike GABA, both
muscimol and THIP pass the blood-brain barrier on systemic
administration [105]. Muscimol is psychoactive, while THIP
is a potent analgesic. Side effects of THIP (including
sedation, dizziness, and blurred vision) meant that it had too
low a therapeutic index to be therapeutically useful as an
analgesic [106, 107]. There is renewed interest in THIP with
respect to sleep therapy [19] as it produces slow wave sleep
and reduces spindling activity in non rapid eye movement
sleep in humans [108].
It does appear that receptors other than classical
benzodiazepine-sensitive, bicuculline-sensitive GABAA
receptors are involved in the effects of THIP on pain
perception and sleep. THIP-induced analgesia is not
sensitive to bicuculline indicating that GABAA receptors are
not involved [109]. The GABAC receptor antagonist action
of THIP may contribute to its analgesic action [24]. The
analgesic action of THIP in rats is blocked by subconvulsant
doses of picrotoxinin [110], a known GABAC receptor
antagonist. Benzodiazepine-sensitive GABAA receptors do
not appear to be involved in the effects of THIP on sleep
patterns [108]. The GABAC receptor antagonist TPMPA has
been used to probe the involvement of GABAC receptors in
sleep-waking behaviour [111]. The binding of THIP to rat
brain membranes, unlike that of GABA and muscimol, is not
stimulated by diazepam [112]. THIP was devoid of the
anticonvulsant and antiepileptogenic effects shown by
diazepam and alphaxalone in pentamethylenetetrazole-
kindled mice [113].
Clinical studies with THIP have indicated that sleep
quality improving effects are obtained at plasma
concentrations of the order of 1 µM [108]. THIP shows
considerable variation in potency on recombinant receptors:
THIP acts on α1β3γ2S recombinant GABAA receptors
expressed in oocytes as a partial agonist (EC50 350µM) and
more potently and as a full agonist on α5β3γ3 (EC50 40
µM) and α5β3γ3 (EC50 29 µM) recombinant receptors
[114]. On α4β3γ2 recombinant receptors THIP acts as a
partial agonist (EC50 102 µM) and on α4β3δ as a
‘superagonist’ (EC50 6 µM) [115]. On recombinant GABAC
receptors THIP acts as an antagonist (Kb 32 µM for ρ1 [103]
and 10 µM for ρ3 receptors [116]. On this basis, α4β3δ
GABAA and ρ3 GABAC receptors are the most likely GABA
Graham A.R. Johnston
receptors to respond to clinically relevant 1 µM plasma
concentrations of THIP.
Studies on the interactions between THIP, benzodia-
zepines and ethanol in the rat cortical wedge preparation
provide evidence for THIP acting on benzodiazepine-
insensitive GABAA receptors in intact tissue possibly
containing α4 subunits [56]. Rotarod studies on the effects of
THIP on motor performance in rats showed a lack of cross-
tolerance with benzodiazepines [117]. In neither study did
ethanol show a potentiation of the effects of THIP [56, 117].
This is interesting in view of the recent findings discussed in
section 4.5. of the sensitivity of α4β3δ GABA A receptors to
ethanol [96] and may suggest that this receptor subtype is not
involved in some of the actions of THIP. Studies on the
effects of THIP in δ-subunit knockout mice may help sort
out the role of α4β3δ GABAA receptors in THIP-induced
analgesia and sleep.
The rat cortical wedge preparation has yielded evidence
of the importance of GABAA receptor ligands acting at
extrasynaptic receptors [118]. In this preparation THIP acted
as a full agonist with an EC50 of 8 µM [118]. In contrast, an
unusually low activity of THIP has been reported on GABA
receptors on isolated rat dorsal roots, a tissue that certainly
does not contain any synapses [119]. THIP was 20 times
weaker than GABA in depolarising these dorsal roots but at
least 20 times more potent than GABA is depressing overall
spontaneous synaptic activity in the rat hemisected spinal
cord as recorded in the ventral roots. As in the rat cortical
wedge preparation, the actions of THIP in the hemisected
spinal cord may involve extrasynaptic GABA receptors, but
the results in isolated dorsal roots clearly show that not all
extrasynaptic GABA receptors show high sensitivity to
activation by THIP. Interestingly, extrasynaptic GABAA
receptor channels in hippocampal slices are known to be
modulated by diazepam [120].
OH OH
H2N HN
N N
O O
Muscimol THIP
OH
OH
N
O N
HN O
HN
4-PIOL 4-Naphthyl-Me-4-PIOL
H
O
N NH 2
H2N
N
H
O
Compound 5b - "superagonist"
Fig. (3). GABAA receptor agonists and partial agonists.
GABAA Receptor Channel Pharmacology
4-PIOL as a Low Efficacy Partial GABAA Receptor
Agonist
Partial agonists offer certain advantages over full
agonists [19]. Full agonists may induce receptor desensitisa-
tion that can lead to tolerance and subsequent withdrawal
symptoms. The non-fused THIP analogue 4-PIOL, Fig. (3)
(5-(4-piperidyl)isoxazol-3-ol), is a low efficacy GABAA
receptor partial agonist that exhibits a predominantly
antagonist profile [19]. Its activity varies with different
recombinant GABAA receptor subtypes and in the rat cortical
wedge preparation, 4-PIOL behaves as a high efficacy partial
agonist [118]. Patch clamp studies on hippocampal neurones
show that 4-PIOL is a non-desensitising partial agonist
whose action can be potentiated by benzodiazepines and
barbiturates [121]. Studies on analogues of 4-PIOL
substituted in the 4-position of the isoxazole ring yielded
GABAA receptor antagonists of increased potency, e.g. 4-
naphthyl-methyl-4-PIOL, Fig. (3), with a linear correlation
between the lipophilicity of the 4-subsitutent and antagonist
activity, providing evidence for a hydrophobic binding
pocket at the GABA recognition site [122].
“Superagonists” at GABAA Receptors
Studies on a series of GABA amides revealed substances
that could act as partial, full or superagonists as assessed by
the stimulation of chloride influx into mouse brain
synaptoneurosomes in a bicuculline- and picrotoxinin-sensi-
tive manner [123]. Compound 5b (N, N’-1, 4-butanediylbis
[4-aminobutanamide]), Fig. (3), produced a maximum
response that was 150% that of GABA and was thus descried
as a “superagonist”. It showed similar affinity to THIP, a
partial agonist with a maximum response 65% that of
GABA. The apparent ‘superagonist’ action of compound 5b
could mean that GABA is in fact a partial agonist in these
experiments. While the exact nature of this “superagonist”
action remains to be determined in functional assays using
recombinant GABAA receptors of known subunit
composition, the concept of ‘superagonists’ opens up another
possible approach to therapeutic agents acting at GABAA
receptors. THIP has been shown to display superagonist
behaviour at α4β3δ receptors with a maximum response
160% that of GABA [124].
STEROIDS THAT INFLUENCE GABAA RECEPTORS
A variety of steroids are known to influence GABAA
receptors via non-genomic actions that are rapid in onset and
offset. These neuroactive steroids include neurosteroids (i.e.
steroids that are synthesised in the brain), sex steroids that
originate in the gonads, and corticosteroids that are made in
the adrenal cortex, together with a range of synthetic steroids
and steroid analogues [16]. Most interest is centred on
neuroactive steroids that act as potent positive allosteric
modulators and have anxiolytic, anticonvulsant, analgesic,
anaesthetic and sedative actions [17].
The CNS depressant action of steroids has been known
since 1927 when it was shown that injection of a colloidal
suspension of cholesterol into cats caused deep anaesthesia
[125]. Subsequently, cholesterol was found to potentiate the
anaesthetic actions of pentobarbitone [126], but it was not
until the extensive investigations of Seyle [127] that it
Current Pharmaceutical Design, 2005, Vol. 11, No. 15 1873
became apparent that a wide range of natural and synthetic
steroids have anaesthetic actions.
The synthetic steroid anaesthetic alphaxalone, Fig. (4),
was the first steroid shown to act as a positive modulator of
GABAA receptors [128]. This was followed by the discovery
that steroid hormone metabolites, e.g. 3α-hydroxy-5α-
pregnan-20-one, Fig, (4), that occur in the brain are
‘barbiturate-like modulators’ of the GABAA receptor [129].
This led to the concept that neurosteroids can directly
modulate GABAA receptors on the cell surface rather than
acting on receptors in the nucleus regulating gene
expression. Steroids produced outside the brain are also
important modulators of GABAA receptors. For example,
THDOC, Fig (4), 3α, 21-dihydroxy-5α-pregnan-20-one, is a
‘neuroactive steroid’ because the sole source of this steroid
appears to be the adrenals. Nonetheless, THDOC is found in
the brain where its concentration is increased during stress
[130]. 3α-Hydroxy-5α-pregnan-20-one and THDOC are
among the most potent known steroid modulators of GABA A
receptors.
O O
OH
HO HO
H H
5α-Pregnan-3α-ol-20-one THDOC
O O
O
HO HO
H H
Alphaxalone Ganaxolone
O
OH
HO OH
OH
O
O
Cortisol Nandrolone
Fig. (4). Steroids that act on GABAA receptor function.
Studies using 3α-hydroxy-5α-pregnan-20-one indicate
that its positive modulatory actions on GABAA receptors are
only modestly influenced by the α-, β- or γ-subunits, [131].
The inclusion of either an ε or δ subunit however dramati-
cally alters the response with the ε subunit reducing and the
δ subunit augmenting the efficacy of modulation by 3α-
hydroxy-5α-pregnan-20-one [131]. The enhanced efficacy of
δ subunit containing GABAA receptors has also been
reported for THDOC [132]. As noted above, knocking out
1874 Current Pharmaceutical Design, 2005, Vol. 11, No. 15
the δ subunit gene in mice reduces their sensitivity to neuro-
steroids [100]. These findings clearly distinguish steroid
positive modulation of GABAA receptors from flumazenil-
sensitive positive modulation by benzodiazepines but do
suggest some similarities with the modulation induced by
volatile anaesthetics and ethanol. Experiments with alphax-
alone on chimeric GABAA receptors indicate that the site of
action for steroids is not the same as that for volatile
anaesthetics and ethanol [133]. Studies comparing the
positive modulator effects on α6β3γ2L GABA A receptors of
4 structurally distinct general anaesthetics – propofol,
pentobarbitone, etomidate and 3α-hydroxy-5α-pregnan-20-
one – showed that the action of all but 3α-hydroxy-5α-
pregnan-20-one depended critically on a single amino acid in
TM2 [134].
A novel neuroactive steroid, 6-aza-3α-hydroxy-5β-
pregnan-20-one, has been used to photoaffinity label rat
brain membranes [135]. It labelled a protein identified as
voltage-dependant anion channel-1 (VDAC-1) that co-
immunoprecipitated with the β2 and β3 subunits of the
GABAA receptor, suggesting that neuroactive steroids may
modulate GABA A receptor function by binding to VDAC-1
as an accessory protein [135]. Further studies using VDAC-1
deficient mice have suggested that VDAC-1 is unlikely to be
involved in steroid modulation of GABAA receptors [136].
In addition to alphaxalone, there are a number of
synthetic steroids that are known to modulate GABAA
receptor function. Anabolic steroids such as nandrolone, Fig.
(4), and stanazolol induce region- and subunit-specific rapid
modulation of GABAA receptor-mediated currents in the rat
forebrain [137]. The antiepileptic agent, ganaxolone, Fig.
(4), belongs to a novel class of neuroactive steroids called
epalons which specifically modulate GABAA receptors in the
central nervous system (CNS). Chemically related to
progesterone but devoid of any hormonal activity, the
epalons have potent antiepileptic, anxiolytic, sedative and
hypnotic activities in animals [138]. Ganaxolone has
demonstrated outstanding efficacy and better tolerability in
children with intractable infantile spasms [139]. It has,
however, been reported to exacerbate absence seizures in
animal models [140].
Cortisol, Fig. (4), is a potent bidirectional modulator of
the action of GABA on GABAA receptors in the guinea-pig
ileum enhancing at low (1-10 pM) concentrations and
inhibiting at higher (10-1000 nM) concentrations [141].
Cortisone is a potent non-competitive inhibitor of these
GABAA receptors acting at concentrations as low as 1 pM
[142]. These corticosteroids are thus the most potent agents
modulating GABAA receptors. The actions of cortisol may
be restricted to particular GABAA receptor subtypes since
cortisol has little effect on GABAA responses in the rat
cuneate nucleus [143]. Biphasic effects of corticosteroids
have been described on TBPS binding to rat brain
membranes, low (nM) concentrations enhancing binding and
higher (µM) concentrations inhibiting, the effect of nM
concentrations indicative of an antagonist action as observed
at these concentrations on GABA responses in the guinea-
pig ileum [144]. Cortisol (10 µM) has been shown to rapidly
increase the spontaneous firing frequency of neurones in rat
paraventricular nucleus and to inhibit whole cell potassium
Graham A.R. Johnston
currents, suggesting the cortisol may act indirectly via
inactivating potassium channels [145]. High affinity binding
sites (nM) for corticosterone have been described on brain
membranes [146], and corticosterone is known to influence
the expression and activity of GABAA receptors in the
hippocampus [147]. Given the risk of memory decline in
patients on corticosteroids [148], further investigations of the
effects of these steroids on GABAA receptors seem
warranted.
NATURAL PRODUCTS AND GABAA RECEPTORS
With increasing community acceptance of herbal
medicines and functional foods there is increasing interest in
natural products that may influence brain function. There is a
view that natural substances are inherently safer than
unnatural substances, i.e. synthetic chemicals. This view is
mistaken as many of the most toxic chemicals are in fact
natural products and the majority of therapeutically
beneficial drugs are synthetic. It is the molecular structure
and dose that determine the effects of substances on human
health, not whether they are of natural or synthetic origin
[149]. There is now an impressive array of natural products
in addition to steroids that are known to influence GABAA
receptor function including substances found in beverages
such as tea, red wine and whiskey, and in herbal preparations
including Ginkgo biloba and Ginseng [57]. Natural
substances represent a rich diversity in chemical structures
that can lead to the development of new therapeutic agents.
Flavonoids and GABAA Receptors
Flavonoids are found in all plants in high abundance and
exhibit a considerable chemical diversity with more than
5,000 different flavonoids having been described. Fruits,
vegetables, and beverages such as tea and red wine are major
sources of flavonoids our diet [150]. It has been estimated
that the average daily intake of flavonoids is 1-2 g [151].
Many flavonoids are polyphenolic and are thus strongly
antioxidant [152]. They have a wide variety of biological
activities and are being studied intensively as anticancer
agents [153]. Flavonoids have a range of activities on
GABAA receptors [154].
Flavonoids were first linked to GABAA receptors when
three isoflavans isolated from bovine urine were shown to
inhibit diazepam binding to brain membranes [155]. The
most potent compound was 3’, 7-dihydroxyisoflavan, Fig.
(5), with an IC50 of 45 µM. Further studies searching for
diazepam-like substances using benzodiazepine binding
assays led to the discovery that the biflavonoid amento-
flavone, Fig. (5), sometimes known as biapigenin, was
capable of displacing benzodiazepine binding to rat brain
membranes with a nM affinity comparable to that of
diazepam [156]. These investigations used amentoflavone
isolated from Karmelitter Geist, an alcoholic tincture of
various plants used to treat anxiety and epilepsy. However it
was concluded that amentoflavone cannot be responsible for
any pharmacological effects of the plant extract as
amentoflavone did not influence flunitrazepam binding in the
brain in vivo following i.v. administration to mice [156]. It
was suggested that amentoflavone was either rapidly
metabolised or did not cross the blood brain barrier, but a
GABAA Receptor Channel Pharmacology
recent study does indicate that amentoflavone does cross the
blood brain barrier [157]. Amentoflavone occurs in a variety
of herbal preparations including St John’s wort [158] and
Ginkgo biloba [159]. A comprehensive battery of in vitro
binding assays has shown that amentoflavone influences a
variety of G-protein coupled receptors for serotonin,
dopamine and opioids at nM concentrations while having no
effect on the binding of muscimol to GABAA receptors
[160]. Using recombinant α1β2γ2L GABAA receptors
expressed in oocytes, amentoflavone has been shown
recently to be a relatively weak (4 µM) negative allosteric
modulator of GABA action acting independently of classical
flumazenil-sensitive benzodiazepine modulatory sites [159].
These studies on amentoflavone illustrate the difficulties of
studying flavonoid actions – the variety of effects, the lack of
selectivity, the need for functional assays and the mismatch
between in vitro and in vivo findings.
Apigenin, Fig. (5), a component of Matricicaria recutita
flowers (chamomile), has been characterised as a centrally-
acting benzodiazepine ligand with anxiolytic effects [161].
Infusions of chamomile flowers are widely used as a tea to
promote sleep. Apigenin competitively inhibited (Ki 4 µM)
the binding of flunitrazepam to brain membranes without
influencing the binding of muscimol to GABAA receptors.
Apigenin was described as having ‘a clear anxiolytic effect
in mice in the elevated plus maze without evidencing
sedation or muscle relaxation effects at doses similar to those
HO O HO
OH
3',7-Dihydroxyisoflavan
HO O
HO
OH O
Amentoflavone
R2
HO O O
R1
OH O O
R1=H, R2=OH Apigenin
R1=MeO, R2=OH Hispidulin
R1=MeO, R2=H Oroxylin A
R1=Me, R2=OH 6-Methylapigenin
Fig. (5). Flavonoids that act on GABAA receptors.
Current Pharmaceutical Design, 2005, Vol. 11, No. 15 1875
used for classical benzodiazepines’ and it was devoid of
anticonvulsant effects [161]. These finding are in contrast to
a later study in rats where apigenin was shown to reduce the
latency of onset of picrotoxin-induced convulsions and to
reduce locomotor activity but was devoid of anxiolytic or
muscle relaxant activities [162]. This later study showed that
apigenin could reduce GABA-activated chloride currents in
cultured cerebellar granule cells, an action that could be
blocked by flumazenil and thus likely to involve classical
benzodiazepine allosteric sites on GABAA receptors. The
inhibitory action of apigenin on locomotor behaviour,
however, could not be blocked by flumazenil and thus could
not ‘be ascribed to an interaction with GABAA-benzodia-
zepine receptors, but to other neurotransmitter systems’
[162]. Another study from the same group reported that
apigenin exerted sedative effects on locomotor activity in
rats in a flumazenil-insensitive manner, whereas chrysin, a
structurally related flavonoid lacking the 4’-hydroxy
substituent of apigenin, showed a clear flumazenil-sensitive
anxiolytic effect in addition to the flumazenil-insensitive
sedation [163]. The apparent discrepancy between the
behavioural effects of apigenin on mice [161] and rats [162]
may be due to mice having higher baseline levels of anxiety.
Recent studies on recombinant receptors in oocytes have
shown that µM apigenin inhibited the activation of α1β1γ2S
GABAA receptors in a flumazenil-insensitive manner and
had a similar effect on ρ1 GABAC receptors [164]. Other
O
OH O
OH
Genistein
OH
OH
O
O
OH
NO 2
R1 Q
R=NO2 3',6-Dinitroflavone
R=Cl 6-Chloro-3'-nitroflavone
1876 Current Pharmaceutical Design, 2005, Vol. 11, No. 15
studies on recombinant α1β2γ2L GABA A receptors describe
an inhibitory effect of apigenin on GABA responses and, in
addition, describe an enhancement of the diazepam-induced
positive allosteric modulation of GABA responses by
apigenin [57, 165]. Such a second order modulation by
apigenin of benzodiazepine modulation of the activation by
GABA of GABAA receptors may indicate that apigenin
needs to work through an endogenous benzodiazepine
system to influence behaviour in a flumazenil-sensitive
manner. Overall, it seems that the effects of apigenin on
GABAA receptors are complex and involve both flumazenil-
sensitive and flumazenil–insensitive components, and that
other receptors could be involved in the behavioural effects
of apigenin. Genistein, Fig. (5), the isoflavone equivalent of
apigenin, is a phytoestrogen with a wide variety of
pharmacological effects on animal cells [166]. It is widely
used as a tyrosine kinase inhibitor but its action as a negative
modulator of the action of GABA on recombinant GABAA
receptors is the result of a direct action on the receptors and
is independent of tyrosine kinase [167, 168].
Hispidulin, Fig. (5), 4’, 5, 7-trihydroxy-6-methoxyflav-
one, i.e. the 6-methoxy derivative of apigenin), was isolated
together with apigenin from Salvia officinalis (Sage) recently
using a benzodiazepine binding assay-guided fractionation
[169]. Hispidulin was some 30 times more potent that
apigenin in displacing flumazenil binding. Preparations of
sage have been used in herbal medicine to assist memory
[170, 171] and an extract of Salvia lavandulaefolia (Spanish
sage) has been shown to enhance memory in healthy young
volunteers [172]. Hispidulin has been shown to act as a
positive allosteric modulator of α1, 3, 5, 6β2γ2S GABAA
receptor subtypes showing little subtype selectivity being a
little more potent at α1, 2, 5β2γ2S subtypes than at α3,
6β2γ2S subtypes [173]. The positive modulatory action of 10
µM hispidulin at α1β2γ2S receptors was reduced from 47%
to 17% by flumazenil, indicating that sites other than
classical flumazenil-sensitive benzodiazepine sites were
involved in the action of hispidulin. As hispidulin did not
influence the action of GABA on α1β2 GABAA receptors,
hispidulin does not interact with low affinity flumazenil-
insensitive benzodiazepine sites [174] in contrast to other
flavonoids such as 6-methylflavone [175]. Of significance is
the ability of hispidulin to act as a positive modulator at
α6β2γ2L GABAA receptors unlike diazepam; 10 µM
hispidulin enhanced the action of GABA at these receptors
by 65%, this action being reduced by 1 µM flumazenil to
37% [173]. Hispidulin was shown to have an anticonvulsant
action in seizure prone Mongolian gerbils and to pass the
blood brain barrier [173]. Flavonoids structurally related to
hispidulin, and that influence benzodiazepine binding, have
been isolated from Scutellaria baicalensis, an important herb
in traditional Chinese medicine [176]. Oroxylin A, Fig. (5),
5, 7-dihydroxy-6-methoxyflavone, i.e. hispidulin lacking the
4’-hydroxy group), inhibits flunitrazepam binding at 1 µM
and on oral administration as a neutralising allosteric
modulator blocking the anxiolytic, myorelaxant and motor
incoordination effects, but not the sedative and anticon-
vulsant effects elicited by diazepam [177]. 6-Methyla-
pigenin, Fig. (5), 4’,5,7-dihydroxy-6-methylflavone) isolated
from Valeriana wallichii, a known sedative herb, influences
benzodiazepine binding at 0.5 µM in manner suggesting it
Graham A.R. Johnston
may be a positive modulator of GABAA receptors [178].
Thus, flavones substituted in the 6-position with a methoxy
or methyl substituent have interesting effects on GABAA
receptor function and may contribute to the properties of
some herbal preparations. Natural and synthetic 2’-hydroxy-
substituted flavones are also of interest [179]. Several
flavonoid glycosides including goodyerin [180], linarin and
hesperidin [181] are also being studied as sedative and
anticonvulsant agents likely to interact with GABAA
receptors.
Using a combinatorial chemistry approach, a range of
relatively simple flavones have been synthesised and
evaluated initially for activity in a benzodiazepine binding
assay [182]. This approach led to some very interesting
compounds that were further evaluated in a variety of
pharmacological tests as GABA A receptor ligands [154]. The
most active anxiolytic flavone was 3’, 6-dinitroflavone, Fig.
(5), which was 30 times more potent than diazepam. It was
orally active and had minimal sedative action at anxiolytic
doses. In contrast, 6-chloro-3’-nitroflavone, Fig. (5), had no
anxiolytic properties and abolished the anxiolytic,
anticonvulsant and amnesic effects of diazepam [154].
There have been extensive structure-activity studies
aimed as developing models of flavonoid pharmacophores
for their interaction with GABAA receptors [183-187]. A
problem common to most of these studies is that the activity
data is based on ligand binding studies to what is now known
to be a mixture of benzodiazepine binding sites. Given our
increased knowledge of the diversity of benzodiazepine and
flavonoid actions on cloned receptors of defined subunit
composition, future structure-activity studies need to be
based on data from functional studies on GABA receptors of
known subunit composition [175].
Terpenoids and GABAA Receptors
Terpenoids are widespread in plants, especially in what
are known as essential oils that can be extracted from plants
and have a wide range of uses from perfume constituents to
paint thinners. Terpenoids are oxygenated products formally
derived from C5 isoprene units and are classified by the
number of C5 units in their structure. Thus monoterpenoids
have 2xC5 units, sesquiterpenoids 3xC5 units, diterpenoids
4xC5 units and triterpenoids 6xC5 units. The most widely
used terpenoid in studies on GABAA receptors is the
sesquiterpenoid lactone picrotoxinin, Fig. (6), a non-
competitive antagonist at GABAA receptors [1]. A number of
other terpenoids, however, are of interest for their actions on
GABAA receptors.
Bilobalide, Fig. (6), a sesquiterpenoid lactone from
Ginkgo biloba that bears some structural similarities to
picrotoxinin, including a lipophilic side chain and a
hydrophilic cage, is also a non-competitive antagonist at
GABAA receptors [188]. Both bilobalide and picrotoxinin
appear to act at sites in the chloride channel of GABAA
receptors and are thus negative allosteric modulators. The
cognition-enhancing effects of Ginkgo extracts may be partly
mediated by bilobalide acting to enhance hippocampal
pyramidal neuronal excitability [189]. While picrotoxinin is
a convulsant, bilobalide is an anticonvulsant [189, 190]. As
with the α5 subunit preferring negative allosteric modulator
GABAA Receptor Channel Pharmacology
O O
OH O
O O
O
Picortoxinin
O O
HO
α-Thujone
HO
(+)-Borneol Valerenic acid
Fig. (6). Terpenoids that act on GABAA receptors.
mentioned in Section 4.2, the lack of convulsant action in an
agent that reduces GABA action may be important for
enhancement of cognition. The lack of convulsant action of
bilobalide may result from subunit selectivity but this has yet
to be established. The structurally-related ginkgolides,
especially ginkgolide B, also act as negative modulators at
GABAA receptors [191]. They also inhibit strychnine-
sensitive glycine receptors and platelet activating factor
[191, 192]. Bilobalide and the ginkgolides reduce
barbiturate-induced sleeping time in mice, an effect perhaps
relevant to the clinically observed ‘vigilance-enhancing’ and
antidepressant-like actions of Ginkgo extracts [193].
The monoterpenoid α-thujone, Fig. (6), is a psychoactive
component of absinthe, a liqueur popular in France in the
19th and early 20th centuries. It is found in extracts of
woodworm and some other herbal medicines and beverages
since ancient Egyptian times [194]. α-Thujone is a convul-
sant that acts as a negative allosteric modulator of GABAA
receptors [195]. It also acts as an antagonist of 5HT3 recep-
tors by influencing agonist-induced desensitisation [194].
The structurally-related substance thymol, Fig. (6), a
constituent of thyme essential oil, is a flumazenil-insensitive
positive allosteric modulator of GABAA receptors [196]. At
higher concentrations, thymol had a direct action on GABAA
receptors similar to that of the anaesthetic propofol and other
phenols [197]. The anticonvulsant effects of thymoquinone,
the major constituent of Nigella sativa seeds, may be due to
positive modulation of GABAA receptors [198].
Current Pharmaceutical Design, 2005, Vol. 11, No. 15 1877
O
O
H
HO H
OH
O
O O H
Bilobalide
O
Thymol Thymoquinone
COOH
O
HO
lsocurcumenol
(+)-Borneol, Fig. (6), a monoterpenoid found in many
essential oils, is a flumazenil-insensitive positive allosteric
modulator of recombinant GABAA receptors of low affinity
but very high efficacy producing 12 fold enhancement of the
action of 10 µM GABA at a concentration of 450 µM [57,
199]. (+)-Borneol is found in high concentrations in extracts
of Valerian officinalis that are widely used to reduce the
latency of sleep onset, the depth of sleep and the perception
of well-being. Extracts of Valerian are known to contain a
large number of constituents including flavonoids and
terpenoids, many of which are considered to be active at
GABAA receptors. The sesquiterpenoid valerenic acid, Fig.
(6), has a direct partial agonist action on GABAA receptors
[200]. Isocurcumenol, Fig. (6), a sesquiterpenoid from
Cyperus rotundus, was found to inhibit [H-3]Ro15-1788
binding and enhance [H-3]flunitrazepam binding in the
presence of GABA in a manner consistent with it acting as a
positive allosteric modulator [201].
Ginsenosides, triterpenoid glycosides that are the major
active constituents of Panax ginseng, are known to
negatively modulate nicotinic and NMDA receptor activity.
Of a series of ginsenosides, ginsenoside Rc was the most
potent (EC50 53 µM) in enhancing the action of GABA on
recombinant α1β1γ2S GABAA receptors expressed in
oocytes [202].
Sage and GABAA Receptors
Sage has been used widely to treat memory deficits and
extracts of Salvia lavandulaefolia (Spanish Sage) have been
1878 Current Pharmaceutical Design, 2005, Vol. 11, No. 15
shown to enhance memory in healthy young volunteers
[172].
In addition to the flavonoids apigenin, hispidulin and
linarin (see section 7.1.), a number of terpenoids have been
extracted from varieties of Salvia (sage) that influence
benzodiazepine binding [169]. The diterpenoid lactone
galdosol, Fig. (7) from the common sage Saliva officinalis,
inhibited flumazenil binding at 0.8 µM [169].
The diterpenoid quinone miltirone, Fig. (7), from the
Chinese medicinal herb Salvia miltriorrhiza, inhibited
flunitrazepam binding at 0.3 µM and was orally active in
animal models as a tranquilliser without muscle relaxant
properties [203]. Structure-activity studies on miltirone led
to the development of a synthetic compound that was much
more potent than miltirone on flunitrazepam binding (IC50
0.05 µM) [204].
The structurally-related diterpenoids carnosic acid and
carnosol, Fig. (7), extracted from Salvia officinalis, while not
influencing diazepam or muscimol binding, did inhibit TBPS
binding [205]. This suggests that, like flavonoids,
diterpenoids can influence GABAA receptors in a manner
independent of classical benzodiazepine sites and could be
missed in benzodiazepine binding assays. The structures of
galdosol, carnosic acid and carnosol, Fig. (7), contain the o-
isopropylphenolic moiety that is present in thymol, Fig. (6),
and the anaesthetic agent propofol.
O OH
O responses [211]. When applied to mice through respiration,
HO
O drinks such as wine, sake, brandy, and shochu also
O potentiated GABA responses to varying degrees [211].
Miltirone Galdosol
OH OH
HO HO
HO2 C
O thousands times as potent as ethanol in the potentiation of
O GABAA receptor-mediated responses [211]. The aging of
Carnosic acid Garnosol
Fig. (7). Diterpenoids from Salvia that influence GABAA receptors.
Sage also contains α-thujone, Fig. (6), a known GABAA
receptor antagonist as noted above, which may influence the
GABA enhancing effects of hispidulin, galdosol, miltirone,
carnosic acid, carnosol and related compounds in sage
extracts. The levels of α-thujone in individual sage plants are
known to vary considerably [206].
Dietary and Environmental Chemicals that may
Influence GABAA Receptors
There are many chemicals in food and beverages,
together with other chemicals in our environment, that are
known to be capable of influencing the function of GABAA
Graham A.R. Johnston
receptors in addition to the flavonoids, terpenoids and
ethanol discussed in previous sections. GABA itself occurs
widely in plants being involved in pH regulation, nitrogen
storage, plant development and defence [207]. High levels of
GABA in plants extracts may be a confusing factor in
evaluating the effects of such extracts on GABAA receptors
in binding or functional assays, but such GABA will not
normally influence GABAA receptors in the brain on
ingestion due to the blood brain barrier.
Tea and coffee contain a range of chemicals in addition
to GABA that have been shown to influence recombinant
bovine α1β1 GABA A receptors. Extracts of green, oolong or
black tea contained catechins, especially (-)-epicatechin
gallate and (-)-epigallocatechin gallate, that inhibited GABA
responses and alcohols, such as leaf alcohol and linalool,
Fig. (8), that enhanced GABA responses at concentrations of
1 mM [208]. Coffee extracts contained theophylline, which
inhibited GABA responses in a non-competitive mechanism
(Ki 0.55 mM), and theobromine, which inhibited in a
competitive manner (Ki 3.8 mM), while a number of
compounds including 1-octen-3-ol and sotolone, Fig. (8),
enhanced GABA responses [209]. When 1-octen-3-ol (100
mg/kg) was orally administered to mice prior to
intraperitoneal administration of pentobarbitone, the sleeping
time of mice induced by pentobarbital increased significantly
[209]. Sotolone is a key component in the “nutty” and
“spicy-like” aroma of oxidative aged port wine [210]. Many
components in the fragrance of whiskey, in particular ethyl
3-phenylpropanoate, Fig. (8), strongly enhanced GABAA
ethyl 3-phenylpropanoate delayed the onset of convulsions
induced by pentylenetetrazole. The extract of other alcoholic
Although these fragrant components are present in alcoholic
drinks at low concentrations (extremely small quantities
compared with ethanol), they may also modulate the mood
or consciousness through the potentiation of GABAA
responses after absorption into the brain because these
hydrophobic fragrant compounds are easily absorbed into the
brain through the blood-brain barrier and are several
whiskey results in enhanced potency of the fragrance in
potentiating GABAA responses and in prolonging
pentobarbitone-induced sleeping time in mice [212]. As all
of the above tests on chemicals from tea, coffee and whiskey
were carried out on recombinant bovine α1β1 GABAA
receptors, the observed effects are independent of classical
benzodiazepine-sensitive sites. Several perfume constituents
have been shown to act as positive modulators of GABAA
receptors including the terpenoids eugenol, citronellol and
hinokitol, Fig. (8) [213]. The tea flavonoid (-)-epigallo-
catechin gallate has been shown to be some 10 times more
potent than apigenin as a second order modulator of the
positive modulation by diazepam of α1β2γ2L human
recombinant GABAA receptors expressed in oocytes [165].
Simple disaccharides are able to enhance TBOB binding
to GABAA receptors [214]. Lactose (EC50 1.5 µM) was 100-
600 fold more potent than maltose or sucrose. Lactose did
not influence flunitrazepam binding and its effects on TBOB
GABAA Receptor Channel Pharmacology
HO
HO
Leaf alcohol Citronellol
OMe O
HO
EtO
Eugenol Ethyl 3-phenylpropanoate
Fig. (8). Volatile substances in beverages and perfumes that influence GABAA receptor.
binding could be blocked by GABA. Regional differences in
the potency of lactose enhancement of TBOB binding
suggest that the effect might be GABAA receptor subtype
selective [214].
As noted in section 6, cholesterol has been long known to
produce deep anaesthesia in cats following injection of a
colloidal suspension [125] and to potentiate pentobarbitone-
induced anaesthesia [126]. Dietary cholesterol and agents
that alter cholesterol levels may influence GABAA receptor
function in the brain. Alterations in membrane cholesterol in
dissociated hippocampal neurones alters GABAA receptor
properties [215]. Cholesterol enrichment increased the
positive modulatory effects of the nonsteroidal agents
propofol, flunitrazepam and pentobarbitone but reduced the
positive modulatory effects of the steroids pregnanolone and
alphaxalone. Depletion of membrane cholesterol increased
the effects of pregnanolone and alphaxalone without
influencing the effects of the nonsteroidal modulators.
Increases in dietary cholesterol in rats has been shown to
depress brain waves as measured by EEG [216], an effect
that may be attributable to changes in GABAA receptor
function. There has been speculation about an association
between brain cholesterol and Alzheimer’s disease and the
suggestion that cholesterol-lowering strategies influence the
progression of this disease [217]. There is some evidence
that statins can reduce cholesterol turnover in the brain thus
enabling statins to reduce the incidence of Alzheimer’s
disease [218]. Such treatments might influence GABAA
receptor function through alteration of cholesterol levels in
the brain.
Inhaled drugs of abuse such as the solvents toluene, 1, 1,
1-trichloroethane and trichlorethylene act as positive
modulators of GABAA receptors, acting in a manner
suggesting that their sites of action may overlap with those
of ethanol and volatile anaesthetics [219]. Another study,
however, found toluene to be an antagonist of the activation
of GABAA receptors [220].
CONCLUSIONS
When I reviewed GABAA receptor pharmacology in
1996 [8] the then literature prompted a conclusion that there
appeared to be at least 11 distinct sites on GABAA receptors
for interactions with specific ligands. The likely sites were:
Current Pharmaceutical Design, 2005, Vol. 11, No. 15 1879
OH OH
Linalool 1-Octen-3-ol
O O
O
HO
OH
Hinekitol Sotolone
(1) agonist/partial agonist/competitive antagonist recognition
sites; (2) picrotoxinin sites; (3) sedative-hypnotic barbiturate
sites; (4) neuroactive steroid sites; (5) benzodiazepine sites;
(6) ethanol sites; (7) sites for inhalation anaesthetics; (8) sites
for furosemide associated with α6 subunits; (9) sites for
Zn2+; (10) sites for a variety of divalent cations, such as
Ca2+, Sr2+, Ba2+, Cd2+, Mn 2+, and Mg2+; and (11) sites for
La3+. I noted that it was likely that there were subtypes of
neuroactive steroid sites and that there were certainly
subtypes of benzodiazepine sites. In addition, I noted that
there were possibly sites associated with (a) phospholipids
interacting with GABAA receptor protein subunits, (b)
cyclic nucleotide protein kinase activity involved
phosphorylation of the intracellular loop of some GABAA
receptor protein subunits, and (c) the interaction of GABAA
receptors and microtubules that may anchor receptor clusters
at postsynaptic membranes.
The situation has become even more complex since 1996.
Thanks to the use of genetically modified mice we now
know the importance of the different types of GABAA
receptor subunits for the actions of particular agents – e.g.
α1 and α2 subunits for the sedative and anxiolytic actions of
benzodiazepines respectively, α5 subunits for agents
influencing spatial memory, and δ subunits for the potent
action of ethanol. Thanks to the use of recombinant receptor
technology, expressing GABAA receptors of known subunit
composition, we now have detailed knowledge of the actions
of the increasingly chemically diverse range of natural
products on GABAA receptor function. We know of
flavonoids that influence GABAA receptor function as
positive and negative allosteric modulators, some of these
actions of flavonoids being sensitive to the classical
benzodiazepine antagonist flumazenil and other actions
being insensitive. It is a similar story with terpenoids. Thus it
is likely that there are at least two distinct sites, flumazenil-
sensitive and flumazenil-insensitive, on GABAA receptors
for flavonoids and terpenoids of which the flumazenil-
sensitive sites may overlap with classical benzodiazepine
sites.
It appears likely that the various proposed sites on
GABAA receptors overlap and interact making it difficult to
put a meaningful figure on the number of distinct sites
though my 1996 figure of 11 now seems very conservative.
We await detailed three-dimensional structural information
1880 Current Pharmaceutical Design, 2005, Vol. 11, No. 15
on the various subtypes of GABAA receptors in order to
work out exactly where all of these chemically diverse
ligands interact. Such information is starting to emerge
through homology modelling based on a 4Å resolution
structure of a nicotinic receptor [221], e.g. the structure of
the proposed propofol binding site involving the M2 and M3
regions of GABAA receptors [222].
The diversity of sites on GABAA receptors represent
targets for the further development of specific agents acting
on particular GABAA receptor subtypes. The structures of
the various ligands described in this and other reviews serve
as leads for the discovery of new chemical entities for the
treatment of disorders involving specific GABAA receptors.
ACKNOWLEDGEMENTS
The author is grateful to his many colleagues who have
contributed to his studies on GABAA receptors including
Robin Allan, Erica Campbell, Mary Chebib, Rujee Duke,
Renee Granger, Belinda Hall, Jane Hanrahan, Shelley
Huang, Povl Krogsgaard-Larsen, Ken Mewett, Hue Tran and
Paul Whiting, and to the Australian National Health and
Medical Research Council and Polychip Pharmaceuticals for
financial support.
REFERENCES
[1] Chebib M, Johnston GAR, GABA-activated ligand gated ion
channels: Medicinal chemistry and molecular biology. J Med Chem
2000; 43: 1427-1447.
[2] Bormann J. The 'ABC' of GABA receptors, Trends Pharmacol Sci
2000; 21: 16-19.
[3] Le Novere N, Changeux JP. The Ligand Gated Ion Channel
database: an example of a sequence database in neuroscience, Phil.
Trans. Roy Soc London - Series B: Biol Sci 2001; 356: 1121-1130.
[4] Barnard EA, Skolnick P, Olsen RW, Möhler H, Sieghart W, Biggio
G, et al. International Union of Pharmacology - XV - Subtypes of
γ-aminobutyric acidA receptors - classification on the basis of
subunit structure and receptor function. Pharmacol Rev 1998; 50:
291-313.
[5] Vacher CM, Bettler B, GABAB receptors as potential therapeutic
targets, Curr. Drug Targ. CNS Neurol Disord 2003; 2: 248-259.
[6] Rotolo TC, Dacheux RF. Two neuropharmacological types of
rabbit ON-alpha ganglion cells express GABAC receptors. Vis
Neurosci 2003; 20: 373-84.
[7] Whiting PJ. GABA A receptor subtypes in the brain: a paradigm for
CNS drug discovery? Drug Discovery Today 2003; 8: 445-450.
[8] Johnston GAR. GABA A receptor pharmacology. Pharmacol Therap
1996; 69: 173-198.
[9] McKernan RM, Whiting PJ. Which GABAA-receptor subtypes
really occur in the brain?. Trends Neurosci 1996; 19: 139-143.
[10] Burt DR. Reducing GABA receptors. Life Sci 2003: 73: 1741-
1758.
[11] Smith AJ, Simpson PB. Methodological approaches for the study
of GABA A receptor pharmacology and functional responses. Anal
Bioanal Chem 2003; 377: 843-851.
[12] Rosahl TW. Validation of GABAA receptor subtypes as potential
drug targets by using genetically modified mice. Curr Drug Targ
CNS Neurol Disord 2003; 2: 207-212.
[13] Möhler H, Fritschy JM, Rudolph U. A new benzodiazepine
pharmacology. J. Pharmacol Exp Therap 2002; 300: 2-8.
[14] Rudolph U, Crestani F, Möhler H. GABAA receptor subtypes:
dissecting their pharmacological functions. Trends Pharmacol Sci
2001; 22: 188-194.
[15] Sieghart W. Unravelling the function of GABAA receptor subtypes.
Trends Pharmacol Sci 2000; 21: 411-413.
[16] Hamilton NM. Interaction of steroids with the GABAA receptor.
Curr Top Med Chem 2002; 2: 887-992.
Graham A.R. Johnston
[17] Lambert JJ, Belelli D, Peden DR, Vardy AW, Peters JA.
Neurosteroid modulation of GABAA receptors. Prog. Neurobiol
2003; 71: 67-80.
[18] Korpi ER, Grunder G, Luddens H. Drug interactions at GABAA
receptors, Prog Neurobiol 2002; 67: 113-159.
[19] Krogsgaard-Larsen P, Frølund B, Liljefors T, Specific GABA-A
agonists and partial agonists. The Chemical Record 2002; 2: 419-
430.
[20] Kneussel M, Dynamic regulation of GABAA receptors at synaptic
sites. Brain Res. - Brain Res Rev 2002; 39: 74-83.
[21] Sigel E, Dodd RH. Novel positive allosteric modulators of GABAA
receptors. Drugs of the Future 2001; 26: 1191-1197.
[22] Frølund B, Ebert B, Kristiansen U, Liljefors T, Krogsgaard-Larsen
P. GABAA receptor ligands and their therapeutic potentials. Curr
Top Med Chem 2002; 2: 817-832.
[23] Rudolph U, Möhler H. Analysis of GABAA receptor function and
dissection of the pharmacology of benzodiazepines and general
anesthetics through mouse genetics. Ann Rev Pharmacol Toxicol
2004; 44: 475-498.
[24] Johnston GAR, Chebib M, Hanrahan JR, Mewett KN. GABAC
receptors as drug targets. Curr Drug Targ CNS Neurol Disord
2003; 2: 260-268.
[25] Christopoulos A. Allosteric binding sites on cell-surface receptors:
Novel targets for drug discovery. Nature Rev Drug Disc 2002; 1:
198-210.
[26] Vafa B, Schofield PR. Heritable mutations in the glycine, GABAA,
and nicotinic acetylcholine receptors provide new insights into the
ligand-gated ion channel superfamily. Int Rev Neurobiol 1998; 42:
285-332.
[27] Holopainen IE, Metsahonkala EL, Kokkonen H, Parkkola RK,
Manner TE, Nagren K, et al. Decreased binding of [11C]flumazenil
in Angelman syndrome patients with GABAA receptor β3 subunit
deletions. Ann Neurol 2001; 49: 110-113.
[28] Homanics GE, Delorey TM, Firestone LL, Quinlan JJ, Handforth
A, Harrison NL, et al. Mice devoid of γ-aminobutyrate type A
receptor β3 subunit have epilepsy, cleft palate, and hypersensitive
behavior. Proc Natl Acad Sci USA 1997; 94: 4143-4148.
[29] Sinkkonen ST, Homanics GE, Korpi ER. Mouse models of
Angelman syndrome, a neurodevelopmental disorder, display
different brain regional GABAA receptor alterations. Neurosci Lett
2003; 340: 205-208.
[30] Feusner J, Ritchie T, Lawford B, Young RM, Kann B, Noble EP.
GABAA receptor beta 3 subunit gene and psychiatric morbidity in a
post-traumatic stress disorder population. Psych Res 2001; 104:
109-117.
[31] Taylor M, Bhagwagar Z, Cowen PJ, Sharp T. GABA and mood
disorders. Psych Med 2003; 33: 3873-93.
[32] Loh EW, Ball D. Role of the GABA-Abeta 2, GABA-Aalpha 6,
GABA-Aalpha 1 and GABA-Agamma 2 receptor subunit genes
cluster in drug responses and the development of alcohol
dependence. Neurochem Int 2000; 37: 413-423.
[33] Parsian A, Zhang ZH. Human chromosomes 11p15 and 4p12 and
alcohol dependence: Possible association with the GABRB1 gene.
Am J Med Genet 1999; 88: 533-538.
[34] Neu A, Neuhoff H, Trube G, Fehr S, Ullrich K, Roeper J, et al.
Activation of GABAA receptors by guanidinoacetate: A novel
pathophysiological mechanism. Neurobiol Dis 2002; 11: 298-307.
[35] Leung JW, Xue H. GABAergic functions and depression: from
classical therapies to herbal medicine. Curr Drug Targ CNS Neurol
Disord 2003; 2: 363-374.
[36] Wassef AA, Dott SG, Harris A, Brown A, O'Boyle M, Meyer WJ,
et al. Critical review of GABA-ergic drugs in the treatment of
schizophrenia. J Clin Psychopharmacol 1999; 19: 222-232.
[37] Wassef A, Baker J, Kochan LD. GABA and schizophrenia: a
review of basic science and clinical studies. J Clin Psycho-
pharmacol 2003; 23: 601-640.
[38] Green AR, Hainsworth AH, Jackson DM. GABA potentiation: a
logical pharmacological approach for the treatment of acute
ischaemic stroke. Neuropharmacology 2000; 39: 1483-1494.
[39] Shulman A, Strashun AM, Goldstein BA. GABA A-benzodiazepine-
chloride receptor-targeted therapy for tinnitus control: preliminary
report. Int Tinnitus J 2002; 8: 30-6.
[40] Maubach K. GABAA receptor subtype selective cognition
enhancers. Curr Drug Targ CNS Neurol Disord 2003; 2: 233-239.
[41] Scheffer IE, Berkovic SF. The genetics of human epilepsy. Trends
Pharmacol Sci 2003; 24: 428-433.
GABAA Receptor Channel Pharmacology
[42] Jones-Davis DM, Macdonald RL. GABAA receptor function and
pharmacology in epilepsy and status epilepticus. Curr Opin
Pharmacol 2003; 3: 12-18.
[43] Baulac S, Huberfeld G, Gourfinkel-An I, Mitropoulou G, Beranger
A, Prud'homme JF, et al. First genetic evidence of GABAA receptor
dysfunction in epilepsy: a mutation in the gamma 2-subunit gene.
Nature Genetics 2001; 28: 46-48.
[44] Wallace RH, Marini C, Petrou S, Harkin LA, Bowser DN, Panchal
RG, et al. Mutant GABAA receptor gamma 2-subunit in childhood
absence epilepsy and febrile seizures. Nature Genetics 2001; 28:
49-52.
[45] Bianchi MT, Song LY, Zhang H, Macdonald RL. Two different
mechanisms of disinhibition produced by GABAA receptor
mutations linked to epilepsy in humans. J Neurosci 2002; 22: 5321-
5327.
[46] Macdonald RL, Bianch MT, Feng H. Mutations linked to
generalized epilepsy in humans reduce GABAA receptor current.
Exp Neurol 2003; 184: S58-67.
[47] Kananura C, Haug K, Sander T, Runge U, Gu WL, Hallmann K, et
al. A splice-site mutation in GABRG2 associated with childhood
absence epilepsy and febrile convulsions. Arch Neurol 2002; 59:
1137-1141.
[48] Chou IC, Peng CT, Huang CC, Tsai JJ, Tsai FJ, Tsai CH.
Association analysis of gamma 2 subunit of gamma-aminobutyric
acid type A receptor polymorphisms with febrile seizures. Pediat
Res 2003; 54: 26-29.
[49] Cossette P, Liu LD, Brisebois K, Dong HH, Lortie A, Vanasse M,
et al. Mutation of GABRA1 in an autosomal dominant form of
juvenile myoclonic epilepsy. Nature Genetics 2002; 31: 184-189.
[50] Fisher JL. A mutation in the GABAA receptor α1 subunit linked to
human epilepsy affects channel gating properties. Neuropharma-
cology 2004; 46: 629-637.
[51] Gottesmann C. GABA mechanisms and sleep. Neuroscience 2002;
111: 231-239.
[52] Laposky AD, Homanics GE, Basile A, Mendelson WB. Deletion of
the GABA A receptor beta 3 subunit eliminates the hypnotic actions
of oleamide in mice. NeuroReport 2001; 12: 4143-4147.
[53] Buhr A, Bianchi MT, Baur R, Courtet P, Pignay V, Boulenger JP,
et al. Functional characterization of the new human GABAA
receptor mutation beta 3(R192H). Hum Genet 2002; 111: 154-160.
[54] Smith AJ, Alder L, Silk J, Adkins C, Fletcher AE, Scales T, et al.
Effect of alpha subunit on allosteric modulation of ion channel
function in stably expressed human recombinant gamma-
aminobutyric acid(A) receptors determined using Cl-36 ion flux.
Mol Pharmacol 2001; 59: 1108-1118.
[55] Sanna E, Busonero F, Talani G, Carta M, Massa F, Peis M, et al.
Comparison of the effects of zaleplon, zolpidem, and triazolam at
various GABAA receptor subtypes. Eur J Pharmacol 2002; 451:
103-110.
[56] Storustovu S, Ebert B. Gaboxadol: in vitro interaction studies with
benzodiazepines and ethanol suggest functional selectivity. Eur J
Pharmacol 2003; 467: 49-56.
[57] Johnston GAR. Dietary chemicals and brain function. J Proc Roy
Soc NSW 2003; 135: 57-71.
[58] Cherubini E, Conti F. Generating diversity at GABAergic
synapses. Trends Neurosci 2001; 24: 155-162.
[59] Smart TG. Regulation of excitatory and inhibitory
neurotransmitter-gated ion channels by protein phosphorylation.
Curr Opin Neurobiol 1997; 7: 358-367.
[60] Hosie AM, Dunne EL, Harvey RJ, Smart TG. Zinc-mediated
inhibition of GABAA receptors: discrete binding sites underlie
subtype specificity. Nature Neurosci 2003; 6: 362-369.
[61] Benson JA, Low K, Keist R, Möhler H, Rudolph U. Pharmacology
of recombinant γ-aminobutyric acid A receptors rendered diazepam-
insensitive by point-mutated α-subunits. FEBS Letters 1998; 431:
400-404.
[62] Rudolph U, Crestani F, Benke D, Brunig I, Benson JA, Fritschy
JM, et al. Benzodiazepine actions mediated by specific gamma-
aminobutyric acid(A) receptor subtypes. Nature 1999; 401: 796-
800.
[63] McKernan RM, Rosahl TW, Reynolds DS, Sur C, Wafford KA,
Atack JR, et al. Sedative but not anxiolytic properties of
benzodiazepines are mediated by the GABA A receptor alpha(1)
subtype. Nature Neurosci 2000; 3: 587-592.
Current Pharmaceutical Design, 2005, Vol. 11, No. 15 1881
[64] Low K, Crestani F, Keist R, Benke D, Brunig I, Benson JA, et al.
Molecular and neuronal substrate for the selective attenuation of
anxiety. Science 2000; 290: 131-134.
[65] Reynolds DS, McKernan RM, Dawson GR. Anxiolytic-like action
of diazepam: which GABAA receptor subtype is involved? Trends
Pharmacol Sci 2001; 22: 402-403.
[66] Atack JR. Anxioselective compounds acting at the GABAA
receptor benzodiazepine binding site. Curr Drug Targ CNS Neurol
Disord 2003; 2: 213-232.
[67] Collins I, Moyes C, Davey WB, Rowley M, Bromidge FA, Quirk
K, et al. 3-Heteroaryl-2-pyridones: Benzodiazepine site ligands
with functional selectivity for α2/α3-subtypes of human GABAA
receptor-ion channels. J Med Chem 2002; 45: 1887-1900.
[68] Carling RW, Moore KW, Street LJ, Wild D, Isted C, Leeson PD, et
al. 3-Phenyl-6-(2-pyridyl)methoxy-1, 2, 4-triazolo[3, 4-
a]phthalazines and analogues: High-affinity γ-aminobutyric acid-A
benzodiazepine receptor ligands with α2, α3 and α5-subtype
binding selectivity over α1. J Med Chem 2004; 47: 1807-1822.
[69] Crestani F, Martin JR, Möhler H, Rudolph U. Mechanism of action
of the hypnotic zolpidem in vivo. Br J Pharmacol 2000; 131: 1251-
1254.
[70] Dodd PR, Davies LP, Watson WE, Nielsen B, Dyer JA, Wong LS,
et al. Neurochemical studies on quinolone antibiotics: effects on
glutamate, GABA and adenosine systems in mammalian CNS.
Pharmacol Toxicol 1989; 64: 404-411.
[71] Johnstone TB, Hogenkamp DJ, Coyne L, Su J, Halliwell RF, Tran
MB, et al. Modifying quinolone antibiotics yields new anxiolytics.
Nat Med 2004; 10: 31-32.
[72] Caraiscos VB, Elliott EM, You-Ten KE, Cheng VY, Belelli D,
Newell JG, et al. Tonic inhibition in mouse hippocampal CA1
pyramidal neurons is mediated by α5 subunit-containing gamma-
aminobutyric acid type A receptors. Proc Natl Acad Sci USA 2004;
101: 3662-3667.
[73] Collinson N, Kuenzi FM, Jarolimek W, Maubach KA, Cothliff R,
Sur C, et al. Enhanced learning and memory and altered
GABAergic synaptic transmission in mice lacking the alpha 5
subunit of the GABAA receptor. J Neurosci 2002; 22: 5572-5580.
[74] Chambers MS, Atack JR, Broughton HB, Collinson N, Cook S,
Dawson GR, et al. Identification of a novel, selective GABAA α5
receptor inverse agonist which enhances cognition. J Med Chem
2003; 46: 2227-2240.
[75] Jackel C, Kleinz R, Makela R, Hevers W, Jezequel S, Korpi ER, et
al. The main determinant of furosemide inhibition on GABA(a)
receptors is located close to the first transmembrane domain. Eur J
Pharmacol 1998; 357: 251-256.
[76] Knoflach F, Benke D, Wang Y, Scheurer L, Luddens H, Hamilton
BJ, et al. Pharmacological modulation of the diazepam-insensitive
recombinant gamma-aminobutyric acid(a) receptors alpha-4-alpha-
2-gamma-2 and alpha-6-beta-2-gamma-2. Mol Pharmacol 1996;
50: 1253-1261.
[77] Thompson SA, Whiting PJ, Wafford KA. Barbiturate inrteractions
at the human GABAA receptor: dependnece on receptor subunit
combination. Br J Pharmacol 1996; 117: 521-527.
[78] Thompson SA, Arden SA, Marshall G, Wingrove PB, Whiting PJ,
Wafford KA. Residues in transmembrane domains I and II
determine gamma-aminobutyric acid type A receptor subtype-
selective antagonism by furosemide. Mol Pharmacol 1999; 55:
993-999.
[79] Luddens H, Lang HJ, Korpi ER. Structure-activity relationship of
furosemide-derived compounds as antagonists of cerebellum-
specific GABAA receptors. Eur J Pharmacol 1998; 344: 269-277.
[80] Thomet U, Baur R, Razet R, Dodd RH, Furtmuller R, Sieghart W,
et al. A novel positive allosteric modulator of the GABAA receptor:
the action of (+)-ROD188. Br J Pharmacol 2000; 131: 843-850.
[81] Wall MJ. Cis-4-amino-crotonic acid activates alpha 6 subunit-
containing GABAA but not GABAC receptors in granule cells of
adult rat cerebellar slices. Neurosci Lett 2001; 316: 37-40.
[82] Sinkkonen ST, Mansikkamaki S, Moykkynen T, Luddens H, Uusi-
Oukari M, Korpi ER. Receptor subtype-dependent positive and
negative modulation of GABA A receptor function by niflumic acid,
a nonsteroidal anti-inflammatory drug. Mol Pharmacol 2003; 64:
753-63.
[83] Jones A, Korpi ER, Mckernan RM, Pelz R, Nusser Z, Makela R, et
al. Ligand-gated ion channel subunit partnerships - GABA(a)
receptor alpha(6) subunit gene inactivation inhibits delta subunit
expression. J Neurosci 1997; 17: 1350-1362.
1882 Current Pharmaceutical Design, 2005, Vol. 11, No. 15
[84] Homanics GE, Ferguson C, Quinlan JJ, Daggett J, Snyder K,
Lagenaur C, et al. Gene knockout of the α6 subunit of the γ-
aminobutyric acid type A receptor - lack of effect on responses to
ethanol, pentobarbital, and general anesthetics. Mol Pharmacol
1997; 51: 588-596.
[85] Korpi ER, Koikkalainen P, Vekovischeva OY, Makela R, Kleinz R,
Uusi-Oukari M, et al. Cerebellar granule-cell-specific GABAA
receptors attenuate benzodiazepine-induced ataxia: evidence from
α6-deficient mice. Eur J Neurosci 1999; 11: 233-240.
[86] Sigel E, Baur R. Electrophysiological evidence for the coexistence
of alpha 1 and alpha 6 subunits in a single functional GABAA
receptor. J Neurochem 2000; 74: 2590-2596.
[87] Wall MJ. Furosemide reveals heterogeneous GABAA receptor
expression at adult rat Golgi cell to granule cell synapses,
Neuropharmacology 2002; 43: 737-749.
[88] Leao RM, Mellor JR, Randall AD. Tonic benzodiazepine-sensitive
GABAergic inhibition in cultured rodent cerebellar granule cells.
Neuropharmacology 2000; 39: 990-1003.
[89] Rosmond R, Bouchard C, Bjorntorp P. Allelic variants in the
GABAA alpha 6 receptor subunit gene (GABRA6) is associated
with abdominal obesity and cortisol secretion. Int J Obes Rel
Metabol Disord 2002; 26: 938-941.
[90] Hoffman WE, Balyasnikova IV, Mahay H, Danilov SM, Baughman
VL. GABA alpha 6 receptors mediate midazolam-induced
anxiolysis. J Clin Anes 2002; 14: 206-209.
[91] Reynolds DS, Rosahl TW, Cirone J, O'Meara GF, Haythornthwaite
A, Newman RJ, et al. Sedation and anesthesia mediated by distinct
GABAA receptor isoforms. J Neurosci 2003; 23: 8608-8617.
[92] Cirone J, Rosahl TW, Reynolds DS, Newman RJ, O'Meara GF,
Hutson PH, et al. Gamma-aminobutyric acid type A receptor beta 2
subunit mediates the hypothermic effect of etomidate in mice.
Anesthesiology 2004; 100: 1438-1445.
[93] Halliwell RF, Thomas P, Patten D, James CH, Martinez TA, Miledi
R, et al. Subunit-selective modulation of GABAA receptors by the
non-steroidal anti-inflammatory agent, mefenamic acid. Eur J
Neurosci 1999; 11: 2897-2905.
[94] Thompson SA, Wheat L, Brown NA, Wingrove PB, Pillai GV, et
al. Salicylidene salicylhydrazide, a selective inhibitor of β1-
containing GABAA receptors. Br J Pharmacol 2004; 142: 97-106.
[95] Quinlan JJ, Firestone LL, Homanics GE. Mice lacking the long
splice variant of the gamma 2 subunit of the GABAA receptor are
more sensitive to benzodiazepines. Pharmacol Biochem Behav
2000; 66: 371-374.
[96] Wallner M, Hanchar HJ, Olsen RW. Ethanol enhances alpha 4 beta
3 delta and alpha 6 beta 3 delta gamma-aminobutyric acid type A
receptors at low concentrations known to affect humans. Proc Natl
Acad Sci USA 2003; 100: 15218-15223.
[97] Harris RA. Transfected cells for study of alcohol actions on
GABA(a) receptors. Addict Biol 1996; 1: 157-163.
[98] Sur C, Farrar SJ, Kerby J, Whiting PJ, Atack JR, McKernan RM.
Preferential coassembly of alpha 4 and delta subunits of the
gamma-aminobutyric acid(A) receptor in rat thalamus. Mol
Pharmacol 1999; 56: 110-115.
[99] Peng Z, Hauer B, Mihalek RM, Homanics GE, Sieghart W, Olsen
RW, et al. GABA A receptor changes in delta subunit-deficient
mice: Altered expression of alpha 4 and gamma 2 subunits in the
forebrain. J Comp Neurol 2002; 446: 179-197.
[100] Mihalek RM, Banerjee PK, Korpi ER, Quinlan JJ, Firestone LL,
Mi ZP, et al. Attenuated sensitivity to neuroactive steroids in
gamma-aminobutyrate type A receptor delta subunit knockout
mice. Proc Natl Acad Sci USA 1999; 96: 12905-12910.
[101] Spigelman I, Li Z, Liang J, Cagetti E, Samzadeh S, Mihalek RM, et
al. Reduced inhibition and sensitivity to neurosteroids in
hippocampus of mice lacking the GABAA receptor delta subunit. J
Neurophysiol 2003; 90: 903-910.
[102] Johnston GAR. GABA chemistry: analogs of GABA as therapeutic
and investigational agents. In GABA In The Nervous System: The
View At Fifty Years, DL. Martin R. Olsen, eds.; Lippincott
Williams and Wilkins: Philadelphia 2000; pp. 65-80.
[103] Woodward RM, Polenzani L, Miledi R. Characterization of
bicuculline/baclofen-insensitive (rho-like) gamma-aminobutyric
acid receptors expressed in Xenopus oocytes. II. Pharmacology of
gamma-aminobutyric acidA and gamma-aminobutyric acidB
receptor agonists and antagonists. Mol Pharmacol 1993; 43: 609-
625.
Graham A.R. Johnston
[104] Krogsgaard-Larsen P, Johnston GAR, Lodge D, Curtis DR. A new
class of GABA agonist. Nature 1977; 268: 53-55.
[105] Krogsgaard-Larsen P, Frølund B, Kristiansen U, Frydenvang K,
Ebert B. GABAA and GABAB receptor agonists, partial agonists,
antagonists and modulators - design and therapeutic prospects. Eur
J Pharm Sci 1997; 5: 355-384.
[106] Kjaer M, Neilson H. The analgesic effect of the GABA agonist
THIP in patients with chronic pain of malignant origin. Br J Clin
Pharmacol 1983; 16: 477-485.
[107] Vaught JL, Pelley K, Costa LG, Setler P, Enna SJA. A comparison
of the antinociceptive responses to the GABA-receptor agonists
THIP and baclofen. Neuropharmacology 1985; 24: 211-216.
[108] Faulhaber J, Steiger A, Lancel M. The GABAA agonist THIP
produces slow wave sleep and reduces spindling activity in NREM
sleep in humans. Psychopharmacology 1997; 130: 285-291.
[109] Zorn SH, Enna SJ. The GABA agonist THIP attentuates
antinociception in the mouse by modifying central cholinergic
transmission. Neuropharmacology 1987; 26: 433-437.
[110] Tatsuo M, Yokoro CM, Salgado JV, Pesquero S, Santana M,
Francischi JN. Hyperalgesic effect induced by barbiturates,
midazolam and ethanol - pharmacological evidence for GABA-a
receptor involvement. Brazil J Med Biol Res 1997; 30: 251-256.
[111] Arnaud C, Gauthier P, Gottesmann C. Study of a GABAC receptor
antagonist on sleep-waking behavior in rats. Psychopharmacology
2001; 154: 415-419.
[112] Skerritt JH, Johnston GAR. Diazepam stimulates the binding of
GABA and muscimol but not THIP to rat brain membranes.
Neurosci Lett 1983; 38: 315-320.
[113] Hansen SL, Sperling BB, Sanchez C. Anticonvulsant and
antiepileptogenic effects of GABAA receptor ligands in
pentylenetetrazole-kindled mice. Prog Neuropsychopharm Biol
Psych 2004; 28: 105-13.
[114] Ebert B, Whiting PJ, Krogsgaard-Larsen P, Kemp JA. Molecular
pharmacology of γ-aminobutyric acid type A receptor agonists and
partial agonists in oocytes injected with different α, β, and γ
receptor subunit combinations. Mol Pharmacol 1994; 46: 957-963.
[115] Brown N, Kerby J, Bonnert TP, Whiting PJ, Wafford KA.
Pharmacological characterization of a novel cell line expressing
human alpha(4)beta(3)delta GABAA receptors. Br J Pharmacol
2002; 136: 965-974.
[116] Vien J, Duke RK, Mewett KN, Johnston GAR, Shingai R, Chebib
M. trans-4-Amino-2-methylbut-2-enoic acid (2-MeTACA) and (+/-
)-trans-2-aminomethylcyclopropanecarboxyic acid (+/-)-TAMP)
can differentiate rat rho 3 from human rho 1 and rho 2 recombinant
GABAC receptors. Br J Pharmacol 2002; 135: 883-890.
[117] Voss J, Sanchez C, Michaelsen S, Ebert B. Rotarod studies in the
rat of the GABAA receptor agonist gaboxadol: lack of ethanol
potentiation and benzodiazepine cross-tolerance. Eur J Pharmacol
2003; 482: 215-222.
[118] Ebert B, Storustovu SI, Mortensen M, Frølund B. Characterization
of GABAA receptor ligands in the rat cortical wedge preparation:
evidence for action at extrasynaptic receptors?. Br J Pharmacol
2002; 137: 1-8.
[119] Allan RD, Evans RH, Johnston GAR. γ-Aminobutyric acid
agonists: an in vitro comparison between depression of spinal
synaptic activity and depolarization of spinal root fibres in the rat.
Br J Pharmacol 1980; 70: 609-615.
[120] Lindquist CE, Ebert B, Birnir B. Extrasynaptic GABAA channels
activated by THIP are modulated by diazepam in CA1 pyramidal
neurons in the rat brain hippocampal slice. Mol Cell Neurosci
2003: 24: 250-257.
[121] Kristiansen U, Lambert J. Benzodiazepine and barbiturate ligands
modulate responses of cultured hippocampal neurones to the
GABAA receptor partial agonist, 4-piol. Neuropharmacology 1996;
35: 1181-1191.
[122] Mortensen M, Frølund B, Jorgensen AT, Liljefors T, Krogsgaard-
Larsen P, Ebert B. Activity of novel 4-PIOL analogues at human
alpha(1)beta(2)gamma(2S) GABAA receptors - correlation with
hydrophobicity. Eur J Pharmacol 2002; 451: 125-132.
[123] Carlier PR, Chow ESH, Barlow RL, Bloomquist JR. Discovery of
non-zwitterionic GABA A receptor full agonists and a superagonist.
Bioorg Med Chem Lett 2002; 12: 1985-1988.
[124] Adkins CE, Pillai GV, Kerby J, Bonnert TP, Haldon C, McKernan
RM, et al. α4β3δ GABA A receptors characterized by fluorescence
resonance energy transfer-derived measurements of membrane
potential. J Biol Chem 2001; 276: 38934-38939.
GABAA Receptor Channel Pharmacology
[125] Cashin MF, Moravek V. The physiological action of cholesterol.
Am J Physiol 1927; 82: 294-298.
[126] Starkenstein E, Weden H. The physiology and pharmacology of
sterols. IV. Influence of cholesterol in the action of hypnotics and
narcotics. Arch exp Pathol Pharmak 1936; 182: 700-714.
[127] Seyle H. Correlations between the chemical structure and the
pharmacological actions of the steroids. Endocrinology 1942; 30:
437-453.
[128] Harrison NL, Simmonds MA. Modulation of the GABA receptor
complex by a steroid anaesthetic. Brain Res 1984; 323: 287-292.
[129] Majewska MD, Harrison NL, Schwartz RD, Barker JL, Paul SM.
Steroid hormone metabolites are barbiturate-like modulators of the
GABA receptor. Science 1986; 232: 1004-1007.
[130] Purdy RH, Morrow AL, Moore PH, Paul SM. Stress-induced
elevations of γ-aminobutyric acid type A receptor-active steroids in
the rat brain. Proc Natl Acad Sci USA 1991; 88: 4553-4557.
[131] Belelli D, Casula A, Ling A, Lambert JJ. The influence of subunit
composition on the interaction of neurosteroids with GABAA
receptors, Neuropharmacology 2002; 43: 651-661.
[132] Wohlfarth KM, Bianchi MT, Macdonald RL. Enhanced
neurosteroid potentiation of ternary GABAA receptors containing
the delta subunit. J Neurosci 2002; 22: 1541-1549.
[133] Rick CE, Ye Q, Finn SE, Harrison NL. Neurosteroids act on the
GABAA receptor at sites on the N-terminal side of the middle of
TM2. NeuroReport 1998; 9: 379-383.
[134] Pistis M, Belelli D, McGurk K, Peters JA, Lambert JJ.
Complementary regulation of anaesthetic activation of human
(alpha(6)beta(3)gamma(2L)) and Drosophila (RDL) GABA
receptors by a single amino acid residue. J Physiol 1999; 515: 3-18.
[135] Darbandi-Tonkabon R, Hastings WR, Zeng CM, Akk G, Manion
BD, Bracamontes JR, et al. Photoaffinity labeling with a
neuroactive steroid analogue. 6-azi-pregnanolone labels voltage-
dependent anion channel-1 in rat brain. J Biol Chem 2003; 278:
13196-13206.
[136] Darbandi-Tonkabon R, Manion BD, Hastings WR, Craigen WJ,
Akk G, Bracamontes JR, et al. Neuroactive steroid interactions
with voltage-dependent anion channels: lack of relationship to
GABAA receptor modulation and anesthesia. J Pharmacol Exp
Therapeut 2004; 308: 502-511.
[137] Jorge-Rivera JC, McIntyre KL, Henderson LP. Anabolic steroids
induce region- and subunit-specific rapid modulation of GABAA
receptor-mediated currents in the rat forebrain. J Neurophysiol
2000; 83: 3299-3309.
[138] Monaghan EP, Navalta LA, Shum L, Ashbrook DW, Lee DA.
Initial human experience with ganaxolone, a neuroactive steroid
with antiepileptic activity. Epilepsia 1997; 38: 1026-1031.
[139] Reddy DS, Newer gabaergic agents for pharmacotherapy of
infantile spasms. Drugs Today 2002; 38: 657-675.
[140] Snead OC, Ganaxolone. a selective, high-affinity steroid modulator
of the γ-aminobutyric acid-A receptor, exacerbates seizures in
animal models of absence. Ann Neurol 1998; 44: 688-691.
[141] Ong J, Kerr DI, Johnston GAR. Cortisol: a potent biphasic
modulator at GABAA-receptor complexes in the guinea pig isolated
ileum. Neurosci Lett 1987; 82: 101-106.
[142] Ong J, Kerr DI, Capper HR, Johnston GAR. Cortisone: a potent
GABAA antagonist in the guinea-pig isolated ileum. J Pharm
Pharmacol 1990; 42: 662-664.
[143] Andres-Trelles F, Bibby V, Lustman S, Simmonds MA. Effects of
cortisol on GABAA receptor-mediated responses compared in
guinea-pig ileum and rat cuneate nucleus. Neuropharmacology
1989; 28: 705-708.
[144] Majewska MD. Antagonist-type interaction of glucocorticoids with
the GABA receptor-coupled chloride channel. Brain Res 1987;
418: 377-382.
[145] Zaki A, Barrett-Jolley R. Rapid neuromodulation by cortisol in the
rat paraventricular nucleus: an in vitro study. Br J Pharmacol 2002;
137: 87-97.
[146] Orchinik M, Hastings N, Witt D, McEwen BS. High affinity
binding of corticosterone to mammalian neuronal membranes -
possible role of corticosteroid binding globulin. J Steroid Biochem
Mol Biol 1997; 60: 229-236.
[147] Orchinik M, Carroll SS, Li YH, McEwen BS, Weiland NG.
Heterogeneity of hippocampal GABAA receptors: Regulation by
corticosterone. J Neurosci 2001; 21: 330-339.
[148] Keenan PA, Kuhn TW. Do glucocorticoids have adverse effects on
brain function? CNS Drugs 1999; 11: 245-251.
Current Pharmaceutical Design, 2005, Vol. 11, No. 15 1883
[149] Topliss JG, Clark AM, Ernst E, Hufford CD, Johnston GAR,
Rimoldi JM, et al. Natural and synthetic substances related to
human health - (IUPAC Technical Report). Pure Appl Chem 2002;
74: 1957-1985.
[150] Aherne SA, O'Brien NM. Dietary flavonols: Chemistry, food
content, and metabolism. Nutrition 2002; 18: 75-81.
[151] Havsteen BH. The biochemistry and medical significance of the
flavonoids. Pharmacol Therap 2002; 96: 67-202.
[152] Heim KE, Tagliaferro AR, Bobilya DJ. Flavonoid antioxidants:
chemistry, metabolism and structure-activity relationships. J Nutrit
Biochem 2002; 13: 572-584.
[153] Le Marchand L. Cancer preventive effects of flavonoids - a review.
Biomed Pharmacotherap 2002; 56: 296-301.
[154] Marder M, Paladini AC. GABAA receptor ligands of flavonoid
structure. Curr Top Med Chem 2002; 2: 853-867.
[155] Luk KC, Stern L, Weigele M, O'Brien RA, Spirst N. Isolation and
identification of "diazepam-like" compounds in bovine brain. J Nat
Prod 1983; 46: 852-861.
[156] Nielsen M, Frokjaer S, Braestrup C. High affinity of the naturally-
occurring biflavonoid, amentoflavone, to brain benzodiazepine
receptors in vitro. Biochem Pharmacol 1988; 37: 3285-3287.
[157] Gutmann H, Bruggisser R, Schaffner W, Bogman K, Botomino A,
Drewe J. Transport of amentoflavone across the blood-brain barrier
in vitro. Plant Med 2002; 68: 804-807.
[158] Baureithel KH, Büter KB, Engesser A, Burkard W, Schaffer W.
Inhibition of benzodiazepine binding in vitro by amentoflavone, a
constituent of various species of Hypericum. Pharm Acta Helv
1997; 72: 153-157.
[159] Hanrahan JR, Chebib M, Davucheron NM, Hall BJ, Johnston GAR.
Semisynthetic preparation of amentoflavone: a negative modulator
at GABAA receptors. Bioorg Med Chem Lett 2003; 13: 2281-2284.
[160] Butterweck V, Nahrstedt A, Evans J, Hufeisen S, Rauser L, Savage
J, Poedak B, et al. In vitro receptor screening of pure constitutents
of St. John's wort reveals novel interactions with a number of
GPCRs. Psychopharmacology 2002; 162: 193-202.
[161] Viola H, Wasowski C, Levi de Stein M, Wolfman C, Silvera R,
Medina AE, et al. a component of Matricaria recutita flowers, is a
central benzodiazepine receptors-ligand with anxiolytic effects.
Plant Med 1995; 61: 213-216.
[162] Avallone R, Zanoli P, Puia G, Kleinschnitz M, Schreier P, Baraldi
M. Pharmacological profile of apigenin, a flavonoid isolated from
Matricaria chamomilla. Biochem Pharmacol 2000; 59: 1387-1394.
[163] Zanoli P, Avallone R, Baraldi M. Behavioural characterisation of
the flavonoids apigenin and chrysin. Fitoterapia 2000; 71: S117-
S123.
[164] Goutman JD, Waxemberg MD, Donate-Oliver F, Pomata PE,
Calvo DJ. Flavonoid modulation of ionic currents mediated by
GABAA and GABAC receptors. Eur J Pharmacol 2003; 461: 79-87.
[165] Campbell EL, Chebib M, Johnston GAR. The dietary flavonoids
apigenin and (-)-epigallocatechin gallate enhance the positive
modulation by diazepam of the activation by GABA of
recombinant GABAA receptors. Biochem Pharmacol 2004; 68:
1631-1638.
[166] Dixon RA, Ferreira D. Genistein Phytochemistry 2002; 60: 205-
211.
[167] Dunne EL, Moss SJ, Smart TG. Inhibition of GABAA receptor
function by tyrosine kinase inhibitors and their inactive analogues.
Mol Cell Neurosci 1998; 12: 300-310.
[168] Huang RQ, Fang MJ, Dillon GH. The tyrosine kinase inhibitor
genistein directly inhibits GABAA receptors. Mol Brain Res 1999;
67: 177-183.
[169] Kavvadias D, Monschein V, Sand P, Riederer P, Schreier P.
Constituents of sage (Salvia officinalis L.) with in vitro affinity to
human brain benzodiazepine receptor. Plant Med 2003; 69: 113-
117.
[170] Perry EK, Pickering AT, Wang WW, Houghton PJ, Perry NS.
Medicinal plants and Alzheimer's disease: from ethnobotany to
phytotherapy. J Pharm Pharmacol 1999; 51: 527-534.
[171] Perry NS, Howes M-J, Houghton P, Perry E. Why sage may be a
wise memory remedy: Effects of Salvia on the nervous system.
Med Arom Plants Indust Profiles 2000; 14: 207-223.
[172] Tildesley NT, Kennedy DO, Perry EK, Ballard CG, Savelev S,
Wesnes KA, et al. Salvia lavandulaefolia (Spanish Sage) enhances
memory in healthy young volunteers. Phamaco Biochem Behav
2003; 75: 669-674.
1884 Current Pharmaceutical Design, 2005, Vol. 11, No.15
[173] Kavvadias D, Sand P, Youdim KA, Qaiser MZ, Rice-Evans C,
Baur R, et al. The flavone hispidulin, a benzodiazepine receptor
ligand with positive allosteeric properties, traverses the blood-brain
barrier and exhibits anti-convulsive effects. Br J Pharmacol 2004,
142: 811-820
[174] Walters RJ, Hadley SH, Morris KDW, Amin J. Benzodiazepines
act on GABAA receptors via two distinct and separable
mechanisms. Nature Neurosci 2000; 3: 1274-1281.
[175] Hall BJ, Chebib M, Hanrahan JR, Johnston GAR. Flumazenil-
independent positive modulation of γ-aminobutyric acid by 6-
methylflavone at human recombinant α1β2γ2L and α1β2 GABA A
receptors. Eur J Pharmacol 2004; 491: 1-8.
[176] Wang HY, Hui KM, Chen YJ, Xu SX, Wong JTF, Xue H.
Structure-activity relationships of flavonoids, isolated from
Scutellaria baicalensis, binding to benzodiazepine site of GABAA
receptor complex. Plant Med 2002; 68: 1059-1062.
[177] Huen MS, Leung JW, Ng W, Lui WS, Chan MN, Wong JT, et al.
5, 7-Dihydroxy-6-methoxyflavone, a benzodiazepine site ligand
isolated from Scutellaria baicalensis Georgi, with selective
antagonistic properties. Biochem Pharmacol 2003; 66: 125-32.
[178] Wasowski C, Marder M, Viola H, Medina JH, Paladini AC.
Isolation and identification of 6-methylapigenin, a competitive
ligand for the brain GABAA receptors, from Valeriana wallichii.
Plant Med 2002; 68: 934-936.
[179] Huen MS, Hui KM, Leung JW, Sigel E, Baur R, Wong JT, et al.
Naturally occurring 2'-hydroxyl-substituted flavonoids as high-
affinity benzodiazepine site ligands. Biochem Pharmacol 2003; 66:
2397-2407.
[180] Du XM, Sun NY, Takizawa N, Guo YT, Shoyama Y. Sedative and
anticonvulsant activities of goodyerin, a flavonol glycoside from
Goodyera schlechtendaliana. Phytother Res 2002; 16: 261-263.
[181] Fernandez S, Wasowski C, Paladini AC, Marder M. Sedative and
sleep-enhancing properties of linarin, a flavonoid-isolated from
Valeriana officinalis. Pharmacol Biochem Behav 2004; 77: 399-
404.
[182] Marder M, Viola H, Bacigaluppo JA, Colombo MI, Wasowski C,
Wolfman C, et al. Detection of benzodiazepine receptor ligands in
small libraries of flavone derivatives synthesized by solution phase
combinatorial chemistry. Biochem Biophys Res Comm 1998; 249:
481-485.
[183] Dekermendjian K, Kahnberg P, Witt MR, Sterner O, Nielsen M,
Liljefors T. Structure-activity relationships and molecular modeling
analysis of flavonoids binding to the benzodiazepine site of the rat
brain GABAA receptor complex. J Med Chem 1999; 42: 4343-
4350.
[184] Hong X, Hopfinger AJ. 3D-pharmacophores of flavonoid binding
at the benzodiazepine GABAA receptor site using 4D-QSAR
analysis. J Chem Inf Comput Sci 2003; 43: 324-336.
[185] Huang XQ, Liu T, Gu JD, Luo XM, Ji RY, Cao Y, et al. 3D-QSAR
model of flavonoids binding at benzodiazepine site in GABAA
receptors. J Med Chem 2001; 44: 1883-1891.
[186] Kahnberg P, Lager E, Rosenberg C, Schougaard J, Camet L,
Sterner O, et al. Refinement and evaluation of a pharmacophore
model for flavone derivatives binding to the benzodiazepine site of
the GABAA receptor. J Med Chem 2002; 45: 4188-4201.
[187] Marder M, Estiu G, Blanch LB, Viola H, Wasowski C, Medina JH,
et al. Molecular modelling and QSAR analysis of the interaction of
flavone derivatives with the benzodiazepine site of GABAA
receptor complex. Bioorg Med Chem 2001; 9: 323-335.
[188] Huang SH, Duke RK, Chebib M, Sasaki K, Wada K, Johnston
GAR. Bilobalide, a sesquiterpene trilactone from Ginkgo biloba, is
an antagonist at recombinant alpha(1) beta(2)gamma(2L) GABAA
receptors. Eur J Pharmacol 2003; 464: 1-8.
[189] Sasaki K, Oota I, Wada K, Inomata K, Ohshika H, Haga M. Effects
of bilobalide, a sesquiterpene in Ginkgo biloba leaves, on
population spikes in rat hippocampal slices. Comp Biochem
Physiol C-Toxicol Pharmacol 1999; 124: 315-321.
[190] Sasaki K, Hatta S, Haga M, Ohshika H. Effects of bilobalide on
gamma-aminobutyric acid levels and glutamic acid decarboxylase
in mouse brain. Eur J Pharmacol 1999; 367: 165-173.
[191] Ivic L, Sands TT, Fishkin N, Nakanishi K, Kriegstein AR,
Stromgaard K. Terpene trilactones from Ginkgo biloba are
antagonists of cortical glycine and GABAA receptors. J Biol Chem
2003; 278: 49279-85.
[192] Chatterjee SS, Kondratskaya EL, Krishtal OA. Structure-activity
studies with Ginkgo biloba extract constituents as receptor-gated
Graham A.R. Johnston
chloride channel blockers and modulators. Pharmacopsychiatry
2003; 36: S68-77.
[193] Brochet D, Chermat R, DeFeudis FV, Drieu K. Effects of single
intraperitoneal injections of an extract of Ginkgo biloba (EGb 761)
and its terpene trilactone constituents on barbital-induced narcosis
in the mouse. Gen Pharmacol 1999; 33: 249-256.
[194] Deiml T, Haseneder R, Zieglgansberger W, Rammes G,
Eisensamer B, Rupprecht R, et al. α-Thujone reduces 5-HT3
receptor activity by an effect on the agonist-induced
desensitization. Neuropharmacology 2004; 46: 192-201.
[195] Hold KM, Sirisoma NS, Ikeda T, Narahashi T, Casida JE. a-
thujone (the active component of absinthe): γ-aminobutyric acid
type A receptor modulation and metabolic detoxification. Proc Natl
Acad Sci USA 2000; 97: 3826-3831.
[196] Priestley CM, Williamson EM, Wafford K, Sattelle DB. Thymol, a
constituent of thyme essential oil, is a positive allosteric modulator
of human GABAA receptors and a homo-oligomeric GABA
receptor from Drosophila melanogaster. Br J Pharmacol 2003;
140: 1363-1372.
[197] Mohammadi B, Haeseler G, Leuwer M, Dengler R, Krampfl K,
Bufler J. Structural requirements of phenol derivatives for direct
activation of chloride currents via GABAA receptors. Eur J
Pharmacol 2001; 421: 85-91.
[198] Hosseinzadeh H, Parvardeh S. Anticonvulsant effects of
thymoquinone, the major constituent of Nigella sativa seeds, in
mice. Phytomedicine 2004; 11: 56-64.
[199] Granger RE, Campbell EL, Johnston GAR. Valerian monoterpenes
produce unprecedented enhancememnt of GABA action at GABAA
receptors. J Neurochem 2004; 88: (Suppl. 1) 99.
[200] Yuan CS, Mehendale S, Xiao Y, Aung HH, Xie JT, Ang-Lee MK.
The gamma-aminobutyric acidergic effects of valerian and
valerenic acid on rat brainstem neuronal activity. Anesth Analg
2004; 98: 353-358.
[201] Ha JH, Lee KY, Choi HC, Cho J, Kang LS, Lim JC, et al.
Modulation of radioligand binding to the GABAA-benzodiazepine
receptor complex by a new component from Cyperus rotundus.
Biol Pharmaceut Bull 2002; 25: 128-130.
[202] Choi SE, Choi S, Lee JH, Whiting PJ, Lee SM, Nah SY. Effects of
ginsenosides on GABAA receptor channels expressed in Xenopus
oocytes. Arch Pharmac Res 2003; 26: 28-33.
[203] Lee CM, Wong HN, Chui KY, Choang TF, Hon PM, Chang HM.
Miltirone, a central benzodiazepine receptor partial agonist from a
Chinese medicinal herb Salvia miltiorrhiza. Neurosci Lett 1991;
127: 237-241.
[204] Chang HM, Chui KY, Tan FW, Zhong ZP, Lee CM, Sham HL, et
al. Structure-activity relationship of miltirone, an active central
benzodiazepine ligand isolated from Salvia miltiorrhiza Bunge
(Danshen). J Med Chem 1991; 34: 1675-1692.
[205] Rutherford DM, Nielsen MPC, Hansen SK, Witt MR, Bergendorff
O, Sterner O. Isolation from salvia officinalis and indentification of
two diterpenes which inhibit t-butylbicyclophosphoro[35S]thionate
binding to chloride channel of rat cerebrocortical membranes in
vitro. Neurosci Lett 1992; 135: 224-226.
[206] Perry NB, Anderson RE, Brennan NJ, Douglas MH, Heaney AJ,
McGimpsey JA, et al. Essential oils from Dalmation sage (Salvia
officinalis l.): vaiations among individuals, plant parts, seasons, and
sites. J Ag Food Chem 1999; 47: 2048-2054.
[207] Shelp BJ, Bown AW, McLean MD. Metabolism and functions of γ-
aminobutyric acid. Trends Plant Sci 1999; 4: 446-452.
[208] Hossain SJ, Hamamoto K, Aoshima H, Hara Y. Effects of tea
components on the response of GABAA receptors expressed in
Xenopus oocytes. J Ag Food Chem 2002; 50: 3954-3960.
[209] Hossain SJ, Aoshima H, Koda H, Kiso Y. Effects of coffee
components on the response of GABAA receptors expressed in
Xenopus oocytes. J Ag Food Chem 2003; 51: 7568-7575.
[210] Ferreira ACS, Barbe J-C, Bertrand A. 3-Hydroxy-4, 5-dimethyl-
2(5H)-furanone: a key odorant of the typical aroma of oxidative
aged port wine. J Ag Food Chem 2003; 51: 4356-4363.
[211] Hossain SJ, Aoshima H, Koda H, Kiso Y. Potentiation of the
ionotropic GABA receptor response by whiskey fragrance. J Ag
Food Chem 2002; 50: 6828-6834.
[212] Koda H, Hossain SJ, Kiso Y, Aoshima H. Aging of whiskey
increases the potentiation of GABAA receptor response. J Ag Food
Chem 2003; 51: 5238-44.
 GABAA Receptor Channel Pharmacology
[213] Aoshima H, Hamamoto K. Potentiation of GABAA receptors
expressed in Xenopus oocytes by perfume and phytoncid. Biosci
Biotech Biochem 1999; 63: 743-748.
[214] Rezai N, Duggan C, Cairns D, Lees G, Chazot PL. Modulation of
[3H] TBOB binding to the rodent GABAA receptor by simple
disaccharides. Biochem Pharmacol 2003; 65: 619-23.
[215] Sooksawate T, Simmonds MA. Influence of membrane cholesterol
on modulation of the GABAA receptor by neuroactive steroids and
other potentiators. Br J Pharmacol 2001; 134: 1303-1311.
[216] Agar A, Yargicoglu P, Senturk KU, Oner G. The role of diet
cholesterol changes on EEG. Int J Neurosci 1994; 75: 103-109.
[217] Simons M, Keller P, Dichgans J, Schulz JB. Cholesterol and
Alzheimer's disease: is there a link?. Neurology 2002; 57: 1089-
1093.
[218] Locatelli S, Lutjohann D, Schmidt HH, Otto C, Beisiegel U, von
Bergmann K. Reduction of plasma 24S-hydroxycholesterol
View publication stats
Current Pharmaceutical Design, 2005, Vol. 11, No. 15 1885
(cerebrosterol) levels using high-dosage simvastatin in patients
with hypercholesterolemia: evidence that simvastatin affects
cholesterol metabolism in the human brain. Arch Neurol 2002; 59:
213-216.
[219] Beckstead MJ, Weiner JL, Eger EI, Gong DH, Mihic SJ. Glycine
and gamma-aminobutyric acid(A) receptor function is enhanced by
inhaled drugs of abuse. Mol Pharmacol 2000; 57: 1199-1205.
[220] Meulenberg CJ, Vijverberg HP. Selective inhibition of γ-
aminobutyric acid type A receptors in human IMR-32 cells by low
concentrations of toluene. Toxicology 2003; 190: 243-248.
[221] Miyazawa A, Fujiyoshi Y, Unwin N. Structure and gating
mechanism of the acetylcholine receptor pore. Nature 2003; 423:
949-955.
[222] Bali M, Akabas MH. Defining the propofol binding site location on
the GABAA receptor. Mol Pharmacol 2004; 65: 68-76.