See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/314109989

The Direct Actions of Cannabidiol and 2-Arachidonoyl Glycerol at GABAA

Receptors

Article  in  Pharmacological Research · February 2017

DOI: 10.1016/j.phrs.2017.02.022

CITATIONS READS

37 1,018

6 authors, including:

Tim Bakas Petra S Van Nieuwenhuijzen

The University of Sydney The University of Sydney
3 PUBLICATIONS   39 CITATIONS    20 PUBLICATIONS   485 CITATIONS   

SEE PROFILE SEE PROFILE

Iain S Mcgregor Jonathon C Arnold

The University of Sydney The University of Sydney
679 PUBLICATIONS   13,299 CITATIONS    98 PUBLICATIONS   2,624 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Synthetic Cannabinoid Receptor Agonists View project

Animal Behavior, psychopharmacology View project

All content following this page was uploaded by Tim Bakas on 11 January 2018.

The user has requested enhancement of the downloaded file.

 Pharmacological Research 119 (2017) 358–370

Contents lists available at ScienceDirect

Pharmacological Research
journal homepage: www.elsevier.com/locate/yphrs

Original research article

The direct actions of cannabidiol and 2-arachidonoyl glycerol at

GABAA receptors
T. Bakas a , P.S. van Nieuwenhuijzen a , S.O. Devenish a,b , I.S. McGregor b , J.C. Arnold c,d ,
M. Chebib a,∗
a
Faculty of Pharmacy, The University of Sydney, Sydney, NSW 2006, Australia
b
School of Psychology, The University of Sydney, Sydney, NSW 2006, Australia
c
Discipline of Pharmacology, The University of Sydney, Sydney, NSW 2006, Australia
d
Brain and Mind Centre, The University of Sydney, Sydney, NSW 2006, Australia

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

Article history: Cannabidiol (CBD) is a major non-intoxicating component of cannabis and possesses anti-epileptic, anx-
Received 28 October 2016 iolytic and anti-hyperalgesic properties. The mechanism of action of CBD in producing such effects
Received in revised form 23 February 2017 remains unclear. Despite evidence that some endogenous and synthetic cannabinoids interact with
Accepted 24 February 2017
GABAA receptors, no-one has yet investigated the effects of CBD. Here we used two-electrode voltage
Available online 27 February 2017
clamp electrophysiology to compare the actions of CBD with those of the major central endocannabi-
noid, 2-arachidonoyl glycerol (2-AG) on human recombinant GABAA receptors (synaptic ␣1-6␤␥2 and
Chemical compounds studied in this article:
extrasynaptic ␣4␤2␦) expressed on Xenopus oocytes. CBD and 2-AG were positive allosteric modulators at
Cannabidiol (PubChem CID: 644019)
2-Arachidonoyl Glycerol (PubChem CID:
␣1-6␤␥2 receptors, with low micromolar potencies. The maximal level of enhancement seen with either
5282280) CBD or 2-AG were on ␣2-containing GABAA receptor subtypes, with approximately a 4-fold enhancement
of the GABA EC5 evoked current, more than twice the potentiation seen with other ␣-subunit receptor
Keywords: combinations. Further we observed ␤-subunit selectivity, whereby modulatory activity was higher at
Cannabinoids ␤2/␤3 over ␤1 subunits. The ␤1-subunit homologous mutant ␤2(V436T) substantially diminished the
Cannabidiol
efficacy of both drugs to a third of that obtained with wild-type ␤2 subunit combinations, but without
GABAA receptors
Endocannabinoids
changing potency. The potency of CBD increased and efficacy preserved in binary ␣1/␣2␤2 receptors
2-Arachidonoyl glycerol indicating that their effects do not involve the classic benzodiazepine site. Exploration of extrasynaptic
␣4␤2␦ receptors revealed that both compounds enhanced GABA EC5 evoked currents at concentrations
ranging from 0.01–1 ␮M. Taken together these results reveal a mode of action of CBD on specifically
configured GABAA receptors that may be relevant to the anticonvulsant and anxiolytic effects of the
compound.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction tors. GABAA receptors exhibit high receptor subtype heterogeneity,

GABA mediates fast inhibitory neurotransmission in the mam-
malian central nervous system (CNS) via its actions on GABAA
receptors. GABAA receptors form part of the Cys-loop receptor
family of ion channels which include the nicotinic acetylcholine,
serotonin 3 (5-HT3 ) and strychnine-sensitive glycine gated recep-
Abbreviations: CBD, cannabidiol; 2-AG, 2-arachidonoyl glycerol; GABAA recep-
tor, ␥-amino butyric acid receptor type A; EC, effective concentration; CNS, central
nervous system; 5-HT, 33 serotonin 3; AEA, anandamide; CB1, cannabinoid receptor
1; CB2, cannabinoid receptor 2; THC, 9-tetrahydrocannabinol; DMSO, dimethyl
sulphoxide; DS2, delta selective compound 2; BDZ, benzodiazepines.
∗ Corresponding author.
E-mail address: mary.collins@sydney.edu.au (M. Chebib).
assembling from a combination of subunits derived from a family
of 19 genes, and include ␣1–6, ␤1–3, ␥1–3, ␦, ␲, ␧, ␪ and ␳1–3 sub-
units [1,2]. These subunits co-assemble to form heteropentamers
that allow the conductance of chloride ions through a central pore.
The majority of GABAA receptors are composed of two ␣, two ␤ and
either one ␥ or a ␦ subunit [1,2].
GABAA receptors are the target of numerous clinically-relevant
drugs such as benzodiazepines (BDZ), barbiturates and general
anaesthetics [1,2], as well as natural products including kavalac-
tones and flavonoids [3,4], with the specific subunit configuration
determining the pharmacological properties of these receptors.
Additionally, the brain distribution of these receptors is regionally
specific and each combination mediates a distinct physiologi-
cal role. For example, GABAA receptors containing ␣1–3 subunits

http://dx.doi.org/10.1016/j.phrs.2017.02.022
1043-6618/© 2017 Elsevier Ltd. All rights reserved.
 T. Bakas et al. / Pharmacological Research 119 (2017) 358–370
are localised in the synapse and mediate fast synaptic inhibition
(termed phasic inhibition). In contrast, ␣4–6 subunit containing
GABAA receptors are found largely extrasynaptically and medi-
ate a form of tonic inhibition [5,6]. Most ␥ containing receptors
are localised in the synapse whilst the ␦ subunit, co-assembles
predominantly with ␣ 4 /␣6 subunits to reside at perisynaptic or
extrasynaptic sites [5,6]. Thus GABAA receptors play a major role
in the maintenance of synchronised, well-orchestrated communi-
cations between relevant neural networks, and as such, changes
in GABAA receptor neurotransmission have implications for neu-
rological and psychiatric disorders including epilepsy, insomnia,
anxiety, stroke and schizophrenia [7,8].
Distinct GABAA receptor subtypes have been implicated in these
various disorders and may represent useful therapeutic targets.
Indeed, preclinical studies using transgenic mice and/or subtype
selective ligands support the attribution of receptor subunits to
specific disorders and/or to the pharmacological actions of estab-
lished therapeutics. These include: ␣1 receptor subtypes which are
implicated in epilepsy and in the sedative/hypnotic effects of drugs;
␣2/␣3 receptor subtypes which are involved in anxiolytic actions of
drugs; and ␣5 receptor subtypes which are implicated in learning,
memory and the action of nootropic drugs [9]. Further, different ␤-
subunits mediate distinct functional associations, including ␤2 /␤3
receptor subtypes being implicated in sedation, sleep, epilepsy, and
anaesthesia [10].
Synthetic, plant-based and endogenous cannabinoids comprise
diverse families of compounds, some of which exert their main
effects via G-protein coupled CB1 and/or CB2 cannabinoid recep-
tors [11]. CB1 receptors are ubiquitously expressed in the CNS
and are involved in the presynaptic regulation of GABA and gluta-
mate release [12]. 2-Arachidonoyl glycerol (2-AG) and anandamide
(N-arachidonoyl ethanol amide, AEA) are the major endogenous
lipid neurotransmitters (endocannabinoids) that are synthesised
on demand and act upon presynaptic CB1 receptors via retrograde
signalling mechanisms [13]. 2-AG is more abundant than AEA in
the CNS, where it acts with higher affinity and intrinsic activity on
both CB1 and CB2 receptors [14,15].
The phytocannabinoid, cannabidiol (CBD), is a major non-
intoxicating component of cannabis and is a structural isomer of
the main intoxicating compound 9-tetrahydrocannabinol (THC).
Unlike THC, CBD displays low affinity for the orthosteric site of
CB1 receptors and acts as a neutralising modulator with high affin-
ity on an allosteric site [16,17]. Despite an apparent lack of overt
psychoactive effects, CBD has a range of functional effects and can
reduce anxiety, depression, pain, psychotic symptoms and seizures,
including treatment-resistant pediatric epilepsies such as Dravet
syndrome [11,18]. Dravet syndrome typically arises from a genetic
mutation of the sodium channel Nav1.1. This channel is expressed
predominantly on inhibitory GABAergic interneurons, such that the
mutation leads to abnormal excitability and seizures [19]. Recent
clinical trials indicate that CBD can have a profound effect in reduc-
ing seizure numbers in a substantial subset of Dravet sufferers
[18,20]. However, no known mechanism of action accounts for the
anticonvulsant effects, or for many of the other clinically relevant
actions of CBD [18].
Recently, the endocannabinoids 2-AG and AEA were char-
acterised as positive allosteric modulators of GABAA receptors,
potentially accounting for some of their physiological effects
[21,22]. However, no studies to date have evaluated the effects
of CBD on GABAA receptors. CBD, THC, 2-AG and other fatty acid
neurotransmitters have been found to act as allosteric modulators
on a variety of Cys-loop gated receptors other than GABAA recep-
tors, including glycine receptors, 5-HT3 receptors and ␣7 nicotinic
acetylcholine receptors [23–33]. Accordingly, here we performed
a full characterisation of both CBD and 2-AG on 10 human
359
recombinant GABAA receptors expressed in Xenopus oocytes using
two-electrode voltage clamp methods.
The data show that CBD and 2-AG were positive allosteric mod-
ulators at all ␣ containing GABAA receptors but with a higher
efficacy for the ␣2-containing receptor. CBD and 2-AG exhibited
higher efficacy at ␣2␤2␥2L and ␣2␤3␥2L receptors compared to
␣2␤1␥2L GABAA receptors, indicating preference for ␤2/␤3 over
␤1-containing receptors. We then assessed whether the point
mutation ␤2V436T found in the TM4 region affects the actions
of either CBD or 2-AG on ␣2␤2(V436T)␥2L receptors. Whilst the
mutation attenuated the ability of these compounds to enhance
GABA currents, the potencies were similar to that of wild-type
␣2␤2␥2L receptors. Additionally, the modulatory actions of CBD
and 2-AG were not mediated via the classical benzodiazepine ␣-␥2L
interface. Finally, characterization of CBD and 2-AG mediated GABA
enhancement on extrasynaptic, ␦-containing receptors is reported.
These actions of CBD on GABAA receptors may contribute to the
anti-seizure and anxiolytic effects seen with this compound.
2. Materials and methods
2.1. Compounds
GABA, zinc chloride, delta selective 2 (DS2) and dimethyl
sulphoxide (DMSO) were purchased from Sigma-Aldrich (Syd-
ney, Australia). CBD was obtained from THC Pharm (Frankfurt,
Germany) as the crystalline solid. 2-AG was obtained from Cayman
Chemicals (Ann Arbor, MI, USA) as a 9:1 mixture of 2-AG and 1-AG (a
spontaneous isomer) in acetonitrile. 2-AG and CBD were prepared
as 100 mM stock solutions in DMSO prior to use. The final exper-
imental solutions of CBD and 2-AG contained no more than 0.8%
DMSO which when applied alone did not produce any alteration in
electrophysiological recordings.
2.2. GABAA receptor subunit constructs
Human complementary DNA (cDNA) for ␣1, ␤2 and ␥2L GABAA
receptor subunits subcloned into pCDM8 vectors were provided by
Dr Paul Whiting (Merck, Sharpe and Dohme Research Labs, Har-
low, UK). cDNA for human ␣3, ␣4, ␣5, ␣6, ␤1, ␤3 and ␦ subunits
(vectors: pGEMHE, pCDNA3, pCDM8. pCDNA1) were a gift from Dr
Bjarke Ebert (H. Lundbeck A/S,Valby, Denmark). Human ␣2 cDNA
subcloned into pCMV6-XL5 was purchased from OriGene Technolo-
gies (Rockville, MD, USA). The cloned DNA underwent complete
sequencing to confirm the sequence (Australian Genome Research
Facility, Westmead, NSW, Australia).
Each cDNA containing vector were linearised with restriction
endonucleases as follows; ␣1, ␣5, ␣6, ␤1, ␤2, ␥2L using NotI; ␣2
using SmaI; ␣3 and ␤3 using NheI; ␣4 and ␦ using HpaI. Capped RNA
transcripts were synthesised from linearised plasmids using the
mMessage mMachine T7 transcript kit (Ambion, Austin, TX, USA).
Lithium chloride precipitated cRNA was diluted in nuclease-free
water and stored at −20 ◦ C. The quality of cRNA was determined by
0.45% agarose gel electrophoresis and mRNA concentrations were
®
measured by NanoDrop ND-1000 UV–vis Spectrophotometer.
2.3. Xenopus laevis oocytes preparation
Surgical procedures on Xenopus laevis were approved by the
Animal Ethics Committee of the University of Sydney (reference
number: 2013/5915). The procedure for the harvesting and enzy-
matic separation of X. laevis oocytes was identical to that described
previously [34–36]. Briefly, oocytes were harvested by anaesthetis-
ing a sexually mature female X. laevis by immersion in 0.17% tricaine
with 0.02% NaCl for 10–15 min. Immediately after and performed
on ice, the anaesthetised animal had a lobe of the ovaries surgically
360 T. Bakas et al. / Pharmacological Research 119 (2017) 358–370

removed. The lobe was cut into smaller pieces and rinsed in oocyte Table 1
Estimated parameters from best-fit to a three parameter logistic equation for GABA
releasing buffer 2 (OR2; 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2 ,
activation at GABAA receptors.
5 mM HEPES, pH 7.4) and treated with collagenase A (2 mg·mL−1 in
OR2; Boehringer Manheim, Germany) for 1 h at 18◦ C, by shaking to Drug/Receptor type EC50 (␮M) nH n
defoliculate and separate oocytes from connective tissue. Released ␣1␤2 5.6 (3.6–6.9)b 1.0 (0.8–1.2) 5
oocytes were rinsed in ND96 wash solution (96 mM NaCl, 2 mM ␣1␤2␥2L 181.3 (127.0–258.7) 0.8 (0.6–0.9) 12
KCl, 1 mM MgCl2 , 1.8 mM CaCl2 , 5 mM HEPES, pH 7.4). ␣2␤2 22.9 (15.4–34.1) 0.6 (0.5–0.7) 4
␣2␤2␥2L 214.5 (157.7–255.3) 1.0c 5
␣2␤2␥2L + CBD 93.0 (76.3–113.2) 1.0 5
␣2␤2␥2L + 2-AG 114.7 (97.9–134.4) 1.0 5
2.4. X. laevis oocyte injection ␣2␤2(V436T)␥2L 219.2 (185.5–259.0) 1.0 4
␣3␤2␥2L 155.1 (96.1–250.6) 0.8 (0.6–0.9) 5
␣4␤2␥2L 84.6 (62.7–114.1) 0.9 (0.7–1.1) 10
Stage V–VI oocytes were collected and injected in the cyto-
␣5␤2␥2L 24.2 (21.4–27.3) 1.1 (1.0–1.3) 6
plasm with a 15–20 ␮m diameter tip micropipette (micropipette ␣6␤2␥2L 13.4 (7.5–24.3) 0.6 (0.4–0.8) 5
puller, Sutter Instruments, USA). Using a Nanoject injector (Drum- ␣2␤1␥2L 142.2 (105.4–191.7) 0.8 (0.6–1.0) 5
mond Scientific Co., Broomall, PA, USA) set to deliver a volume of ␣2␤3␥2L 50.8 (32.7–78.9) 0.9 (0.7–1.1) 5
50.6 nL/oocyte, a total of 3–5 ng of cRNA was used when express- ␣4␤2␦ 2.2 (1.8–2.6) 1.0 5
␣4␤2␦ + CBDa 0.1(0.0–0.2) 1.0 5
ing ␣:␤:␥ subunits and 15–20 ng of cRNA when expressing ␣:␤:␦
23.6 (9.3–59.7) 1.0
subunit in ratios of 1:1:3 and 5:1:5 respectively. Injected oocytes ␣4␤2␦ + 2-AGa 0.1(0.0–0.2) 1.0 5
were incubated for 2–6 days on an oscillator at 18◦ C in ND96 solu- 20.6 (13.2–32.1) 1.0
tion supplemented with 2.5 mM pyruvate, 0.5 mM theophylline, a
For the biphasic fits, the bottom fraction composed 42.9% and 34.0% with CBD
50 ␮g/mL gentamycin and 50 ␮g/mL tetracycline. and 2-AG respectively.
b
Values in brackets represents 95% Confidence Intervals.
c
Hill coefficients without confidence intervals have slopes fixed to 1.

2.5. Electrophysiology: two-electrode voltage clamp recordings

Receptor activities were assessed by recording whole cell
currents using the two-electrode voltage clamp technique as pre-
viously described [34,35]. Briefly, microelectrodes fabricated with
a micropipette puller (Sutter instruments, USA), were filled with
3 M KCl solution (0.2–2 M). Oocytes were continuously super-
fused with ND96 buffer in a cell chamber at an approximate rate
of 5 mL/min, and a solution change up to 5–10 s upon drug appli-
cation. The microelectrode impaled cells were voltage clamped at
−60 mV and the currents elicited in response to the application of
drugs recorded using a Geneclamp 500 B amplifier (Axon Instru-
ment, Foster City, CA, USA), a Power Lab 2/25 analog-digital data
acquisition system (AD Instruments, Sydney, NSW, Australia) and
Lab Chart version 5.0.2 program.
To ensure ␥ subunit incorporation, oocytes injected with ␣␤␥2
subunits were screened with 10 ␮M Zn2+ in the presence of 100 ␮M
GABA. Oocytes that displayed <10% Zn2+ inhibition were utilized in
subsequent experiments [37]. Such pharmacological testing ascer-
tains that the majority of receptors expressed are composed of ␣␤␥
subunits. To further ascertain ␥2 incorporation, oocytes expressing
␣1␤␥2 or ␣2␤␥2 were screened with 1 ␮M diazepam co-applied
with a GABA EC5 .
To ensure ␦ subunit incorporation, oocytes injected with ␣4␤2␦
subunits were first screened with 300 nM DS2 alone and co-applied
with a GABA EC5 [38,39]. To assess the modulatory effects of 2-AG or
CBD, oocytes were incubated with 2-AG or CBD for 75–90 s before
co-applying with the GABA EC5 (as determined for each cell). A
maximum concentration of 100 ␮M 2-AG and 100 ␮M CBD in 0.8%
DMSO was used in this study and represents the limit of solubil-
ity. No alteration in recordings was observed with 0.8% DMSO. A
3–15 min washout period was allowed between drug applications
depending on dose and receptor combination to avoid receptor
desensitization.
2.6. Data analysis and statistical procedures
The responses obtained with GABA applied alone or in the pres-
ence of a fixed drug concentration, namely 10 ␮M CBD or 2-AG,
were normalized as a percentage of the maximum GABA response.
For modulation studies, the GABA concentration eliciting an EC5
was co-applied with increasing concentrations of either CBD or 2-
AG. Current responses were normalized as potentiation of the GABA
EC5 current and calculated as follows (1);
% potentiation = 100 × (I drug − I GABA )/I GABA , (1)
where Idrug is the current in the presence of a given concentration
of drug, and IGABA is the amplitude of the control GABA current
(EC5 ± 0.5).
Normalised responses were pooled and graphed (mean ± SEM)
from at least two different batches of oocytes. Concentration-
response curves were fitted to either a monophasic (2) or biphasic
(3) Hill equation, where;
I = I max /(1 + 10(logEC50−log[A])nH) ), (2)
I = (I max /(1 + 10(logEC50 1−log[A])nH)
))(Frac)
+ (I max /(1 + 10 (logEC50 2−log[A])nH)
))(1-Frac) (3)
where I is the peak amplitude of the current elicited by a given
concentration of agonist [A], Imax is the maximum amplitude of
the current, EC50 is the concentration required for half-maximal
response (there are two EC50 s for a biphasic curve), and nH is the
Hill coefficient (fixed to 1). Frac represents the proportion of the
maximal response mediated by the more potent component in a
biphasic curve. In all cases, the bottom was constrained to 0.
The best-fit parameters were contrasted using an extra-sum of
squares F-test to detect whether an estimated parameter differs
among data sets. Statistical significance was attained at p < 0.05 and
all data analyses were conducted using GraphPad Prism version
7.00.
3. Results
3.1. Potentiation of GABA by either CBD or 2-AG is selective for
the ˛2 subunit
To determine the potency and selectivity of CBD and 2-AG at
GABAA receptors, we first evaluated receptors that differed in their
␣ subunit composition i.e. ␣x␤2␥2L (where x = 1, 2, 3, 4, 5, 6). Full
GABA concentration response curves were obtained for each recep-
tor subtype and the EC50 values for GABA are summarized in Table 1.
These values were within the concentration ranges from previously
 T. Bakas et al. / Pharmacological Research 119 (2017) 358–370 361

Table 2 In comparison, 2-AG was approximately 8-fold more potent at

Estimated parameters from best-fit to a three parameter model for positive modu-
␣1␤2␥2L (EC50 = 2.4 ␮M) compared to ␣2␤2␥2L (EC50 = 15.7 ␮M)
lation by CBD at GABAA receptors.
and ␣3␤2␥2L (EC50 = 14.7 ␮M) receptors (Fig. 2C; Table 3). No
Receptor type Maximal effect (%)a EC50 (␮M) n significant difference in EC50 values was found for 2-AG at
␣1␤2 217.3 (174.7–259.9)b 3.7 (1.7–8.0) 5 ␣4␤2␥2L (EC50 = 3.9 ␮M), ␣5␤2␥2L (EC50 = 1.5 ␮M) and ␣6␤2␥2L
␣1␤2␥2L 153.9 (132.6–175.1) 6.5 (3.5–12.3) 5 (EC50 = 6.4 ␮M) (Fig. 2D; Table 3).
␣2␤2 262.6 (219.2–305.9) 2.0 (1.0–4.1) 5 The greatest levels of enhancement were on ␣2␤2␥2L GABAA
␣2␤2␥2L 331.5 (277.2–391.1) 16.1 (9.0–28.6) 6
receptors by either CBD (332%; Table 2) or 2-AG (291%; Table 3)
␣2␤2(V436T)␥2L 123.6 (79.9–167.2) 13.3 (4.6–35.8) 5
␣3␤2␥2L 129.4 (109.9–148.8) 10.0 (6.0–16.7) 6 and was statistically significant when compared to all other ␣ sub-
␣4␤2␥2L 90.4 (74.8–105.9) 0.9 (0.4–1.8) 5 unit containing GABAA receptors (CBD: F = 12.0, p < 0.0001; 2-AG:
␣5␤2␥2L 73.6 (57.0–90.3) 1.4 (0.6–3.2) 5 F = 13.3, p < 0.0001). This data infers that CBD and 2-AG are positive
␣6␤2␥2L 71.9 (42.6–101.2) 8.2 (2.4–28.1) 5
allosteric modulators of all ␣-containing GABAA receptors but with
␣2␤1␥2L 151.4 (123.5–189.9) 17.4 (9.4–23.0) 5
␣2␤3␥2L 268.4 (249.6–287.6) 4.4 (3.1–6.3) 5 significantly higher efficacy at the ␣2-containing receptor subtype.
␣4␤2␦ 752.4 (649.2–881.5) 23.1 (15.8–33.7) 5
a
Note that the normalisation method is expressed as potentiation of the GABA
EC5 , where 100% represents a doubling of this current. 3.2. CBD and 2-AG significantly decrease the GABA EC50 on
b
Values in brackets represents 95% Confidence Intervals. ˛2ˇ22L GABAA receptors

Table 3 Positive allosteric modulation of GABA can either increase the

Estimated parameters from best-fit to a three parameter model for positive modu- apparent potency of GABA without affecting the maximal GABA
lation by 2-AG at GABAA receptors. effect, as observed with BDZs, or can increase the maximal GABA
Receptor type Maximal effect (%)a EC50 (␮M) n effect without significantly affecting GABA potency as observed
with general anaesthetics [44–46]. In order to assess how CBD and
␣1␤2␥2L 88.9 (70.6–99.4)b 2.4 (0.8–6.7) 5
␣2␤2␥2L 290.6 (223.5–357.7) 15.7 (8.2–30.0) 6
2-AG affects different GABA concentrations, we performed GABA
␣2␤2(V436T)␥2L 99.1 (74.1–124) 6.0 (1.4–11.9) 5 concentration-response curves in the presence of a fixed concen-
␣3␤2␥2L 127.0 (93.0–161.0) 14.7 (6.8–31.3) 5 tration of CBD or 2-AG (10 ␮M). Fig. 3A and B show representative
␣4␤2␥2L 114.7 (95.3–134.1) 3.9 (2.1–7.3) 6 traces for the GABA concentration response curve in the pres-
␣5␤2␥2L 98.3 (64.1–132.5) 1.5 (0.3–8.2) 5
ence of CBD (10 ␮M) and 2-AG (10 ␮M) respectively. CBD (10 ␮M)
␣6␤2␥2L 118.6 (62.8–174.4) 6.4 (1.2–35.5) 5
␣2␤1␥2L 142.1 (113.2–169.4) 13.3 (8.2–21.3) 5 significantly left-shifted the GABA concentration-response curve
␣2␤3␥2L 239.7 (204.4–275.1) 9.8 (6.5–14.6) 5 (F = 28.1, p < 0.0001) without affecting maximal GABA concentra-
␣4␤2␦ 479.6 (436.3–527.4) 4.8 (3.4–6.8) 5 tions (Fig. 3C). The GABA EC50 decreased from 214.5 ␮M in the
a
Note that the normalisation method is expressed as potentiation of the GABA absence of CBD to 93.0 ␮M in the presence of CBD (Table 1).
EC5 , where 100% represents a doubling of this current. Similarly, 2-AG (10 ␮M) produced a significant left-ward shift
b
Values in brackets represents 95% Confidence Intervals. of the GABA concentration response curve (F = 18.6, p < 0.0001) and
did not affect the maximal GABA current (Fig. 3C). The mean GABA
reported studies using whole cell recordings from Xenopus oocytes EC50 was 114.7 ␮M in the presence of 2-AG (Table 1). Additionally,
expressing various recombinant GABAA receptors [3,39–41]. CBD and 2-AG significantly enhanced GABA evoked currents below
In previous studies with cannabinoids on glycine receptors, the GABA EC50 but evoked less potentiation of GABA induced cur-
it was found that incubating cells resulted in greater enhance- rents at concentrations above the EC50 . This demonstrates that low
ment than simple co-application [27,42]. As such, we evaluated the GABA concentrations are potentiated selectively by these agents
enhancement of GABA elicited currents by incubating cells in either and thus enhance GABA in a manner similar to that of BDZ.
CBD or 2-AG for 75–90 s before co-applying the GABA EC5 (which
was determined for each individual oocyte). We noted that in addi-
tion to enhancing GABA, CBD (10 ␮M) and 2-AG (10 ␮M) showed 3.3. The 2L subunit is not required for CBD activity
small direct responses in the absence of GABA on ␣1–5␤2␥2L
receptors ranging from 0.5 to 5.0% of the maximal GABA response, As the modulation of GABA by CBD displayed a hallmark feature
whilst on ␣6␤2␥2L receptors a more pronounced direct activation similar to benzodiazepines, we wanted to determine whether the
up to 15% of the maximal GABA response was observed (Figs. 1 A effect of CBD was mediated via the classical benzodiazepine bind-
and B, 2 A and B). However, these direct responses with CBD or 2-AG ing site, located at the ␣–␥2L subunit interface. Thus we evaluated
were not concentration dependent and did not differ with increased the modulatory effects of CBD on binary ␣1␤2 and ␣2␤2 GABAA
concentrations of either CBD or 2-AG (p > 0.05). This result differs receptors that lack the classical benzodiazepine ␣–␥2L interface.
from previous observations on glycine receptors, where signifi- As observed with ternary ␣␤␥ receptors, incubating with CBD,
cant concentration dependent direct activations with CBD were a small direct activation of less than 5% of the maximal GABA
reported [43]. As the direct effects were small and concentration response was observed on binary receptors as well as a signifi-
independent, these effects were not further evaluated. cant enhancement of GABA (Fig. 4A and B). We then evaluated the
To assess the potency of CBD and 2-AG in modulating potency of CBD at ␣1␤2 and ␣2␤2 GABAA receptors. The maxi-
GABA evoked currents, full concentration response curves for mum enhancement elicited by CBD was 217% at ␣1␤2 (Fig. 4C) and
CBD and 2-AG against GABA EC5 induced currents were per- 263% at ␣2␤2 receptors (Fig. 4D; Table 2). The EC50 value for CBD
formed and are shown in Figs. 1 and 2 respectively. The at ␣1␤2 was 3.7 ␮M and was not significantly different to the EC50
EC50 values ranged from 0.9–16.1 ␮M for CBD (Fig. 1C and D; at ␣1␤2␥2L receptors (p > 0.05; Table 2). In contrast, the potency
Table 2) and 1.5–15.7 ␮M for 2-AG (Fig. 2C and D; Table 3). of CBD at ␣2␤2 GABAA receptors was 2.0 ␮M (EC50 ) and was sig-
CBD was approximately 10-fold more potent on ␣4␤2␥2L nificantly more potent than on ternary ␣2␤2␥2L receptors (F = 6.5,
(EC50 = 0.9 ␮M) and ␣5␤2␥2L (EC50 = 1.4 ␮M) compared with p < 0.01). The data infers that the modulation of GABA by CBD is not
␣1␤2␥2L (EC50 = 6.5 ␮M), ␣2␤2␥2L (EC50 = 16.1 ␮M), ␣3␤2␥2L mediated via the ␣-␥2L subunit interface. This observation is simi-
(EC50 = 10.0 ␮M) and ␣6␤2␥2L (EC50 = 8.2 ␮M) GABAA receptors lar to that previously reported for 2-AG, where 2-AG also enhanced
(Table 2). binary ␣1␤2 receptors [21].
362 T. Bakas et al. / Pharmacological Research 119 (2017) 358–370

Fig. 1. Effect of CBD at ␣-containing GABAA receptors expressed in Xenopus oocytes. Representative current traces (nA vs. s) illustrating the enhancing effects of CBD at: A.
␣1–3␤2␥2L and B. ␣4–6␤2␥2L GABAA receptors. GABA EC5 corresponds to the GABA concentration that elicits 5% of the maximal GABA response as determined for each
oocyte. The EC5 values were approximately 6 ␮M for ␣1␤2␥2L (black traces), and ␣3␤2␥2L (blue traces), 10 ␮M for ␣2␤2␥2L (red traces), 5 ␮M for ␣4␤2␥2L (black traces),
2.5 ␮M for ␣5␤2␥2L (red traces) and 0.1 ␮M for ␣6␤2␥2L (blue traces). Horizontal bars represent duration of drug application. Oocytes are perfused with 10 ␮M CBD for a
75–90 s incubation period prior to co-application with a GABA EC5 . Small direct responses by CBD (10 ␮M) were observed during the incubation period. Full concentration-
response curves for CBD in the presence of GABA EC5 at C. ␣1–3␤2␥2L and D. ␣4–6␤2␥2L GABAA receptors. Data points represent mean ± SEM (n ≥ 5) of peak current response
normalized to the current elicited by GABA EC5 as described in Methods. Data were fitted using three-parameter logistic equation and the best-estimated values for each
parameter are detailed in Tables 2 and 3. Overall, CBD is significantly (p < 0.0001) more efficacious on ␣2␤2␥2L subtypes compared to all other receptor combinations.

Fig. 2. Effect of 2-AG at ␣-containing GABAA receptors expressed in Xenopus oocytes. Representative current traces (nA vs. s) illustrating the enhancing effects of 2-AG at:
A. ␣1–3␤2␥2L and B. ␣4–6␤2␥2L GABAA receptors. GABA EC5 corresponds to the GABA concentration that elicits 5% of the maximal GABA response as determined for each
oocyte. The EC5 values were approximately 6 ␮M for ␣1␤2␥2L (black traces), and ␣3␤2␥2L (blue traces), 10 ␮M for ␣2␤2␥2L (red traces), 5 ␮M for ␣4␤2␥2L (black traces),
2.5 ␮M for ␣5␤2␥2L (red traces) and 0.1 ␮M for ␣6␤2␥2L (blue traces). Horizontal bars represent duration of drug application. Oocytes are perfused with 10 ␮M 2-AG for
a 75–90 s incubation period prior to co-application with a GABA EC5 . Small direct responses by 2-AG (10 ␮M) were observed during the incubation period. Concentration-
response curves for 2-AG in the presence of GABA EC5 at C. ␣1–3␤2␥2L and D. ␣4–6␤2␥2L GABAA receptors. Data points represent mean ± SEM (n ≥ 5) of peak current
response normalized to the current elicited by GABA EC5 as described in Methods. Data were fitted using three-parameter logistic equation and the best-estimated values
for each parameter are detailed in Tables 2 and 3. Overall, 2-AG was significantly (p<0.0001) more efficacious on ␣2␤2␥2L receptor subtypes.
 T. Bakas et al. / Pharmacological Research 119 (2017) 358–370 363

Fig. 3. Effects of a fixed concentration of CBD or 2-AG on the GABA concentration-response curve at ␣2␤2␥2L GABAA receptors expressed in Xenopus oocytes. Representative
current traces (nA vs. s) illustrating the enhancing effects of A. CBD (10 ␮M; red trace) and B. 2-AG (10 ␮M; blue trace) on increasing GABA concentration at ␣2␤2␥2L GABAA
receptors. Horizontal bars represent duration of drug application and highlight the 75–90 s incubation period and subsequent co-application with GABA. Representative
traces are from a single oocyte recording. Concentration-response curves at C. ␣2␤2␥2L GABAA receptors, depicting GABA alone and in the presence of CBD (10 ␮M; red
curve) or 2-AG (10 ␮M; blue curve). Both CBD and 2-AG significantly shifted the GABA curve to the left (p < 0.001). Data points represent mean ± SEM (n ≥ 5) of peak current
response normalized as described in Methods. Data were fitted using three-parameter logistic equation and the best-estimated values for each parameter are detailed in
Table 1.

Fig. 4. Potentiation of GABA current responses by CBD at ␣1␤2 and ␣2␤2 GABAA receptors expressed in Xenopus oocytes. Representative current traces (nA vs. s) illustrating
the enhancing effects of CBD (10 ␮M) A. at ␣1␤2␥2L and ␣1␤2 GABAA receptors and B. at ␣2␤2␥2L and ␣2␤2 GABAA receptors. GABA concentration corresponds to EC5
as determined for each oocyte expressing each subunit combination, and was approximately 6 ␮M (␣1␤2␥2L), 0.6 ␮M (␣1␤2; Blue traces), 10 ␮M (␣2␤2␥2L) and 0.5 ␮M
(␣2␤2; red traces) receptor subtypes. Horizontal bars represent duration of drug application and highlight both the incubation period and subsequent co-application with
GABA. Small direct responses by CBD (10 ␮M) were observed during the incubation period. Full concentration-response curves depicting the potentiating effects of CBD in
the presence of a GABA EC5 at C. ␣1␤2 (blue curve) and ␣1␤2␥2L and D. ␣2␤2 (red curve) and ␣2␤2␥2L GABAA receptor subtypes. CBD activated both binary and ternary
receptors with similar efficacy and was approximately 8-fold more potent on ␣2␤2 than on ␣2␤2␥2L subtypes (see Table 2 for EC50 values). Data points represent mean ± SEM
(n ≥ 5) of peak current response normalized as described in Methods. Data were fitted using a three-parameter logistic equation.

3.4. Potentiation of GABA evoked currents by either 2-AG or CBD
was significantly greater at GABAA receptors containing a ˇ2 or
ˇ3 subunit
A number of clinically relevant positive allosteric modulators
of GABAA receptors such as general anaesthetics exhibit subtype
selectivity with different levels of enhancement depending on the
type of ␤ subunit [2,5]. As both CBD and 2-AG were significantly
more efficacious at ␣2 containing GABAA receptors than any other ␣
containing receptor, we used the ␣2 subunit to evaluate the effect of
the various ␤ subunits on CBD and 2-AG efficacy and potency· Thus
the modulatory effects of CBD and 2-AG were assessed at ␣2␤x␥2L
364 T. Bakas et al. / Pharmacological Research 119 (2017) 358–370

Fig. 5. Potentiation of GABA currents by CBD or 2-AG at ␤-containing GABAA receptors at recombinant ␣2␤x␥2L (x = 1, 2, 3) GABAA receptors expressed in Xenopus oocytes.
A. Representative current traces (nA vs. s) illustrating the enhancing effects of CBD (10 ␮M; red traces) and 2-AG (10 ␮M; blue traces) at different ␤-subunit containing GABAA
receptors. GABA concentration corresponds to EC5 as determined for each individual oocyte and was approximately 5 ␮M (␣2␤1␥2L), 10 ␮M (␣2␤2␥2L) and 5 ␮M (␣2␤3␥2L).
Horizontal bars represent duration of drug application and highlight both the incubation period and subsequent co-application with GABA. Small direct responses by CBD
(10 ␮M) and 2-AG (10 ␮M) were observed during the incubation period. B. Bar graph illustrating the enhancement of the GABA EC5 with 10 ␮M CBD (red bars) and 10 ␮M
2-AG (blue bars) on GABA-elicited currents (EC5 ) at different ␤-subunit containing GABAA receptors. Full concentration-response curves at ␣2␤1 − 3␥2L GABAA receptors
for the potentiating effects of GABA EC5 for C. CBD and D. 2-AG. The enhancing effects of CBD and 2-AG were very similar on ␤2 3/ containing receptors, whilst the potency of
CBD on ␣2␤3␥2L receptors was greater than on ␤2 containing subtypes· Additionally the enhancement of GABA responses were greatly attenuated on ␤1 receptor subtypes.
Data points represent mean ± SEM (n ≥ 5) of peak current response normalized as described in Methods. Data were fitted using three-parameter logistic equation and the
best-estimated values for each parameter are detailed in Tables 2 and 3.

GABAA receptors (where x = 1 and 3). Similar to ␣2␤2␥2L GABAA
receptors, both CBD and 2-AG exhibited direct effects at ␤1 and
␤3 subunit containing receptors. A direct response of about 10%
of the maximal GABA response was observed during the incuba-
tion period at ␣2␤1␥2L with either CBD (10 ␮M) or 2-AG (10 ␮M)
(Fig. 5A). Albeit small, these were greater than that seen on either
␣2␤2␥2L or ␣2␤3␥2L GABAA receptors where the maximum direct
activation was less than 5% of maximum GABA response (Fig. 5A).
The modulatory effects of CBD (10 ␮M) and 2-AG (10 ␮M) at
␣2␤3␥2L were similar to effects at ␣2␤2␥2L (Fig. 5A and B). In
contrast, at ␣2␤1␥2L GABAA receptors, CBD (10 ␮M) and 2-AG
(10 ␮M) enhanced GABA EC5 induced currents by only 56.8 ± 5.5%
and 57.6 ± 8.7% respectively (Fig. 5B). Full concentration response
curves for CBD and 2-AG against GABA EC5 induced currents at
␣2␤1␥2L, ␣2␤2␥2L and ␣2␤3␥2L receptors are depicted in Fig. 5C
and D, respectively.
The maximal potentiation for CBD and 2-AG against GABA
EC5 induced currents at ␣2␤3␥2L receptors were similar, reach-
ing 268% and 240% respectively (Tables 2 and 3). These values
were not significantly different to that observed at ␣2␤2␥2L recep-
tors (p > 0.05). However, levels of enhancement by CBD and 2-AG
against GABA EC5 induced currents on ␣2␤1␥2L receptors were
lower, reaching maximal values of 151% and 142% respectively
(Tables 2 and 3). These were significantly less than that seen on
␣2␤2␥2L and ␣2␤3␥2L receptors for both CBD (F = 4.5, p < 0.05)
and 2-AG (F = 4.2, p < 0.05).
The EC50 value for CBD at ␣2␤3␥2L receptors was 4.4 ␮M
and was significantly more potent than at either ␣2␤2␥2L
(EC50 = 16.1 ␮M; F = 4.2, p < 0.05; Table 2) or ␣2␤1␥2L receptors
(EC50 = 17.4 ␮M; F = 5.2, p < 0.05). This data shows that CBD exhibits
selectivity for ␤3 over ␤1/␤2 containing receptors with a rank order
of ␤3 > ␤2 > ␤1. In contrast, 2-AG exhibited similar EC50 values at
␣2␤3␥2L (9·8 ␮M; Table 3) and ␣2␤1␥2L (13.3 ␮M; Table 3) com-
pared to ␣2␤2␥2L receptors (p > 0.05). This data differs from studies
by Sigel et al. [21] using ␣1 containing receptors, where there was
significantly less modulatory effect by 2-AG at ␤1 and ␤3 compared
to ␤2 containing receptors. Thus, there appears to be differences in
 T. Bakas et al. / Pharmacological Research 119 (2017) 358–370 365

Fig. 6. Effect of CBD and 2-AG at ␣2␤2(V436T)␥2L GABAA receptors expressed in Xenopus oocytes. Representative current traces (nA vs. s) illustrating the enhancing effects of
A. CBD (10 ␮M) and B. 2-AG (10 ␮M) at ␣2␤1␥2L, (blue traces), ␣2␤2␥2L (black traces) and ␣2␤2(V436T)␥2L (red traces) GABAA receptors. GABA concentration corresponds
to EC5 as determined for each oocyte and was approximately 10 ␮M GABA. Horizontal bars represent duration of drug application and highlight both the incubation period
and subsequent co-application with GABA. Small direct responses by CBD (10 ␮M) and 2-AG (10 ␮M) were observed during the incubation period. Concentration-response
curves for C. CBD and D. 2-AG on GABA-elicited currents at ␣2␤1␥2L, ␣2␤2␥2Land ␣2␤2(V436T)␥2L GABAA receptors in blue, black and red lines respectively. The mutation
greatly attenuated the enhancing effects of CBD and 2-AG, and was very similar with that of ␣2␤1␥2L GABAA receptors. Further, the potencies remained similar on both
wild-type and on mutant receptors. Data represent mean ± SEM (n ≥ 5) of peak current response normalized as described in Methods. Data were fitted using three-parameter
logistic equation and the best-estimated values for each parameter are detailed in Tables 2 and 3.

the binding site for 2-AG at ␣1 compared to ␣2 containing recep-
tors.
3.5. The ˇ2V436T significantly reduces enhancement by CBD and
2-AG of GABA induced currents
It has been previously shown that the mutant ␤2(V436T) sub-
unit located in the TM4 region abolishes potentiation of GABA by
2-AG using ␣1 containing receptors [21]. Therefore, in order to
determine whether this mutation affects CBD and 2-AG modula-
tion, we evaluated CBD and 2-AG at recombinant ␣2␤2(V436T)␥2L
receptors expressed in oocytes. This mutation did not affect the
response to GABA, with no statistically significant change in the
EC50 as compared to wild-type receptors (p > 0.05; Table 1).
While both CBD and 2-AG enhanced GABA EC5 induced
currents at ␣2␤2(V436T)␥2L receptors, there was significantly
less enhancement as compared to wild-type ␣2␤2␥2L receptors
(p < 0.05) (Fig. 6C and D). Interestingly, the modulatory effects of
CBD and 2-AG at ␣2␤2(V436T)␥2L receptor subtypes were reduced
to levels similar to that seen at ␣2␤1␥2L receptors (Fig. 6C and D).
The maximum potentiation of CBD and 2-AG were 124% and 99%
at ␣2␤2(V436T)␥2L receptors respectively (Tables 2 and 3). The
EC50 values for CBD and 2-AG were 13.3 and 6.0 ␮M respectively
and these values did not significantly differ from values obtained at
wild-type ␣2␤1/2/3␥2L receptors (p > 0.05). These data infer that
the ␤2(V436T) mutation attenuates the ability of the compounds to
enhance GABA at ␤2 containing receptors, however the potency of
CBD and 2-AG on ␣2␤2(V436T)␥2L receptors remained unaffected,
indicating that neither valine nor threonine at 436 is contributing
to the binding of these compounds at ␣2␤2␥2L receptors. Despite
no change in the potency of CBD and 2-AG, there was a signifi-
cant reduction in the maximal potentiation of GABA currents at the
mutant receptor. This could be the result of changes to the intrinsic
properties of the receptor that govern transitions from the closed to
the open state, or changes to the conformational changes elicited by
the CBD and 2-AG that increase the open probability of the channel.
3.6. CBD and 2-AG are positive allosteric modulators of GABA at
extrasynaptic ı-containing GABAA receptors
We sought to determine the actions of CBD and 2-AG on
extrasynaptic ␦-containing GABAA receptors. Oocytes displaying an
8.2 ± 0.7 fold enhancement by DS2 (300 nM) of GABA EC5 (30 nM)
evoked currents were utilized. Maximal GABA currents ranged
between 55 and 161 nA. The enhancement of the GABA EC5 by
DS2 ranged between 33 and 56% of maximum GABA current and
is similar to previously reported currents [38,39,47]. Addition-
ally, DS2 (300 nM) directly opened the channel in the absence of
GABA and this ranged between 2.5–14% of maximum GABA cur-
rents. The response by DS2 was similar to that reported by Hartiadi
et.al, where with a 5:1:5 injection ratio of ␣4, ␤2, and ␦ subunits,
whereby DS2 significantly enhanced GABA evoked currents and in
addition directly elicited small currents in the absence of GABA [39].
As observed with ternary ␣␤␥ receptors, incubating with either
CBD or 2-AG produced small direct activations on ␣4␤2␦ receptor
subtypes as well as a significant enhancement of GABA EC5 evoked
currents (Fig. 7A and B). To assess the potency of CBD and 2-AG
in modulating GABA evoked currents, full concentration response
curves for CBD and 2-AG on ␣4␤2␦ receptors are shown in Fig. 7C.
Although CBD displayed significantly greater levels of enhance-
366 T. Bakas et al. / Pharmacological Research 119 (2017) 358–370

Fig. 7. Potentiation of GABA current responses by CBD or 2-AG at ␣4␤2␦ extrasynaptic GABAA receptors expressed in Xenopus oocytes. Representative current traces (nA
vs. s) illustrating the enhancing effects of A. CBD (10 ␮M; red traces) and B. 2-AG (10 ␮M; blue traces) at ␣4␤2␦ GABAA receptors. GABA concentration corresponds to EC5
as determined for each oocyte expressing ␣4␤2␦ GABAA receptors, and was approximately 30 nM. Horizontal bars represent duration of drug application and highlight
both the incubation period and subsequent co-application with GABA. Small direct responses by CBD (10 ␮M) or 2-AG (10 ␮M) were observed during the incubation period.
Full concentration-response curves depicting the potentiating effects of C. CBD (red curve) or 2-AG (blue curve) in the presence of a GABA EC5 at ␣4␤2␦ GABAA receptor
subtypes. CBD was approximately 5-fold less potent than 2-AG (p<0.0001), whilst maximal enhancement of GABA EC5 currents was approximately 8-fold and 5-fold with
co-application of either CBD (10 ␮M) or 2-AG (10 ␮M) respectively on ␣4␤2␦ GABAA receptors.

ment than 2-AG, with maximal levels of 752% as compared with
480% at ␣4␤2␦ receptors (Tables 2 and 3; F = 27.0, p < 0.0001), 2-AG
was 5-fold more potent than CBD. The EC50 values for CBD and 2-
AG were 23.1 ␮M and 4.8 ␮M respectively (Tables 2 and 3; F = 32.6,
p < 0.0001). The potency of 2-AG on extrasynaptic ␣4␤2␦ (EC50 of
4.8 ␮M) was similar to that reported by Sigel and colleagues at
␣1␤2␦ receptors (EC50 of 1.7 ␮M) [21].
Finally, GABA concentration response curves were determined
for ␣4␤2␦ receptors in the presence of a fixed concentration of CBD
(10 ␮M) or 2-AG (10 ␮M) (Fig. 8A and B respectively). Both com-
pounds modulated GABA evoked currents ranging from 0.01–1 ␮M,
concentrations below the EC50 but had little effect when GABA
concentrations were above this, resulting in a biphasic curve fit
(Fig. 8C). Neither compound significantly affected the potency of
GABA as the EC50 of GABA was unchanged in the presence of either
CBD or 2-AG.
4. Discussion
The present series of experiments used two-electrode voltage
clamp methods to investigate the direct actions of the phy-
tocannabinoid, CBD and the major endocannabinoid, 2-AG on
human GABAA receptors recombinantly expressed in Xenopus
oocytes. Results showed that CBD and 2-AG are positive allosteric
modulators, with low micromolar potencies at ␣␤␥2 receptor com-
binations. The greatest level of enhancement of GABA EC5 evoked
currents (approximately 4 fold) was observed on ␣2 containing
GABAA receptor subtypes, which was more than double the poten-
tiation seen with the other ␣ subunit receptor combinations. Sigel
and colleagues [21] similarly showed 2-AG enhanced GABA-evoked
currents at ␣-containing GABAA receptors however did not demon-
strate greater enhancement on ␣2 containing receptor subtypes,
most likely due to using only a single submaximal concentration of
2-AG (3 ␮M), a concentration that failed to fully differentiate the
maximum efficacy at each receptor type.
In addition, 2-AG and CBD enhance currents elicited by low
GABA concentrations far more than those of higher concentra-
tions of GABA, resulting in an increase in the apparent affinity
of GABA. The maximal current response to GABA was unaffected.
This pattern of effect is similar to that reported with the synthetic
cannabinoid inverse agonist AM251 at ␣1␤2␥2L receptors [48] and
also mimics that of BDZs, a profile that confers BDZ safety [9]. How-
ever, as previously reported for 2-AG [21], we show that the binding
site for CBD is not located at the classical BDZ binding site [49],
because like 2-AG, CBD similarly enhanced GABA-evoked currents
at binary ␣1␤2 and ␣2␤2 receptor subtypes that lack the ␣-␥2
subunit interface that confers BDZ sensitivity.
Both CBD and 2-AG were more efficacious at ␤2 /␤3 than ␤1
containing GABAA receptors, a pharmacological profile common
with other GABAergic modulators such as etomidate and lorecle-
zole [5,50]. While 2-AG exhibited similar potency at all ␤ containing
receptors possessing the ␣2 subunit, CBD was more potent on ␤3
than either ␤2 or ␤1 containing subtypes. Positive allosteric mod-
ulators that have selectivity for ␤2/␤3 over ␤1 subunits tend to
produce less sedation and ataxia, while anxiolytic activity is often
retained [50]. Indeed, CBD’s profile of ␤ subtype specificity is con-
sistent with its therapeutic actions as an anxiolytic lacking sedative
or ataxic effects [51,52].
Interestingly, Sigel et al. [21] report greater enhancement of
GABA induced currents with 2-AG at ␣1␤2␥2 subtype. This differs
when using ␣2 containing receptors where we observe similar effi-
cacy by 2-AG and CBD at both ␤2 and ␤3 but reduced efficacy at ␤1
containing GABAA receptors. These results resemble that seen with
the cannabinoid inverse agonist AM251, which is also more effica-
cious at ␤2 /␤3 over ␤1 containing GABAA receptors [48]. It would
be of future interest to identify amino acids that differ between
the ␣1 and ␣2 subunits and assess whether such amino acids con-
tribute to the potency and efficacy of 2-AG at ␣1 compared to ␣2
containing receptors.
Mutagenesis and modeling studies have pointed towards a
putative binding site for 2-AG located between the M3 and M4
transmembrane domains of the ␤2 subunit of ␣1 containing
GABAA receptors [21,22]. The most notable of these were ␤2V436T
and ␤1T436V which abolished and introduced, respectively, the
enhancing effects of 3 ␮M 2-AG at these receptors [21]. In agree-
ment with such studies, the putative cannabinoid binding site of
the highly homologous glycine receptor was also suggested to be
between the M3 and M4 domains [42]. Our results similarly show
a large reduction in the modulatory activity of CBD as well as 2-AG
at ␤2V436T containing ␣2 containing GABAA receptors. Whilst this
single residue was previously reported to determine cannabinoid
sensitivities between ␣␤␥ receptors containing a ␤1 or ␤2 subunit,
 T. Bakas et al. / Pharmacological Research 119 (2017) 358–370 367

Fig. 8. Effects of a fixed concentration of CBD or 2-AG on the GABA concentration-response curve at extrasynaptic ␣4␤2␦ GABAA receptors expressed in Xenopus oocytes.
Representative current traces (nA vs. s) illustrating the enhancing effects of A. CBD (10 ␮M; red trace) and B. 2-AG (10 ␮M; blue trace) on increasing GABA concentrations on
␣4␤2␦ subunit containing GABAA receptors. Horizontal bars represent duration of drug application and highlight the 75–90 s incubation period and subsequent co-application
with GABA. Small direct responses by CBD (10 ␮M) and 2-AG (10 ␮M) were observed during the incubation period. Representative traces are from a single oocyte recording.
Full concentration-response curves of C. GABA at ␣4␤2␦ GABAA receptors, either alone or in the presence of a set concentration of CBD (10 ␮M; red curve) and 2-AG (10 ␮M;
blue curve), and where low GABA concentrations of less than 1 ␮M were significantly enhanced by either CBD or 2-AG. Data points represent mean ± SEM (n ≥ 5) of peak
current response normalized as described in Methods. Data were fitted using a six-parameter logistic equation (biphasic model) and the best-estimated values for each
parameter are detailed in Table 1.

it appears to only partially account for the actions of cannabinoids
on receptors with a ␤3 subunit [21]. The ␤3 subunit, like the ␤1 sub-
unit contains a threonine at position 436, and we found the efficacy
of both CBD and 2-AG is significantly higher at ␤3 compared to ␤1
receptors. Further the potency of CBD and 2-AG at ␣2␤2(V436T)␥2L
receptors was unaffected, indicating that neither a valine or threo-
nine at this position is contributing directly to the binding of either
of agent. However this site is contributing to the change in efficacy
between ␤2 and ␤1 containing receptors by 2-AG and CBD, which
most likely is due to the intrinsic properties of the receptors and the
compound’s ability to modulate GABA responses. It is most likely
that this site is important for the gating of the receptor and that
other nearby amino acid residues in M3 and M4 transmembrane
domains may be involved in the binding of CBD and 2-AG.
In addition to the M3 and M4 transmembrane domains, the
M2 transmembrane domain of the ␣1 glycine receptor subunit has
been implicated in the actions of CBD, specifically serine 267 which
when mutated to isoleucine abolished the modulatory activity [53].
The homologous amino acid in the ␤2 and ␤3 GABAA receptor
subunit is asparagine 265 (N265) while the ␤1 subunit contains
a serine (S265). When this asparagine amino acid is mutated to
a serine in the ␤2 or ␤3 subunit, i.e. the equivalent amino acid
in the ␤1 subunit, responses to a variety of structurally unrelated
GABAA allosteric modulators including alcohol and general anaes-
thetics are reduced [54]. Moreover, this mutation has been thought
to affect the gating mechanism of the receptor [55,56], and as such,
we did not assess this mutation.
Finally we assessed the activity of CBD and 2-AG at ␦-containing
GABAA receptors. The potency of CBD or 2-AG at extrasynaptic
␣4␤2␦ receptors was about 10 fold less than on ␣4␤2␥2L. How-
ever, the maximal level of enhancement seen on these ␦-containing
receptors was approximately 8 fold the GABA EC5 evoked current,
an effect similar to that observed with AM251[22]. It must be noted,
however, that GABA is a weak partial agonist at these receptors
and does not elicit maximal chloride currents on these receptor
subtypes. Ahring and colleagues [47] demonstrated that as a result
of GABA eliciting a much lower open probability at ␣4␤2␦ recep-
tors than at ␣4␤2␥ receptors, the modulation of GABA EC5 evoked
currents at ␣4␤2␦ receptors are not directly comparable to the
modulation of GABA EC5 evoked currents at ␣4␤2␥ receptors. Thus,
the modulation of the same effective concentration of GABA (i.e: a
GABA EC5 ) at ␣4␤2␦ receptors by CBD and 2-AG cannot be directly
compared with the other receptors tested, where GABA itself acts
with significantly higher efficacy.
The biphasic GABA concentration-response relationship in the
presence of CBD or 2-AG may indicate effects on a mixed receptor
population. Whilst pharmacological testing with DS2 supports the
presence of the ␦ subunit, it does not preclude the co-expression of
other receptor subunit combinations. Indeed several reports show
differential effects with GABA agonists (e.g. GABA, THIP and mus-
cimol) that lead to biphasic concentration response curves [57,58].
This bi-phasic effect is the result of various ternary ␣4␤␦ and binary
␣4␤ heteropentameric receptor subtypes that differ in subunit sto-
ichiometry, resulting in pools of high and low affinity receptor
combinations. Further, application of low GABA concentrations in
the presence of an allosteric modulator may result in the selective
enhancement of GABA responses and if mixed populations of recep-
tors are expressed would lead to a biphasic concentration-response
whereby one population is affected preferentially.
Recently, it has been demonstrated that different mRNA injec-
tion ratios for ␣4␤2␦ receptors can result in the preferential
expression of receptors that responded to either low concentra-
tions of GABA and were sensitive to direct activation by DS2 (i.e
in the absence of GABA), or higher concentrations of GABA which
were sensitive to GABA modulation by DS2 [39,59]. We used a 5:1:5
RNA injection ratio to favour the expression of ␣4␤2␦ receptors
that were sensitive to higher concentrations of GABA and displayed
GABA modulation by DS2. However this approach does not exclude
the expression a small number of ␣4␤2␦ receptors sensitive to low
concentrations of GABA because we also noted a small direct action
by DS2. Indeed we show that CBD and 2-AG potentiated currents
at ␣4␤2␦ receptors only with very low GABA concentrations and
this may be the result of modulating receptors sensitive to very
low GABA concentrations. Nevertheless our studies indicate that
any effects by CBD or 2-AG at ␦ containing receptors would only
occur when GABA concentrations at extrasynaptic sites are less
than 1 ␮M. It is therefore worth noting that GABA concentrations as
low as 100 nM have been reported at these sites [60], indicating that
368 T. Bakas et al. / Pharmacological Research 119 (2017) 358–370
tonic currents could, in part, be affected with CBD via these recep-
tors. Further, as 2-AG is produced within cell membranes, including
at extrasynaptic regions of neurons, it may suggest a direct role on
tonic currents via ␦-subunit containing GABAA receptors [21,61].
CBD is a promising therapeutic agent with a broad range of anx-
iolytic, antipsychotic, neuroprotective, analgesic and antiepileptic
actions exhibited in preclinical studies and human clinical trials
[18,62–64]. A recent phase III clinical trial reported that CBD was
effective in reducing seizures in many children with Dravet syn-
drome, a severe treatment-resistant form of childhood epilepsy
[65]. Interestingly, 80% of Dravet patients have a mutation in a
voltage gated sodium channel (SCN1A), which is expressed on
inhibitory interneurons, and this results in impaired GABAergic
neurotransmission leading to the hyperexcitability of neurons [19].
The mechanisms responsible for the anticonvulsant effects of
CBD are not well-established and may involve a multimodal action.
For example, CBD may elicit anticonvulsant activity by increas-
ing brain concentrations of anandamide via inhibition of fatty
acid amide hydrolase (FAAH) and might also dampen neuronal
excitability through presynaptic CB1 mediated reduction of glu-
tamate release [62,66–68]. In addition, CBD was recently found to
act on an epilepsy-associated mutated form of Nav1.6 to reduce
neuronal excitability [66]. Here we provide an additional mecha-
nism that may explain the therapeutic effects of CBD, a mechanism
that involves the action of CBD directly on GABAA receptors. This
action occurs in the micromolar range at which CBD also affects
many other known targets including 5HT1A , 5HT3 , ␮ opioid and
glycine receptors [12,69–71]. Notably, the doses of CBD used in
epilepsy treatment are large (as high as 25 mg/kg), reaching micro-
molar CBD concentrations in plasma and in brain [65,72–74]. The
concentration detected in mice have been shown to reach a Cmax
of 1.3-6.9 ␮g/g in brain tissue (approx. 4–21 ␮M), depending on
administration and suggests that the actions of CBD on GABAA
receptors reported here may indeed be relevant to the anticon-
vulsant effects of the drug.
The ␣2-containing GABAA receptors are implicated as targets for
anxiety and analgesia [9]. Mutation of the ␣2 subunit can render
animals insensitive to the anxiolytic and anti-hyperalgesic effects
of compounds [9,75–77]. Intriguingly, CBD in both animal and
human studies has been shown to be both anxiolytic and analgesic
[18,28,64,78–81]. CBD may exert some of its anxiolytic effects via
the 5HT1A receptor [82,83], and analgesic actions via ␣3-containing
glycine receptors [28]. Nonetheless, considering the similar poten-
cies of CBD on GABAA receptors with glycine and 5HT1A receptors,
the present study suggests that the actions on ␣2 containing recep-
tors may also contribute to the anti-anxiety and anti-hyperalgesic
effects of CBD.
2-AG, like CBD, has anxiolytic, analgesic and neuroprotective
effects as found in many preclinical studies [13,25,84–86]. How-
ever, whether the actions of 2-AG on GABAA receptors are relevant
to these actions remains unclear given the large difference in
potency (approximately 1000 fold) between 2-AG actions on CB1
receptors and those on GABAA receptors [68,87]. Endogenous brain
concentrations of 2-AG under physiological conditions are not well
elucidated partly because it is biosynthesised as needed rather than
stored, and also because it undergoes rapid metabolism [13,61,88].
It has been reported however to be greater than 2 nmol/g of brain
tissue (approx. 2 ␮M in brain homogenates) [88]. Interestingly,
2-AG, at physiological concentrations, plays a role in locomotor
suppression via an action on GABAA receptors, an effect that is
independent of CB1 receptors [21].
In conclusion we report that CBD and 2-AG act as positive
allosteric modulators at various GABAA receptor subtypes with
potency in the low micromolar range. The efficacies of 2-AG and
CBD were broadly similar at most GABAA receptors studied with
the exception of the ␣2 containing GABAA receptors, where the
potentiation with either CBD or 2-AG was more pronounced. Our
study suggests that CBD’s direct action at GABAA receptors should
be considered as a relevant mode of action for the therapeutic effect
of CBD, particularly with respect to its anticonvulsant, analgesic and
anxiolytic properties.
Conflict of interest
The authors have no conflicts to declare.
Authorship contribution
TB, PvN & SOD performed the experiments.
TB, JA & MC designed the research study.
JA, IM & MC contributed essential reagents and equipment.
TB and MC analysed the data.
TB & MC wrote the manuscript.
Acknowledgements
T.B. acknowledges the support of an Australian Postgraduate
Award. S.O.D was supported by the Lambert Initiative for Cannabi-
noid Therapeutics, The University of Sydney, Sydney, Australia.
References
[1] R.W. Olsen, W. Sieghart, International Union of Pharmacology. LXX. Subtypes
of gamma-aminobutyric acid(A) receptors: classification on the basis of
subunit composition, pharmacology, and function, Update Pharmacol. Rev. 60
(3) (2008) 243–260.
[2] R.W. Olsen, W. Sieghart, GABA A receptors: subtypes provide diversity of
function and pharmacology, Neuropharmacology 56 (1) (2009) 141–148.
[3] H.C. Chua, E.T. Christensen, K. Hoestgaard-Jensen, L.Y. Hartiadi, I. Ramzan, A.A.
Jensen, N.L. Absalom, M. Chebib, Kavain, the Major Constituent of the
Anxiolytic Kava Extract, Potentiates GABAA Receptors: Functional
Characteristics and Molecular Mechanism, PLoS One 11 (6) (2016) e0157700.
[4] J.R. Hanrahan, M. Chebib, G.A. Johnston, Interactions of flavonoids with
ionotropic GABA receptors, Adv. Pharmacol. 72 (2015) 189–200.
[5] W. Sieghart, Allosteric modulation of GABAA receptors via multiple
drug-binding sites, Adv. Pharmacol. 72 (2015) 53–96.
[6] M. Farrant, Z. Nusser, Variations on an inhibitory theme: phasic and tonic
activation of GABA(A) receptors, Nat. Rev. Neurosci. 6 (3) (2005) 215–229.
[7] S.G. Brickley, I. Mody, Extrasynaptic GABAA receptors: their function in the
CNS and implications for disease, Neuron 73 (1) (2012) 23–34.
[8] A.N. Clarkson, Perisynaptic GABA receptors the overzealous protector, Adv.
pharmacological Sci. 2012 (2012) 708428.
[9] U. Rudolph, F. Knoflach, Beyond classical benzodiazepines: novel therapeutic
potential of GABAA receptor subtypes, Nat. Rev. Drug Discov. 10 (9) (2011)
685–697.
[10] Y. Yanovsky, S. Schubring, W. Fleischer, G. Gisselmann, X.R. Zhu, H. Lubbert, H.
Hatt, U. Rudolph, H.L. Haas, O.A. Sergeeva, GABAA receptors involved in sleep
and anaesthesia: beta1-versus beta3-containing assemblies, Pflugers Arch. Eu.
J. Physiol. 463 (1) (2012) 187–199.
[11] P. Pacher, S. Batkai, G. Kunos, The endocannabinoid system as an emerging
target of pharmacotherapy, Pharmacol. Rev. 58 (3) (2006) 389–462.
[12] R.G. Pertwee, The diverse CB1 and CB2 receptor pharmacology of three plant
cannabinoids: delta9-tetrahydrocannabinol, cannabidiol and
delta9-tetrahydrocannabivarin, Br. J. Pharmacol. 153 (2) (2008) 199–215.
[13] A. Luchicchi, M. Pistis, Anandamide and 2-arachidonoylglycerol:
pharmacological properties, functional features, and emerging specificities of
the two major endocannabinoids, Mol. Neurobiol. 46 (2) (2012) 374–392.
[14] W. Gonsiorek, C. Lunn, X. Fan, S. Narula, D. Lundell, R.W. Hipkin,
Endocannabinoid 2-arachidonyl glycerol is a full agonist through human type
2 cannabinoid receptor: antagonism by anandamide, Mol. Pharmacol. 57 (5)
(2000) 1045–1050.
[15] P.H. Reggio, Endocannabinoid binding to the cannabinoid receptors: what is
known and what remains unknown, Curr. Med. Chem. 17 (14) (2010)
1468–1486.
[16] R.B. Laprairie, A.M. Bagher, M.E. Kelly, E.M. Denovan-Wright, Cannabidiol is a
negative allosteric modulator of the cannabinoid CB1 receptor, Br. J.
Pharmacol. 172 (20) (2015) 4790–4805.
[17] J.M. McPartland, M. Duncan, V. Di Marzo, R.G. Pertwee, Are cannabidiol and
Delta(9) −tetrahydrocannabivarin negative modulators of the
endocannabinoid system? A systematic review, Br. J. Pharmacol. 172 (3)
(2015) 737–753.
[18] O. Devinsky, M.R. Cilio, H. Cross, J. Fernandez-Ruiz, J. French, C. Hill, R. Katz, V.
Di Marzo, D. Jutras-Aswad, W.G. Notcutt, J. Martinez-Orgado, P.J. Robson, B.G.
Rohrback, E. Thiele, B. Whalley, D. Friedman, Cannabidiol: pharmacology and
 T. Bakas et al. / Pharmacological Research 119 (2017) 358–370
potential therapeutic role in epilepsy and other neuropsychiatric disorders,
Epilepsia 55 (6) (2014) 791–802.
[19] A. Brunklaus, S.M. Zuberi, Dravet syndrome–from epileptic encephalopathy to
channelopathy, Epilepsia 55 (7) (2014) 979–984.
[20] S.A. Hussain, R. Zhou, C. Jacobson, J. Weng, E. Cheng, J. Lay, P. Hung, J.T. Lerner,
R. Sankar, Perceived efficacy of cannabidiol-enriched cannabis extracts for
treatment of pediatric epilepsy: a potential role for infantile spasms and
Lennox-Gastaut syndrome, Epilepsy Behav. 47 (2015) 138–141.
[21] E. Sigel, R. Baur, I. Racz, J. Marazzi, T.G. Smart, A. Zimmer, J. Gertsch, The major
central endocannabinoid directly acts at GABA(A) receptors, Proc. Natl. Acad.
Sci. U. S. A. 108 (44) (2011) 18150–18155.
[22] R. Baur, M. Kielar, L. Richter, M. Ernst, G.F. Ecker, E. Sigel, Molecular analysis of
the site for 2-arachidonylglycerol (2-AG) on the beta(2) subunit of GABA(A)
receptors, J. Neurochem. 126 (1) (2013) 29–36.
[23] G.E. Yevenes, H.U. Zeilhofer, Molecular sites for the positive allosteric
modulation of glycine receptors by endocannabinoids, PLoS One 6 (8) (2011)
e23886.
[24] N. Hejazi, C. Zhou, M. Oz, H. Sun, J.H. Ye, L. Zhang,
Delta9-tetrahydrocannabinol and endogenous cannabinoid anandamide
directly potentiate the function of glycine receptors, Mol. Pharmacol. 69 (3)
(2006) 991–997.
[25] L. Zhang, W. Xiong, Modulation of the Cys-loop ligand-gated ion channels by
fatty acid and cannabinoids, Vitam. Horm. 81 (2009) 315–335.
[26] Z. Yang, K.R. Aubrey, I. Alroy, R.J. Harvey, R.J. Vandenberg, J.W. Lynch,
Subunit-specific modulation of glycine receptors by cannabinoids and
N-arachidonyl-glycine, Biochem. Pharmacol. 76 (8) (2008) 1014–1023.
[27] W. Xiong, K. Cheng, T. Cui, G. Godlewski, K.C. Rice, Y. Xu, L. Zhang,
Cannabinoid potentiation of glycine receptors contributes to
cannabis-induced analgesia, Nat. Chem. Biol. 7 (5) (2011) 296–303.
[28] W. Xiong, T. Cui, K. Cheng, F. Yang, S.R. Chen, D. Willenbring, Y. Guan, H.L. Pan,
K. Ren, Y. Xu, L. Zhang, Cannabinoids suppress inflammatory and neuropathic
pain by targeting alpha3 glycine receptors, J. Exp. Med. 209 (6) (2012)
1121–1134.
[29] W. Xiong, B.N. Koo, R. Morton, L. Zhang, Psychotropic and nonpsychotropic
cannabis derivatives inhibit human 5-HT(3A) receptors through a receptor
desensitization-dependent mechanism, Neuroscience 184 (2011) 28–37.
[30] M. Oz, L. Zhang, M. Morales, Endogenous cannabinoid, anandamide, acts as a
noncompetitive inhibitor on 5-HT3 receptor-mediated responses in Xenopus
oocytes, Synapse 46 (3) (2002) 150–156.
[31] M. Oz, L. Zhang, A. Ravindran, M. Morales, C.R. Lupica, Differential effects of
endogenous and synthetic cannabinoids on alpha7-nicotinic acetylcholine
receptor-mediated responses in Xenopus Oocytes, J. Pharmacol. Exp. Ther. 310
(3) (2004) 1152–1160.
[32] M. Mahgoub, S.Y. Keun-Hang, V. Sydorenko, A. Ashoor, N. Kabbani, L. Al Kury,
B. Sadek, C.F. Howarth, D. Isaev, S. Galadari, M. Oz, Effects of cannabidiol on
the function of alpha7-nicotinic acetylcholine receptors, Eur. J. Pharmacol.
720 (1–3) (2013) 310–319.
[33] S.N. Jackson, S.K. Singhal, A.S. Woods, M. Morales, T. Shippenberg, L. Zhang, M.
Oz, Volatile anesthetics and endogenous cannabinoid anandamide have
additive and independent inhibitory effects on alpha(7)-nicotinic
acetylcholine receptor-mediated responses in Xenopus oocytes, Eur. J.
Pharmacol. 582 (1–3) (2008) 42–51.
[34] B.J. Hall, M. Chebib, J.R. Hanrahan, G.A.R. Johnston, 6-Methylflavanone, a more
efficacious positive allosteric modulator of [gamma]-aminobutyric acid
(GABA) action at human recombinant [alpha]2[beta]2[gamma]2L than at
[alpha]1[beta]2[gamma]2L and [alpha]1[beta]2 GABAA receptors expressed in
Xenopus oocytes, Eur. J. Pharmacol. 512 (2–3) (2005) 97–104.
[35] S.P. Fernandez, N. Karim, K.N. Mewett, M. Chebib, G.A. Johnston, J.R.
Hanrahan, Flavan-3-ol esters: new agents for exploring modulatory sites on
GABA(A) receptors, Br. J. Pharmacol. 165 (4) (2012) 965–977.
[36] S.H. Huang, R.K. Duke, M. Chebib, K. Sasaki, K. Wada, G.A. Johnston, A
sesquiterpene trilactone from Ginkgo biloba, is an antagonist at recombinant
alpha1beta2gamma2L GABA(A) receptors, Eur. J. Pharmacol. 464 (1) (2003)
1–8.
[37] A.M. Hosie, E.L. Dunne, R.J. Harvey, T.G. Smart, Zinc-mediated inhibition of
GABAA receptors: discrete binding sites underlie subtype specificity, Nat.
Neurosci. 6 (4) (2003) 362–369.
[38] M.L. Jensen, K.A. Wafford, A.R. Brown, D. Belelli, J.J. Lambert, N.R. Mirza, The
delta selective compound 2 (DS2): a detailed study of subunit selectivity,
mechanism and site of action utilising human recombinant and rodent native
GABA(A) receptors, Br. J. Pharmacol. (2012).
[39] L.Y. Hartiadi, P.K. Ahring, M. Chebib, N.L. Absalom, High and low GABA
sensitivity alpha4beta2delta GABAA receptors are expressed in Xenopus laevis
oocytes with divergent stoichiometries, Biochem. Pharmacol. 103 (2016)
98–108.
[40] N. Karim, P. Wellendorph, N. Absalom, G.A. Johnston, J.R. Hanrahan, M.
Chebib, Potency of GABA at human recombinant GABA(A) receptors expressed
in Xenopus oocytes: a mini review, Amino Acids 44 (4) (2013) 1139–1149.
[41] M. Mortensen, B. Patel, T.G. Smart, GABA potency at GABA(A) receptors found
in synaptic and extrasynaptic zones, Front. Cell. Neurosci. 6 (2011) 1.
[42] W. Xiong, X. Wu, F. Li, K. Cheng, K.C. Rice, D.M. Lovinger, L. Zhang, A common
molecular basis for exogenous and endogenous cannabinoid potentiation of
glycine receptors, J. Neurosci. 32 (15) (2012) 5200–5208.
[43] J. Ahrens, R. Demir, M. Leuwer, J. de la Roche, K. Krampfl, N. Foadi, M. Karst, G.
Haeseler, The nonpsychotropic cannabinoid cannabidiol modulates and
369
directly activates alpha-1 and alpha-1-Beta glycine receptor function,
Pharmacology 83 (4) (2009) 217–222.
[44] E. Sigel, B.P. Luscher, A closer look at the high affinity benzodiazepine binding
site on GABAA receptors, Curr. Top. Med. Chem. 11 (2) (2011) 241–246.
[45] H.J. Feng, R.L. Macdonald, Multiple actions of propofol on alphabetagamma
and alphabetadelta GABAA receptors, Mol. Pharmacol. 66 (6) (2004)
1517–1524.
[46] D.W. Lam, J.N. Reynolds, Modulatory and direct effects of propofol on
recombinant GABAA receptors expressed in xenopus oocytes: influence of
alpha- and gamma2-subunits, Brain Res. 784 (1-2) (1998) 179–187.
[47] P.K. Ahring, L.H. Bang, M.L. Jensen, D. Strobaek, L.Y. Hartiadi, M. Chebib, N.
Absalom, A pharmacological assessment of agonists and modulators at
alpha4beta2gamma2 and alpha4beta2delta GABAA receptors: the challenge
in comparing apples with oranges, Pharmacol. Res. 111 (2016) 563–576.
[48] R. Baur, J. Gertsch, E. Sigel, The cannabinoid CB1 receptor antagonists
rimonabant (SR141716) and AM251 directly potentiate GABA(A) receptors,
Br. J. Pharmacol. 165 (8) (2012) 2479–2484.
[49] C.E. Griffin 3rd, A.M. Kaye, F.R. Bueno, A.D. Kaye, Benzodiazepine
pharmacology and central nervous system-mediated effects, Ochsner J. 13 (2)
(2013) 214–223.
[50] K.W. Gee, M.B. Tran, D.J. Hogenkamp, T.B. Johnstone, R.E. Bagnera, R.F.
Yoshimura, J.C. Huang, J.D. Belluzzi, E.R. Whittemore, Limiting activity at
beta1-subunit-containing GABAA receptor subtypes reduces ataxia, J.
Pharmacol. Exp. Ther. 332 (3) (2010) 1040–1053.
[51] S.M. Todd, J.C. Arnold, Neural correlates of interactions between cannabidiol
and Delta(9) −tetrahydrocannabinol in mice: implications for medical
cannabis, Br. J. Pharmacol. 173 (1) (2016) 53–65.
[52] L.E. Long, R. Chesworth, X.F. Huang, I.S. McGregor, J.C. Arnold, T. Karl, A
behavioural comparison of acute and chronic Delta9-tetrahydrocannabinol
and cannabidiol in C57BL/6JArc mice, Int. J. Neuropsychopharmacol. 13 (7)
(2010) 861–876.
[53] N. Foadi, M. Leuwer, R. Demir, R. Dengler, V. Buchholz, J. de la Roche, M. Karst,
G. Haeseler, J. Ahrens, Lack of positive allosteric modulation of mutated
alpha(1)S267I glycine receptors by cannabinoids, Naunyn-Schmiedeberg’s
Arch. Pharmacol. 381 (5) (2010) 477–482.
[54] M.L. McCracken, C.M. Borghese, J.R. Trudell, R.A. Harris, A transmembrane
amino acid in the GABAA receptor beta2 subunit critical for the actions of
alcohols and anesthetics, J. Pharmacol. Exp. Ther. 335 (3) (2010) 600–606.
[55] I.N. Cestari, K.T. Min, J.C. Kulli, J. Yang, Identification of an amino acid defining
the distinct properties of murine beta1 and beta3 subunit-containing
GABA(A) receptors, J. Neurochem. 74 (2) (2000) 827–838.
[56] R. Desai, D. Ruesch, S.A. Forman, Gamma-amino butyric acid type A receptor
mutations at beta2N265 alter etomidate efficacy while preserving basal and
agonist-dependent activity, Anesthesiology 111 (4) (2009) 774–784.
[57] P. Meera, M. Wallner, T.S. Otis, Molecular basis for the high THIP/gaboxadol
sensitivity of extrasynaptic GABAA receptors, J. Neurophysiol. 106 (4) (2011)
2057–2064.
[58] N. Karim, P. Wellendorph, N. Absalom, L.H. Bang, M.L. Jensen, M.M. Hansen,
H.J. Lee, G.A. Johnston, J.R. Hanrahan, M. Chebib, Low nanomolar GABA effects
at extrasynaptic alpha4beta1/beta3delta GABA(A) receptor subtypes indicate
a different binding mode for GABA at these receptors, Biochem. Pharmacol. 84
(4) (2012) 549–557.
[59] M.M. Eaton, J. Bracamontes, P. Shu, J.H. Mennerick, G. Steinbach, Akk,
gamma-aminobutyric acid type A alpha4, beta2, and delta subunits assemble
to produce more than one functionally distinct receptor type, Mol. Pharmacol.
86 (6) (2014) 647–656.
[60] A. Dvorzhak, O. Myakhar, P. Unichenko, K. Kirmse, S. Kirischuk, Estimation of
ambient GABA levels in layer I of the mouse neonatal cortex in brain slices, J.
Physiol. 588 (Pt. 13) (2010) 2351–2360.
[61] T. Sugiura, Y. Kobayashi, S. Oka, K. Waku, Biosynthesis and degradation of
anandamide and 2-arachidonoylglycerol and their possible physiological
significance, Prostaglandins Leukot. Essent. Fatty Acids 66 (2–3) (2002)
173–192.
[62] A.C. Campos, F.A. Moreira, F.V. Gomes, E.A. Del Bel, F.S. Guimaraes, Multiple
mechanisms involved in the large-spectrum therapeutic potential of
cannabidiol in psychiatric disorders, Philos. Trans R. Soc. Lond. B Biol. Sci. 367
(1607) (2012) 3364–3378.
[63] M.M. Bergamaschi, R.H. Queiroz, M.H. Chagas, D.C. de Oliveira, B.S. De
Martinis, F. Kapczinski, J. Quevedo, R. Roesler, N. Schroder, A.E. Nardi, R.
Martin-Santos, J.E. Hallak, A.W. Zuardi, J.A. Crippa, Cannabidiol reduces the
anxiety induced by simulated public speaking in treatment-naive social
phobia patients, Neuropsychopharmacology 36 (6) (2011) 1219–1226.
[64] S. Zhornitsky, S. Potvin, Cannabidiol in humans-the quest for therapeutic
targets, Pharmaceuticals 5 (5) (2012) 529–552.
[65] O. Devinsky, E. Marsh, D. Friedman, E. Thiele, L. Laux, J. Sullivan, I. Miller, R.
Flamini, A. Wilfong, F. Filloux, M. Wong, N. Tilton, P. Bruno, J. Bluvstein, J.
Hedlund, R. Kamens, J. Maclean, S. Nangia, N.S. Singhal, C.A. Wilson, A. Patel,
M.R. Cilio, Cannabidiol in patients with treatment-resistant epilepsy: an
open-label interventional trial, Lancet Neurol. 15 (3) (2016) 270–278.
[66] R.R. Patel, C. Barbosa, T. Brustovetsky, N. Brustovetsky, T.R. Cummins,
Aberrant epilepsy-associated mutant Nav1.6 sodium channel activity can be
targeted with cannabidiol, Brain 139 (Pt 8) (2016) 2164–2181.
[67] C.W. Vaughan, M.J. Christie, Retrograde signalling by endocannabinoids
Handbook of Experimental Pharmacology, 168, Springer, 2005, pp. 367–383.
[68] R.G. Pertwee, Pharmacological actions of cannabinoids Handbook of
Experimental Pharmacology, 168, Springer, 2005, pp. 1–51.
370 T. Bakas et al. / Pharmacological Research 119 (2017) 358–370
[69] E.B. Russo, A. Burnett, B. Hall, K.K. Parker, Agonistic properties of cannabidiol
at 5-HT1a receptors, Neurochem. Res. 30 (8) (2005) 1037–1043.
[70] M. Kathmann, K. Flau, A. Redmer, C. Trankle, E. Schlicker, Cannabidiol is an
allosteric modulator at mu- and delta-opioid receptors,
Naunyn-Schmiedeberg’s Arch. Pharmacol. 372 (5) (2006) 354–361.
[71] L. Zhang, W. Xiong, Nonpsychoactive cannabinoid action on 5-HT3 and
glycine receptors, in: E.M. Abood, G.R. Sorensen, N. Stella (Eds.),
endoCANNABINOIDS: Actions at Non-CB1/CB2 Cannabinoid Receptors,
Springer New York, New York, NY, 2013, pp. 199–218.
[72] N. Brzozowska, K.M. Li, X.S. Wang, J. Booth, J. Stuart, I.S. McGregor, J.C. Arnold,
ABC transporters P-gp and Bcrp do not limit the brain uptake of the novel
antipsychotic and anticonvulsant drug cannabidiol in mice, PeerJ 4 (2016)
e2081.
[73] A.L. Geffrey, S.F. Pollack, P.L. Bruno, E.A. Thiele, Drug-drug interaction
between clobazam and cannabidiol in children with refractory epilepsy,
Epilepsia 56 (8) (2015) 1246–1251.
[74] S. Deiana, A. Watanabe, Y. Yamasaki, N. Amada, M. Arthur, S. Fleming, H.
Woodcock, P. Dorward, B. Pigliacampo, S. Close, B. Platt, G. Riedel, Plasma and
brain pharmacokinetic profile of cannabidiol (CBD), cannabidivarine (CBDV),
Delta(9)-tetrahydrocannabivarin (THCV) and cannabigerol (CBG) in rats and
mice following oral and intraperitoneal administration and CBD action on
obsessive-compulsive behaviour, Psychopharmacology (Berl) 219 (3) (2012)
859–873.
[75] E. Engin, J. Liu, U. Rudolph, alpha2-containing GABA(A) receptors: a target for
the development of novel treatment strategies for CNS disorders, Pharmacol.
Ther. 136 (2) (2012) 142–152.
[76] J. Paul, G.E. Yevenes, D. Benke, A. Di Lio, W.T. Ralvenius, R. Witschi, L.
Scheurer, J.M. Cook, U. Rudolph, J.M. Fritschy, H.U. Zeilhofer, Antihyperalgesia
by alpha2-GABAA receptors occurs via a genuine spinal action and does not
involve supraspinal sites, Neuropsychopharmacology 39 (2) (2014) 477–487.
[77] W.T. Ralvenius, D. Benke, M.A. Acuna, U. Rudolph, H.U. Zeilhofer, Analgesia
and unwanted benzodiazepine effects in point-mutated mice expressing only
one benzodiazepine-sensitive GABAA receptor subtype, Nat. Commun. 6
(2015) 6803.
[78] F.S. Guimaraes, T.M. Chiaretti, F.G. Graeff, A.W. Zuardi, Antianxiety effect of
cannabidiol in the elevated plus-maze, Psychopharmacology (Berl) 100 (4)
(1990) 558–559.
View publication stats
[79] A.W. Zuardi, J.A. Crippa, J.E. Hallak, S. Bhattacharyya, Z. Atakan, R.
Martin-Santos, P.K. McGuire, F.S. Guimaraes, A critical review of the
antipsychotic effects of cannabidiol: 30 years of a translational investigation,
Curr. Pharm. Des. 18 (32) (2012) 5131–5140.
[80] A.W. Zuardi, J.E. Hallak, S.M. Dursun, S.L. Morais, R.F. Sanches, R.E. Musty, J.A.
Crippa, Cannabidiol monotherapy for treatment-resistant schizophrenia, J.
Psychopharmacol. (Oxf.) 20 (5) (2006) 683–686.
[81] A.W. Zuardi, I. Shirakawa, E. Finkelfarb, I.G. Karniol, Action of cannabidiol on
the anxiety and other effects produced by delta 9-THC in normal subjects,
Psychopharmacology (Berl) 76 (3) (1982) 245–250.
[82] L.B. Resstel, R.F. Tavares, S.F. Lisboa, S.R. Joca, F.M. Correa, F.S. Guimaraes,
5-HT1A receptors are involved in the cannabidiol-induced attenuation of
behavioural and cardiovascular responses to acute restraint stress in rats, Br.
J. Pharmacol. 156 (1) (2009) 181–188.
[83] A.C. Campos, F.S. Guimaraes, Involvement of 5HT1A receptors in the
anxiolytic-like effects of cannabidiol injected into the dorsolateral
periaqueductal gray of rats, Psychopharmacology (Berl) 199 (2) (2008)
223–230.
[84] E. Shohami, A. Cohen-Yeshurun, L. Magid, M. Algali, R. Mechoulam,
Endocannabinoids and traumatic brain injury, Br. J. Pharmacol. 163 (7) (2011)
1402–1410.
[85] D. Panikashvili, C. Simeonidou, S. Ben-Shabat, L. Hanus, A. Breuer, R.
Mechoulam, E. Shohami, An endogenous cannabinoid (2-AG) is
neuroprotective after brain injury, Nature 413 (6855) (2001) 527–531.
[86] A.F. Almeida-Santos, P.H. Gobira, L.C. Rosa, F.S. Guimaraes, F.A. Moreira, D.C.
Aguiar, Modulation of anxiety-like behavior by the endocannabinoid
2-arachidonoylglycerol (2-AG) in the dorsolateral periaqueductal gray, Behav.
Brain Res. 252 (2013) 10–17.
[87] J.M. McPartland, M. Glass, R.G. Pertwee, Meta-analysis of cannabinoid ligand
binding affinity and receptor distribution: interspecies differences, Br. J.
Pharmacol. 152 (5) (2007) 583–593.
[88] N. Stella, P. Schweitzer, D. Piomelli, A second endogenous cannabinoid that
modulates long-term potentiation, Nature 388 (6644) (1997) 773–778.