The major central endocannabinoid directly acts at GABA(A) receptors.
ABSTRACT GABA(A) receptors are the major ionotropic inhibitory neurotransmitter receptors. The endocannabinoid system is a lipid signaling network that modulates different brain functions. Here we show a direct molecular interaction between the two systems. The endocannabinoid 2-arachidonoyl glycerol (2-AG) potentiates GABA(A) receptors at low concentrations of GABA. Two residues of the receptor located in the transmembrane segment M4 of β(2) confer 2-AG binding. 2-AG acts in a superadditive fashion with the neurosteroid 3α, 21-dihydroxy-5α-pregnan-20-one (THDOC) and modulates δ-subunit-containing receptors, known to be located extrasynaptically and to respond to neurosteroids. 2-AG inhibits motility in CB(1)/CB(2) cannabinoid receptor double-KO, whereas β(2)-KO mice show hypermotility. The identification of a functional binding site for 2-AG in the GABA(A) receptor may have far-reaching consequences for the study of locomotion and sedation.
[show abstract] [hide abstract]
ABSTRACT: GABA(A) receptors are the major inhibitory neurotransmitter receptors in the brain and are the site of action of many clinically important drugs. These receptors are composed of five subunits that can belong to eight different subunit classes. Depending on their subunit composition, these receptors exhibit distinct pharmacological and electrophysiological properties. Recent studies on recombinant and native GABA(A) receptors suggest the existence of far more receptor subtypes than previously assumed. Thus, receptors composed of one, two, three, four, or five different subunits might exist in the brain. Studies on the regional, cellular and subcellular distribution of GABA(A) receptor subunits, and on the co-localization of these subunits at the light and electron microscopic level for the first time provide information on the distribution of GABA(A) receptor subtypes in the brain. These studies will have to be complemented by electrophysiological and pharmacological studies on the respective recombinant and native receptors to finally identify the receptor subtypes present in the brain. The distinct cellular and subcellular location of individual receptor subtypes suggests that they exhibit specific functions in the brain that can be selectively modulated by subtype specific drugs. This conclusion is supported by the recent demonstration that different GABA(A) receptor subtypes mediate different effects of benzodiazepines. Together, these results should cause a revival of GABA(A) receptor research and strongly stimulate the development of drugs with a higher selectivity for alpha2-, alpha3-, or alpha5-subunit-containing receptor subtypes. Such drugs might exhibit quite selective clinical effects.Current Topics in Medicinal Chemistry 09/2002; 2(8):795-816. · 4.17 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: GABA(A) (gamma-aminobutyric acid type A) receptors mediate most of the 'fast' synaptic inhibition in the mammalian brain and are targeted by many clinically important drugs. Certain naturally occurring pregnane steroids can potently and specifically enhance GABA(A) receptor function in a nongenomic (direct) manner, and consequently have anxiolytic, analgesic, anticonvulsant, sedative, hypnotic and anaesthetic properties. These steroids not only act as remote endocrine messengers, but also can be synthesized in the brain, where they modify neuronal activity locally by modulating GABA(A) receptor function. Such 'neurosteroids' can influence mood and behaviour in various physiological and pathophysiological situations, and might contribute to the behavioural effects of psychoactive drugs.Nature reviews. Neuroscience 08/2005; 6(7):565-75. · 30.44 Impact Factor
Article: Positioning of the alpha-subunit isoforms confers a functional signature to gamma-aminobutyric acid type A receptors.[show abstract] [hide abstract]
ABSTRACT: Fast synaptic inhibitory transmission in the CNS is mediated by gamma-aminobutyric acid type A (GABA(A)) receptors. They belong to the ligand-gated ion channel receptor superfamily, and are constituted of five subunits surrounding a chloride channel. Their clinical interest is highlighted by the number of therapeutic drugs that act on them. It is well established that the subunit composition of a receptor subtype determines its pharmacological properties. We have investigated positional effects of two different alpha-subunit isoforms, alpha(1) and alpha(6), in a single pentamer. For this purpose, we used concatenated subunit receptors in which subunit arrangement is predefined. The resulting receptors were expressed in Xenopus oocytes and analyzed by using the two-electrode voltage-clamp technique. Thus, we have characterized gamma(2)beta(2)alpha(1)beta(2)alpha(1), gamma(2)beta(2)alpha(6)beta(2)alpha(6), gamma(2)beta(2)alpha(1)beta(2)alpha(6), and gamma(2)beta(2)alpha(6)beta(2)alpha(1) GABA(A) receptors. We investigated their response to the agonist GABA, to the partial agonist piperidine-4-sulfonic acid, to the noncompetitive inhibitor furosemide and to the positive allosteric modulator diazepam. Each receptor isoform is characterized by a specific set of properties. In this case, subunit positioning provides a functional signature to the receptor. We furthermore show that a single alpha(6)-subunit is sufficient to confer high furosemide sensitivity, and that the diazepam efficacy is determined exclusively by the alpha-subunit neighboring the gamma(2)-subunit. By using this diagnostic tool, it should become possible to determine the subunit arrangement of receptors expressed in vivo that contain alpha(1)- and alpha(6)-subunits. This method may also be applied to the study of other ion channels.Proceedings of the National Academy of Sciences 06/2004; 101(20):7769-74. · 9.68 Impact Factor
The major central endocannabinoid directly acts at
Erwin Sigela,1, Roland Baura, Ildiko Ráczb, Janine Marazzia, Trevor G. Smartc, Andreas Zimmerb, and Jürg Gertscha,1
aInstitute of Biochemistry and Molecular Medicine, University of Bern, CH-3012 Bern, Switzerland;bInstitute of Molecular Psychiatry, Life and Brain
Center, University of Bonn, 53127 Bonn, Germany; andcDepartment of Neuroscience, Physiology and Pharmacology, University College London, London
WC1E 6BT, United Kingdom
Edited by Leslie Lars Iversen, University of Oxford, Oxford, United Kingdom, and approved October 3, 2011 (received for review August 17, 2011)
GABAAreceptors are the major ionotropic inhibitory neurotrans-
mitter receptors. The endocannabinoid system is a lipid signaling
network that modulates different brain functions. Here we show
a direct molecular interaction between the two systems. The endo-
cannabinoid 2-arachidonoyl glycerol (2-AG) potentiates GABAA
tor located in the transmembrane segment M4 of β2confer 2-AG
binding. 2-AG acts in a superadditive fashion with the neurosteroid
3α, 21-dihydroxy-5α-pregnan-20-one (THDOC) and modulates
δ-subunit–containing receptors, known to be located extrasy-
naptically and to respond to neurosteroids. 2-AG inhibits motility
in CB1/CB2cannabinoid receptor double-KO, whereas β2-KO mice
show hypermotility. The identification of a functional binding site
for 2-AG in the GABAAreceptor may have far-reaching consequen-
ces for the study of locomotion and sedation.
retrograde signaling|electrophysiology|allosteric modulation
the mammalian brain. A total of 19 different subunit isoforms
have been identified, with the major receptor type in mammalian
adult brain consisting of α1, β2, and γ2subunits (1, 2). GABAA
receptors are the target of numerous sedating and anxiolytic
drugs such as benzodiazepines (3). The currently known endo-
genous ligands include GABA, neurosteroids (4), and possibly
oleamide (5). The pharmacological properties of this chloride
ion channel strictly depend on receptor subunit composition (2)
and arrangement (6).
Synaptic GABAAreceptors mediate phasic inhibition, whereas
extrasynaptic receptors mediate tonic inhibition (7, 8). The δ-
subunit–containing GABAA receptors occur only extrasyn-
aptically and are particularly sensitive to modulation by neuro-
steroids (4, 9, 10). GABAAreceptors containing the δ-subunit
have been implicated in changing seizure susceptibility and
states of anxiety during the ovarian cycle (11) and in postpartal
The endocannabinoid system (ECS) is part of a complex lipid
signaling network involving the G protein-coupled receptors CB1
and CB2(13, 14). The CB1receptor is ubiquitously expressed in
the central nervous system, whereas the CB2receptor is primarily
expressed in peripheral tissues (13, 15). The two best-charac-
terized endogenous cannabinoid ligands are anandamide (AEA)
and 2-arachidonoyl glycerol (2-AG) (15, 16). Endocannabinoids
are released postsynaptically and modulate neurotransmission by
activating presynaptic CB1receptors. Thus, they act as retro-
grade signals. 2-AG is the major CB1receptor agonist in the
brain, where it is found at micromolar concentrations (17).
Physiological and pharmacological studies provide evidence that
the ECS is involved in the regulation of GABA and glutamate
release (18, 19). The major central action of endocannabinoids is
analgesia (20, 21), but the ECS has also been implicated in
anxiety and movement disorders (22).
ABAAreceptors are chloride ion channels composed of five
subunits (1), mediating fast synaptic and tonic inhibition in
Endocannabinoid 2-AG Potentiates GABAAReceptors. Recombinant
α1β2γ2GABAAreceptors were functionally expressed in Xenopus
oocytes. We found that currents elicited by 1 μM GABA were
potentiated by 2-AG in a concentration-dependent way (Fig. 1 A
and B). Fitting of the concentration–response curve indicated
a maximal potentiation of 138 ± 21% (SEM) and an EC50value
of 2.1 ± 0.5 μM (n = 5). The resulting Hill coefficient of 2.2 ± 0.2
indicates that more than one molecule of 2-AG interacts with
one receptor. Potentiation by 3 μM 2-AG was determined at
different GABA concentrations—0.5 μM (EC∼0.9), 1 μM (EC∼2.3),
10 μM (EC∼35), and 100 μM (EC∼92)—and amounted to 86 ± 13%
(n = 3), 82 ± 25% (n = 6), 10 ± 6% (n = 3) and 2 ± 6% (n = 3),
respectively. This shows that only currents elicited by low GABA
concentrations are potentiated by 2-AG.
Identification of Other Endocannabinoids That Target GABAA Re-
ceptors. To determine if this effect was only observed with
2-AG, we also evaluated physiologically relevant compounds
sharing close structural similarity with 2-AG (Fig. 1C). The
spontaneous isomerization product 1(3)-AG (stereoisomeric
mixture produced by acyl migration) showed a similar potentia-
tion as 2-AG. Whereas AEA and 2-methyl-2′-F-AEA (r-met
AEA) only weakly affected the response to 2-AG, 2-arachidonoyl
glyceryl ether (noladin ether; NE), which is a minor putative
endogenous CB1receptor agonist (23), was similarly active at
GABAA receptors (Fig. 1D). Docosatetraenylethanolamide
(DEA) and arachidonic acid (AA) showed only a weak if any
potentiation (Fig. 1D). Not unexpectedly, oleamide, which was
previously shown to only weakly potentiate GABAAreceptors
(5), was ineffective at 3 μM. The phytocannabinoid Δ9-tetrahy-
drocannabinol (THC) at 3 μM only weakly potentiated the re-
sponse to GABA, at 34 ± 10% (Fig. 1D). These data uncover the
significant effect of 2-AG and NE at GABAAreceptors and in-
dicate the importance of the glycerol moiety for the GABAA
Potentiation by 2-AG Is Selective for GABAAReceptors Containing the
β2Subunit. Next, we investigated the GABAAreceptor subunit
selectivity of 2-AG. The α1subunit in α1β2γ2was replaced by α2,
α3, α5, or α6. This had little effect on the current potentiation by 3
μM 2-AG (Fig. 1E). However, drastic effects were observed upon
replacement of the β2subunit by β1or β3(Fig. 1F). Whereas, in
receptors containing β1, potentiation was abolished, it was re-
duced to approximately one third in receptors containing β3. To
test whether 2-AG shares the binding site with loreclezole (24),
another allosteric activator of GABAAreceptors, we evaluated
the point mutation β2N265S in α1β2γ2receptors that abolishes
the potentiation by loreclezole. As shown in Fig. 1F, this muta-
tion only partially reduced the effect of 2-AG, thus indicating
Author contributions: E.S., A.Z., and J.G. designed research; R.B., I.R., and J.M. performed
research; T.G.S. contributed new reagents/analytic tools; E.S., R.B., I.R., A.Z., and J.G.
analyzed data; and E.S. and J.G. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence may be addressed. E-mail: email@example.com or gertsch@
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
www.pnas.org/cgi/doi/10.1073/pnas.1113444108PNAS Early Edition
| 1 of 6
that endocannabinoids act through a different site. Potentiation
was also strongly reduced upon omission of γ2in α1β2γ2to give
the dual combination α1β2. Possibly, in receptors with two adja-
cent β2subunits the binding site for 2-AG is compromised in
these subunits. Replacement of the β2subunit in dual subunit
combinations by β1or β3in both cases led to an additional strong
reduction of the potentiation (Fig. 1F). For all experiments,
GABA was used at a concentration eliciting 1.0% to 3.2% of the
maximal current amplitude in each receptor form.
We then investigated the concatenated receptors (25, 26) α1-
β1-α1/γ2-β1, α1-β2-α1/γ2-β2, α1-β1-α1/γ2-β2, and α1-β2-α1/γ2-β1, in
which the subunit arrangement is predefined by covalent linkage.
These receptors have been described in detail previously (27). As
shown in Fig. 1G, in receptors containing two β1subunits stim-
ulation by 2-AG was strongly reduced compared with receptors
containing two β2subunits. In the receptors containing one β2
subunit, irrespective of the position, the potentiation was re-
duced by approximately 50%, indicating that two sites for 2-AG
may be located on one α1β2γ2receptor.
Identification of Amino Acid Residues Conferring Subunit Selectivity
of 2-AG. Next, we identified the amino acid residues (AARs)
mediating the selectivity of 2-AG for the β2subunits. We aligned
the protein sequences of the three β-subunits and identified
homologous AAR in predicted transmembrane regions that were
identical in β1and β3, but different from those in β2. These were
the AAR corresponding to β2M294 and β2L301 in transmem-
brane region M3, and β2V436 and β2F439 in M4. Mutation of
these residues in β2to the respective residues present in β1and β3
were prepared and coexpressed with α1and γ2subunits, and the
resulting receptors screened for their potentiation by 3 μM of
2-AG (Fig. 2A and SI Appendix, Fig. S1). All mutations signifi-
cantly reduced potentiation by 2-AG. In the mutant receptor
α1β2V436Tγ2, potentiation was even completely abolished, but
the EC50of GABA was not significantly altered. In a second
step, the corresponding residues in β1were mutated to the ho-
mologous residue present in β2(Fig. 2B). A critical residue lo-
cated in the binding site would be expected to lead to a loss of
potentiation in α1β2Mγ2receptors and to a gain in potentiation in
α1β1Mγ2receptors. Our results clearly show that AAR V436 and,
to a smaller extent, also F439 in M4 of β2mediate the functional
effect of 2-AG, thus pinpointing the receptor binding site. Along
the same line, both mutations combined resulted in almost
complete recovery. Although allosteric effects by the mutations
cannot be fully excluded, we consider this possibility unlikely.
Both AARs are predicted to be located in cytoplasmic leaflet of
the M4 α-helix, in an angle of approximately 60° and a distance
in the direction of the α-helix of 4.5 Å. Intriguingly, in a homol-
ogy model described by Ernst et al. (28), all four mentioned
residues face the same cavity in the receptor.
δ-Subunit–Containing Receptors Are Responsive to 2-AG. In agree-
ment with the finding that 2-AG mediates its effect via β2, α1β2δ
receptors in which γ2in α1β2γ2was replaced by the δ-subunit also
responded in a concentration-dependent way to 2-AG (Fig. 3A).
Fitting of the concentration–response curve carried out with
1 μM GABA (EC∼8) indicated a maximal potentiation of 150 ±
51% (n = 3), an EC50value of 2.9 ± 1.8 μM, and a Hill co-
efficient of 1.25 ± 0.15 (Fig. 3B). In additional experiments,
α1β2δ receptors responded with a potentiation of 114 ± 19%
tools. (A) Concentration–response curve of 2-AG at α1β2γ2GABAAreceptors. Increasing concentrations of 2-AG (upper bars) were preapplied for 30 s and
subsequently coapplied with 1 μM GABA (lower bars) after two control applications of GABA. Concentrations of 2-AG are indicated above the upper bars.
Original current traces are shown. (B) Concentration–response curve of 2-AG. Mean values ± SEM are shown for experiments with five oocytes from three
batches of oocytes. (C) Molecular structures of tested compounds. (D) Relative current potentiation by different agents sharing structural or functional
similarity with 2-AG. Current potentiation by 3 μM of 2-AG, 1-AG, AEA, NE, r-met AEA, AA, DEA, oleamide, THC, and 1 μM of AEA. (E) Lack of a role of the
type of α-subunit in recombinant αxβ2γ2(x = 1, 2, 3, 5, 6) GABAAreceptors for 2-AG effect. Relative current potentiation was determined at 3 μM of 2-AG. (F)
Role of the type of β-subunit in recombinant α1βyand α1βyγ2(y = 1, 2, 3) GABAAreceptors. (G) Concatenated receptors, containing two β1, two β2, or one β1and
one β2subunit in different positions, were modulated by 2-AG as earlier. Data are shown as mean values ± SEM (n = 4). For all experiments shown in this
figure, GABA was used at a concentration eliciting 1.0% to 3.2% of the maximal current amplitude [EC(2.1 ± 1.1)] in each receptor form.
2-AG affects the function of GABAAreceptors. Receptors were expressed in Xenopus oocytes. Currents were measured using electrophysiological
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| www.pnas.org/cgi/doi/10.1073/pnas.1113444108Sigel et al.
(n = 4; SI Appendix, Fig. S2) to 3 μM 2-AG. In α1β2δ receptors,
the subunits may be arranged differently to form a pentamer,
and the different receptor isoforms are characterized by distinct
properties, some of them being strictly dependent on the pres-
ence of neurosteroids and low GABA concentrations (29). To-
gether, these results establish 2-AG as an endogenous allosteric
activator of GABAAreceptors and identify M4 of the β2subunit
as the primary molecular target for 2-AG.
2-AG Acts in a Superadditive Manner with Neurosteroids or Di-
azepam. Next, we investigated a possible functional interaction
between 2-AG and neurosteroids at α1β2γ2GABAAreceptors.
Concentration–response curves for the potentiation by 2-AG
were generated in the presence of 0.1 μM 3α, 21-dihydroxy-5α-
pregnan-20-one (THDOC; Fig. 4A). GABA (0.5 μM) was ap-
plied alone and then in combination with 0.1 μM THDOC, fol-
lowedby applications containing
increasing concentrations of 2-AG. As shown in Fig. 4A poten-
tiation by 0.1 μM THDOC alone amounted to 164 ± 44% (n =
4). As mentioned earlier, the maximal potentiation by 2-AG
amounted to 138 ± 21 (n = 5). Combined application of 2-AG
and THDOC resulted in a curve characterized by an EC50of
1.7 ± 0.5 μM and a maximal potentiation of 1,041 ± 505% (n = 4).
This indicates that THDOC acts by increasing the maximal po-
tentiation without significantly affecting the EC50 for 2-AG.
Combined application of 0.1 μM THDOC and 1 μM 2-AG
resulted in a superadditive potentiation, thus suggesting a syner-
gism between these two agents. Further experiments were per-
formed to support a superadditivity between THDOC and 2-AG.
Fig. 4B shows an experiment in which currents were potentiated
by low concentrations of THDOC, 2-AG, or a combination of
both. We observed a strong superadditive effect between the two
endogenous modulators (Fig. 4C). Superadditivity was also ob-
served in three additional experiments carried out at 0.05 μM
THDOC (SI Appendix, Fig. S3). To assess whether the agonist
site or the modulatory site for neurosteroids (30) was involved in
the superadditivity, we applied 0.1 μM THDOC together with
1 μM 2-AG in the absence of GABA. Currents amplitudes
amounting to less than 4 nA were elicited in oocytes expressing
more than 10,000 nA maximal GABA current. It is therefore
unlikely that the agonist site for neurosteroids is involved in the
superadditivity of 2-AG and THDOC effects.
Next, we investigated a possible interaction of 2-AG with the
benzodiazepine diazepam. Concentration–response curves for
the potentiation by 2-AG were generated in the presence of 0.3
μM diazepam (SI Appendix, Fig. S4). GABA (0.5 μM) was ap-
plied alone and then in combination with 0.3 μM diazepam,
followed by several applications containing GABA, diazepam,
and increasing concentrations of 2-AG. In these experiments,
potentiation by 0.3 μM diazepam alone amounted to 128 ± 7%
(n = 3). The EC50for 2-AG in the presence of diazepam was
0.8 ± 0.3 μM, indicating a small increase in apparent affinity for
2-AG, and the maximal additional potentiation relative to this
level achieved with diazepam alone was 210 ± 35% (n = 3). In
additional experiments, currents were potentiated by 0.3 μM di-
azepam or 2-AG, respectively, or by a combination of both.
Again, we observed a significant superadditivity between the two
modulators (SI Appendix, Fig. S5). These results clearly suggest
a superadditivity between the modulation by diazepam and 2-AG.
Locomotor Activity Is Suppressed by 2-AG and NE in CB1/CB2Double-
KO Mice, and β2 KO Mice Show Hypermotility. The sedating effects
of GABAAreceptor activation can be readily determined in vivo
by evaluating exploratory locomotor behaviors (31). To eliminate
the cannabinoid receptor-mediated effects by 2-AG, the experi-
ments were carried out in cannabinoid receptor (Cnr1−/−/Cnr2−/−)
double-KO mice. Additionally, WT mice were investigated. Ve-
hicle-injected control animals of both genotypes showed a char-
acteristic open-field exploratory behavior. Locomotor activity was
initially high and gradually decreased over time, as the animals
habituated to the new environment (Fig. 5 A and B). As antici-
pated, 2-AG–treated animals (10 mg·kg−1i.v.) showed a strong
hypomotility in WT and in KO animals (P < 0.0001; Fig. 5 A and
mutation α1β2V436Tγ2abolishes potentiation by 2-AG. Impact of selected
point mutations in M3 and M4 of the β2subunit of α1β2Mγ2receptors on the
potentiation by 2-AG. The values are given relative to the current elicited by
GABA (mean of 101% and 81%, respectively, in two different experiments).
(B) The β1subunit, which is naturally not able to confer potentiation to
α1β1γ2 receptors, is partially converted to a competent subunit by the
mutations β1T436V and β1L439F alone and to a larger extent by the com-
bined mutation. The mutations did not affect the EC50for GABA. GABA was
used at a concentration eliciting 1.0% to 3.2% of the maximal current am-
plitude [EC(2.1 ± 1.1)]. Data are shown as mean values ± SEM. Measurements of
the mutations in M3 were performed in at least four oocytes, and those of
the mutations in M4 in five to eight oocytes from two independent batches
Point mutations affecting the stimulation by 2-AG. (A) The point
Concentration response curve of 2-AG at α1β2δ GABAA
receptors. Increasing concentrations of 2-AG (upper bars)
were preapplied for 30 s and subsequently coapplied with
1 μM GABA (EC∼8; lower bars) after two control applica-
tions of GABA. Concentrations of 2-AG are indicated
above the upper bars. Original current traces are shown.
Please note that preapplication of 2-AG resulted in small
currents by itself. Currents did not saturate completely
within the time of application and saturation of poten-
tiation was not reached experimentally. Time of applica-
tion and concentrations used were both limited by the
available 2-AG. (B) Three concentration–response curves
of 2-AG were averaged. Mean values ± SD are shown for
Effect of 2-AG on α1β2δ GABAA receptors. (A)
Sigel et al.PNAS Early Edition
| 3 of 6
B). Similarly, NE (10 mg·kg−1i.v.), a metabolically stable ether-
linked analogue of 2-AG (23), led to potent hypomotility (Fig. 5 C
and D). After 2-AG treatment in several cases, a brief anesthetic
effect (i.e., loss of righting reflex) was detected (WT, three of
eight; KO, five of eight). NE treatment in all cases led to a brief
loss of righting reflex. Thus, these data provide evidence for the
cannabinoid receptor-independent sedative effect of 2-AG
Next, we addressed the potential pharmacological super-
additivity between 2-AG and THDOC. Given that 2-AG in-
jection leads to rapid degradation of 2-AG in the brain (32) we
used an indirect approach through pharmacological inhibition of
2-AG metabolism. Treatment of WT and cannabinoid receptor
(Cnr1−/−/Cnr2−/−) double-KO mice with the selective mono-
acylglycerol lipase inhibitor JZL184 (16 mg·kg−1i.p.), a com-
pound that inhibits the enzymatic hydrolysis of 2-AG (32, 33), or
THDOC (2 mg·kg−1i.v.) alone, only partially inhibited loco-
motor activity after habituation to the test cage (P = 0.08 and
P = 0.156, respectively; Fig. 5 E and F). However, when the
animals received THDOC (2 mg·kg−1i.v.) 2 h after the JZL184
treatment, they presented strong hypolocomotion after habitu-
ation (P < 0.0001). These data show that elevated endogenous 2-
(A) Cumulative concentration response curve of 2-AG without (circles) and with potentiation with 0.1 μM THDOC (squares). The dashed line indicates the
stimulation by 0.1 μM THDOC alone. (B) GABA 0.5 μM (EC0.2–0.5) was applied twice alone, followed by the same concentration of GABA in combination with
0.1 μM THDOC, GABA alone, GABA in combination with 1 μM 2-AG, GABA alone, and GABA in combination with 0.1 μM THDOC and 1 μM 2-AG. Responses to
2-AG, alone or in combination, did not reach equilibrium, presumably because 2-AG is hydrophobic and accumulates in the membrane. (C) Four such
experiments were averaged. Data are shown as mean values ± SD (n = 4).
Superadditivity between 2-AG and the neurosteroid THDOC. Recombinant α1β2γ2GABAAreceptors were functionally expressed in Xenopus oocytes.
Cnr1−/−/Cnr2−/−double-KO mice (B) after treatment with 2-AG (10 mg·kg−1i.v.; circles) or vehicle (squares) and in WT (C) and Cnr1−/−/Cnr2−/−double-KO mice
(D) after treatment with NE (10 mg·kg−1i.v.; circles) or vehicle (squares). Data are shown as the mean distance traveled in 1-min time bins ± SEM. Repeated-
measures ANOVA was used to assess the effect of 2-AG and NE treatment on locomotor activity (n ≥ 8; P < 0.0001). Superadditivity between THDOC and NE in
WT and Cnr1−/−/Cnr2−/−double-KO mice. The spontaneous locomotor activity was determined in WT (E) and Cnr1−/−/Cnr2−/−double-KO mice (F) after
treatment with vehicle (closed squares), THDOC (2 mg·kg−1i.v.; open circles), NE (5 mg·kg−1i.v.; open squares), or combined treatment with THDOC and NE
(closed circles). Superadditivity between THDOC and JZL184 in WT and Cnr1−/−/Cnr2−/−double-KO mice. The spontaneous locomotor activity was determined
in WT (G) and Cnr1−/−/Cnr2−/−double-KO mice (H) after treatment with vehicle (closed squares), THDOC (2 mg·kg−1i.v.; open circles), JZL184 (16 mg·kg−1i.p.;
open squares), or combined treatment with THDOC and JZL184 (closed circles). Data are shown as the mean distance traveled in 1-min time bins ± SEM.
Repeated-measures ANOVA was used to assess significance of the superadditive effect after habituation (P < 0.0001).
Behavioral effects of 2-AG and NE in WT and Cnr1−/−/Cnr2−/−double-KO mice. The spontaneous locomotor activity was determined in WT (A) and
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AG in brain mediates a superadditive effect with externally ad-
ministered THDOC, independent of CB receptors (Fig. 5 E and F).
In WT mice, the superadditive effect was more pronounced than
in KO mice, and may also involve CB receptors. JZL184 16
mg·kg−1i.p. did not trigger the full effect on 2-AG elevation,
which is approximately 10-fold (33), and showed significant
hypomotility only during the habituation phase (Fig. 5 G and H).
Upon completion of the experiments, the JZL184-treated
(Cnr1−/−/Cnr2−/−) double-KO mice showed a fourfold increase
of 2-AG in brain, and the free AA was significantly reduced (SI
Appendix, Fig. S6). As expected, a more potent superadditive
effect was observed in the late phase in animals treated with
combinations of low doses of NE (5 mg/kg−1i.v.) and THDOC (2
mg·kg−1i.v.; P < 0.0001; Fig. 5 G and H), as a result of the better
metabolic stability of NE vs. 2-AG (34). It should be noted that
nontreated KO mice (vehicle controls in Fig. 5 B, D, F, and H)
display a lower motility than WT mice (vehicle controls in Fig. 5
A, C, E, and G; P < 0.0001).
As 2-AG acts via the β2subunit, we studied β2KO mice. These
mice displayed a very pronounced hypermotility (P < 0.0001; Fig.
6A), which is in agreement with a previous report (35). This may
indicate the involvement of a tonic activation of β2 subunit-
containing GABAA receptors by 2-AG in WT C57BL6/J/
129SvEv mice. When 2-AG levels were increased by JZL184 and
CB1receptors simultaneously blocked by low concentrations of
the CB1antagonist SR141716 at which GABAAreceptors were
not affected (36), WT mice showed hypomotility, whereas the β2
KO mice showed hypermotility during the habituation phase
(Fig. 6B). This clearly indicates that physiological concentrations
of 2-AG (as modulated by JZL184) can exert hypolocomotion in
these mice via both β2and CB1receptors. However, treatment of
β2KO mice with NE (10 mg·kg−1i.v.) and SR141716 (3 mg·kg−1
i.v.) likewise induced strong hypomotility (SI Appendix, Fig. S7),
thus indicating additional receptors for NE.
In addition to the well known retrograde GABAergic signaling
of endocannabinoids mediated via the CB1receptor at central
synapses (19), we show an unexpected direct molecular interac-
tion between 2-AG and GABAAreceptors. 2-AG is an endog-
enous ester formed from the omega-6 fatty acid AA and glycerol
by different biosynthetic pathways (16). 2-AG, unlike AEA, is
present at high levels in the central nervous system; it is the most
abundant molecular species of monoacylglycerol found in mouse
and rat brain (∼5–10 μmol/kg tissue) (16). Our finding that el-
evated 2-AG levels upon JZL184 treatment inversely correlate
with free AA levels is consistent with a previous study (32) and
clearly indicates that 2-AG is a major AA metabolite in mouse
brain. AEA levels in brain are generally low (up to 0.1 μmol/kg wet
weight) (17), and at 1 μM of AEA, only minor and irrelevant
effects on GABAAreceptors were detected in this study. This is in
line with our previous observation that AEA fails to modulate
motility in Cnr1−/−/Cnr2−/−double-KO mice (37) even though it
inhibits locomotion in CB1KO mice (38). In this study, NE pro-
duced stronger pharmacological effects in Cnr1−/−/Cnr2−/−dou-
ble-KO mice than 2-AG, which may be explained by its better
metabolic stability (34) or additional receptor targets. The latter
is also indicated by the fact that, in β2KO mice, NE showed
similar effects as in WT mice in the presence of CB1receptor
blockage by SR141716 (SI Appendix, Fig. S7). In agreement with
a previous study (39), we were not able to detect NE in mouse
brain. Other groups have reported only low levels (in nmol/kg) of
NE in rat brain (40). This suggests that NE is not a major AA
brain metabolite, and physiological interactions with GABAAre-
ceptors are therefore rather unlikely.
The superadditivity between the effects of THDOC and 2-AG
at GABAAreceptors is intriguing and may suggest that 2-AG can
modulate the action of neurosteroids at GABAAreceptors. A
study showing that 2-AG distribution in rat brain does not match
the CB1 receptor sites describes high amounts of 2-AG in
brainstem and hippocampus (41). We also explored the super-
additivity of 2-AG and THDOC in animal experiments to obtain
additional evidence for the physiological relevance of our finding.
Low concentrations of THDOC in combination with partially
elevated 2-AG levels or low concentrations of NE administered i.
v. resulted in significant superadditive inhibition of locomotion
both in WT and KO mice. Intriguingly, β2KO mice showed an
overall pronounced hyperlocomotion compared with WT mice,
indicating a role for β2in 2-AG–mediated locomotion behavior.
Blocking of 2-AG degradation in the presence of SR141716 in β2
KO mice resulted in hyperlocomotion during the habituation
phase, which was not observed in WT mice. On the contrary, WT
mice showed inhibition of locomotion during this phase, which is
in agreement with the hypothesis that 2-AG can mediate be-
havioral effects also via β2. Nevertheless, the expected lack of
locomotion inhibition by 2-AG in the later phase in β2KO mice
was not observed. Although our data with β2KO mice demon-
strate the involvement of β2-containing GABAAreceptors during
locomotion habituation, the overall strong hypolocomotion in-
duced by NE in the β2KO mice suggests the involvement of
additional receptors, possibly from the cys-loop family.
2-AG is biosynthetically generated in postsynaptic neurons
directly from constituents of the cell membrane where GABAA
receptors are located (42). Given that 2-AG is synthesized in the
membrane and cannot easily cross the aqueous barrier, it is
tempting to speculate that 2-AG accumulates and interacts lo-
cally with GABAAreceptors within the postsynaptic neuron (Fig.
7). Based on our finding that 2-AG acts exclusively at low con-
centrations of GABA and that it synergizes with neurosteroids, it
is likely that extrasynaptic GABAAreceptors may be preferen-
tially targeted by 2-AG. As it has been shown in hippocampus,
not all synapses are silenced by 2-AG to the same degree, and
residual synaptic activity, especially in dendritic synapses, may
The spontaneous locomotor activity was determined before (A) and after (B)
treatment with JZL184 (16 mg·kg−1i.v.) plus SR141716 (3 mg·kg−1i.v.) in WT
(open circles) and β2-KO mice (open squares). Data are shown as the mean
distance traveled in 1-min time bins ± SEM (n = 6 each).
Hypermotility of β2-KO mice and effects of JZL184 and SR141716.Fig. 7.
diffuse laterally and act in the postsynaptic membrane at extrasynaptic and,
to some degree, also at synaptic β2subunit-containing GABAAreceptors.
Only after diffusion or transport to the presynaptic membrane, 2-AG inhibits
the release of GABA upon activation of CB1receptors.
Hypothetical scheme showing how newly synthesized 2-AG may
Sigel et al. PNAS Early Edition
| 5 of 6
additionally underlie modulation by 2-AG (43), at least during
the late phase of the inhibitory postsynaptic current. Fig. 7
summarizes these possible actions of 2-AG. The fact that mod-
ulation of GABAAreceptors by 2-AG is exclusively observed at
low concentrations of GABA may be the reason why we were not
able to establish conditions to measure robust effects of 2-AG in
brain slice experiments.
In this study, we have identified the AAR V436 and F439 in
the inner membrane leaflet of the M4 helix of the β2subunit as
the molecular site of action of 2-AG. The discovery of a mod-
ulatory site for 2-AG on a specific set of GABAAreceptor sub-
types adds another level of complexity to endocannabinoid and
GABA action and provides important insight into their molecular
Materials and Methods
was 92% pure. The remaining 8% was determined as the spontaneous
breakdown product 1(3)-AG. 2-AG was prepared as a 10-mM stock solution
final DMSO concentration of 0.1%. 1-AG/3-AG, AEA, r-met AEA, oleamide, and
DEA were obtained from Cayman and were at least 95% pure. THC was
obtained from THC Pharm (>95% purity). THDOC was purchased from Fluka.
Electrophysiological Experiments. Preparation of RNA, isolation of Xenopus
oocytes, culturing of the oocytes, injection of cRNA, defolliculation, and
two-electrode voltage-clamp measurements were performed as described
earlier (44). 2-AG was preapplied for 30 s. Relative current potentiation by 2-
AG was determined as (I2-AG + GABA / IGABA−1) * 100%. Unless indicated
otherwise, potentiation by 2-AG was determined at all receptors at a similar
relative GABA concentration relative to the maximal current amplitude at
EC(2.1 ± 1.1)in the each receptor form. Concentration response curves were
fitted with the following equation:
PðcÞ ¼ max=ð1 þ ðc=EC50Þ˄nÞ;
where P is the current potentiation, c, the concentration of 2-AG, max is the
maximal current potentiation, the EC50is the concentration of 2-AG at which
half-maximal potentiation was observed, and n is the Hill coefficient. The
perfusion system was cleaned between two experiments by washing with
100% DMSO after application of 2-AG or THDOC to avoid contamination.
Behavioral Experiments and Quantification of 2-AG and AA. Methods of be-
havioral experiments and quantification of 2-AG and AA are provided in the
ACKNOWLEDGMENTS. We thank Dr. V. Niggli for carefully reading the
manuscript and B. P. Lüscher for frog surgery. This work was supported by
Swiss National Science Foundation Grants 31003A_132806/1 (to E.S.) and
31003A_120672 (to J.G.).
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| www.pnas.org/cgi/doi/10.1073/pnas.1113444108 Sigel et al.