Low-dose alcohol actions on ?4?3? GABAA
receptors are reversed by the behavioral
alcohol antagonist Ro15-4513
M. Wallner*†, H. J. Hanchar*, and R. W. Olsen†
Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA 90095-1735
Edited by Richard L. Huganir, Johns Hopkins University School of Medicine, Baltimore, MD, and approved April 7, 2006 (received for review January 9, 2006)
Although it is now more than two decades since it was first
reported that the imidazobenzodiazepine Ro15-4513 reverses be-
havioral alcohol effects, the molecular target(s) of Ro15-4513 and
the mechanism of alcohol antagonism remain elusive. Here, we
show that Ro15-4513 blocks the alcohol enhancement on recom-
binant ‘‘extrasynaptic’’ ?4?6?3? GABAAreceptors at doses that do
not reduce the GABA-induced Cl?current. At low ethanol concen-
trations (<30 mM), the Ro15-4513 antagonism is complete. How-
ever, at higher ethanol concentrations (>100 mM), there is a
Ro15-4513-insensitive ethanol enhancement that is abolished in
receptors containing a point mutation in the second transmem-
brane region of the ?3 subunit (?3N265M). Therefore, ?4?6?3?
GABA receptors have two distinct alcohol modulation sites: (i) a
low-dose ethanol site present in ?4?6?3? receptors that is antag-
onized by the behavioral alcohol antagonist Ro15-4513 and (ii) a
site activated at high (anesthetic) alcohol doses, defined by mu-
tations in membrane-spanning regions. Receptors composed of
?4?3N265M? subunits that lack the high-dose alcohol site show a
saturable ethanol dose-response curve with a half-maximal en-
hancement at 16 mM, close to the legal blood alcohol driving limit
in most U.S. states (17.4 mM). Like in behavioral experiments, the
alcohol antagonist effect of Ro15-4513 on recombinant ?4?3?
receptors is blocked by flumazenil and ?-carboline-ethyl ester
(?-CCE). Our findings suggest that ethanol?Ro15-4513-sensitive
GABAA receptors are important mediators of behavioral alcohol
alcohol intoxication ? alcohol receptor ? anesthetics
concentrations relevant for mild social intoxication are only
beginning to be revealed. Neurotransmitter receptors for
GABA, NMDA and glycine, and G protein-gated K?channels
have been identified as potential alcohol targets that are sensi-
tive to intoxicating alcohol concentrations (1–4). GABAAre-
ceptors (GABAARs) have long been suspected to be important
mediators of alcohol effects (5, 6) because benzodiazepines
(BZs) and barbiturates, classic GABAAR agonists, share com-
mon pharmacological properties with ethanol, such as sedative-
hypnotic, anti-anxiety, and motor in-coordinating and anticon-
vulsant effects and have additive, possibly even synergistic
effects, when taken together with ethanol (7). In addition, BZs,
barbiturates, and ethanol produce tolerance and cross-tolerance
to each other (8), consistent with GABAARs as targets of action.
We have recently identified subtypes of GABAARs, those
containing the ? and the ?3 subunit, that are uniquely sensitive
to low alcohol concentrations (9). Consistent with the view that
? subunit-containing receptors are important mediators of al-
cohol actions is the finding that GABAAR ?-subunit knockout
mice show multiple defects in behavioral responses to ethanol
(10). Receptors containing the ? subunit have an exclusively
nonsynaptic distribution, are sensitive to low ambient extrasyn-
aptic GABA concentrations, and display slow desensitization,
properties that enable them to mediate a persistent (or tonic)
lthough alcohol is one of the most widely used and abused
drugs, the molecular targets that mediate alcohol effects at
form of inhibition (11). GABAAR ? subunits seem to almost
exclusively associate with the GABAAR ?4 and ?6 subunits, two
closely related and somewhat specialized ? subunits that confer
insensitivity to classical BZ agonists (but not the imidazoben-
zodiazepines flumazenil and Ro15-4513), because they carry the
amino acid arginine (?6R100, ?4R100 instead of histidine
present in ?1, -2, -3, and -5) at a site critical for BZ binding (12).
We showed that the ?6-R100Q ‘‘BZ-site’’ mutation, previously
identified in alcohol-nontolerant rats (13), is sufficient for
behavioral alcohol hypersensitivity, confers alcohol supersensi-
tivity to tonic currents in rat cerebellar granule cells, and
dramatically increases the alcohol sensitivity of recombinantly
expressed receptors, if the ?6R100Q subunit is expressed to-
gether with the ?3 and the ? subunit (14). Therefore, an amino
acid residue important for BZ sensitivity is also critical for
low-dose alcohol sensitivity of ?6-containing GABAAR.
Two decades ago it was reported that the imidazobenzodiaz-
epine Ro15-4513, a structural analog of the clinically used
general BZ antagonist flumazenil, is an alcohol antagonist in
mammals (15, 16). The reversal of motor-impairing, sedative,
anxiolytic, amnestic, and rewarding alcohol actions by Ro15-
groups in different species (17–24). However, the debate
whether Ro15-4513’s alcohol antagonism is due to a specific
alcohol counteracting action, or due to nonspecific functional
antagonism, due to the weak inverse agonist activity of Ro15-
4513 on certain GABAAR subtypes (12), so far has not been
resolved (18, 19, 25–28). Three observations argue against a
nonspecific, inverse agonist action of Ro15-4513: (i) other
inverse agonists are not alcohol antagonists (17, 18); (ii)
GABAAR-mediated Cl?flux in synaptoneurosomes is enhanced
by ethanol (17, 29–31) and this enhancement can be blocked by
Ro15-4513 at concentrations that do not inhibit the GABA
current (17); and (iii) in experiments where Ro15-4513 leads to
a partial reversal of sedative-hypnotic alcohol actions, it did not
reduce the hypothermic actions (32). Hypothermic and analgesic
alcohol actions are at least partially mediated by G protein-gated
potassium channels (3, 4, 33, 34).
Here, we show that the enhancement of ? subunit-containing
recombinant GABAAR at low to moderately intoxicating etha-
nol doses (3–30 mM) is antagonized by the behavioral alcohol
antagonist Ro15-4513, suggesting that these subtypes of
GABAARs, previously thought to be completely insensitive to
BZs, have a high-affinity Ro15-4513-binding site. At anesthetic
Conflict of interest statement: M.W., R.W.O. and H.J.H. have filed a U.S. Provisional Patent
Application, Serial No. 60?693,844.
This paper was submitted directly (Track II) to the PNAS office.
methyl-6,7-dimethoxy-4-ethyl-?-carboline-3-carboxylate; BZ, benzodiazepine.
*M.W. and H.J.H. contributed equally to this work.
†To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or
© 2006 by The National Academy of Sciences of the USA
May 30, 2006 ?
vol. 103 ?
and potentially lethal alcohol concentrations (?100 mM), there
is an additional Ro15-4513-insensitive component of ethanol
receptors that contain a mutation in the ?3 subunit (?3N265M).
This mutation has been previously shown to essentially abolish
(in ?3N265M knockin mice) the in vivo anesthetic actions of
this residue in determining the ? subunit selectivity for enhance-
ment of recombinant GABAARs by loreclezole and etomidate
(36, 37). Homologous residues in GABAAR subunits have been
identified as important for high-dose alcohol and volatile anes-
thetic actions on recombinant GABAARs (38). As expected for
a ‘‘true’’ receptor, ?4?3N265M? GABAARs that lack the Ro15-
4513-insensitive high-dose ethanol enhancement now show a
alcohol antagonism on recombinant receptors can be abolished
by flumazenil. Most importantly, the fact that low-dose alcohol,
as well as the BZ alcohol antagonist Ro15-4513, can exert its
effects on common GABAAR subtypes identifies ethanol?Ro15-
4513-sensitive GABAA receptors as important mediators of
alcohol intoxication at low to moderate alcohol concentrations.
Low-Dose Ethanol Enhancement on ?4?6?3? GABAARs Is Antagonized
by Ro15-4513. Inspired by reports that Ro15-4513 antagonizes
most low-dose ethanol actions in animals (17, 19, 32) and that
100 nM Ro15-4513 blocks the low-dose (30 mM) alcohol
enhancement in GABA-induced36Cl?flux in brain homoge-
nate assays (17, 39), we decided to investigate the effect of the
BZ alcohol antagonist Ro15-4513 on the alcohol-induced
current enhancement in ?4?6?3? receptors expressed in oo-
cytes. Fig. 1 shows that 300 nM Ro15-4513 completely reversed
the ethanol enhancement of ?4?3? GABAARs for ethanol
concentrations up to 30 mM. This ‘‘anti-alcohol effect’’ of
Ro15-4513 is surprisingly specific because, at concentrations
where it abolished the alcohol-induced current increase (up to
300 nM), Ro15-4513 did not lead to a reduction in the ‘‘basal’’
GABA-induced current on these receptors; i.e., it is not an
inverse agonist in this assay at concentrations that inhibit the
ethanol-augmentation of GABA currents (Fig. 1c). The dose
dependence of this effect revealed that the concentration of
Ro15-4513 required to inhibit 50% of the 30 mM ethanol
enhancement (IC50) was ?10 nM (Fig. 1b). The alcohol
antagonist action of low-dose Ro15-4513 suggests that, against
common knowledge, ?4?6?3? GABAAR have a high-affinity
Ro15-4513-binding site, with a Kdof ?10 nM.
alcohol-induced enhancement was not blocked by 300 nM
Ro15-4513 (Fig. 1a). This high-dose ethanol enhancement was
not surmountable by increasing the Ro15-4513 concentrations
(data not shown). Therefore, ?4?3? GABAARs have a distinct
Ro15-4513-insensitive component of alcohol enhancement. To
demonstrate the dose dependence, we applied increasing con-
centrations of Ro15-4513 to currents evoked by 300 nM GABA
plus 10, 30, or 50 mM ethanol. Ro15-4513 led to a dose-
dependent block that was complete for the 10- and 30-mM dose
(with 300 nM Ro15-4513) (Fig. 1 b and c). Again, at the 50-mM
ethanol dose, a small fraction of the alcohol enhancement was
not blocked by Ro15-4513 (Fig. 1c). The complete and specific
antagonism of low-dose alcohol effects on these receptors by
Ro15-4513 suggests the intriguing possibility that Ro15-4513
might work by competitively displacing EtOH from its binding
Antagonizing Ro15-4513’s Alcohol Antagonism by Flumazenil and
?-Carboline-Ethyl Ester (?-CCE). Certain BZ site ligands, like the
general BZ antagonist flumazenil (Ro15-1788) and the struc-
turally unrelated BZ-site ligands ?-CCE and FG7142, were
shown to prevent the alcohol antagonist effects of Ro15-4513 in
behavioral assays (17, 39). We reasoned that this result could be
due to displacement of Ro15-4513 from its binding site by these
compounds, which do not show alcohol antagonism by them-
selves (17, 39). We therefore tested four selected BZ site ligands
for their ability to reverse or mimic Ro15-4513 antagonism of
(expressed in oocytes) in the presence of 300 nM (?EC20)
GABA (to mimic tonic GABA current), 30 mM ethanol (to
increase the GABA current), and 100 nM Ro15-4513 (to reverse
the enhancement by 30 mM ethanol). Flumazenil (Ro15-1788)
and ?-CCE at 300 nM reversed the Ro15-4513-induced alcohol
antagonism. However, the classical BZ agonist flunitrazepam, as
well as DMCM (methyl-6,7-dimethoxy-4-ethyl-?-carboline-3-
carboxylate) (a ?-carboline with pronounced inverse agonist
efficacy on ?2 subunit-containing receptors), each at 1 ?M, did
not reverse the effects of Ro15-4513 (Fig. 2a), indicating that the
Ro15-4513?BZ-binding site on ? subunit-containing receptors is
unique and binds only certain BZs and BZ site ligands with high
affinity. None of the four compounds tested blocked ethanol
enhancement on their own (data not shown). These data are
consistent with previous findings that Ro15-4513’s alcohol an-
tagonism can be antagonized by certain BZ-site ligands in36Cl?
ceptor currents. (a and b) To mimic tonic GABA current, 300 nM GABA was
at ?80 mV, and currents were measured with a two-electrode voltage clamp
(300 nM) completely antagonized ethanol enhancement up to an ethanol
concentration of 30 mM. (b) Cumulative Ro15-4513 dose-response curve
were blocked by 30 ?M bicuculline. (c) GABA peak currents with and without
ethanol and the indicated concentrations of Ro15-4513. Ro15-4513 led to a
dose-dependent inhibition of (10, 30, and 50 mM) ethanol enhancement on
basal current on ?4?3? receptors (evoked by 300 nM GABA). (a and b)
Representative recordings of six and five experiments, respectively.
Ro15-4513 antagonizes ethanol effects on recombinant ?4?3? re-
Wallner et al.PNAS ?
May 30, 2006 ?
vol. 103 ?
no. 22 ?
flux assays in synaptoneurosomes and provide an in vitro cor-
relate to the behavioral data that show that flumazenil and
?-CCE can reverse the alcohol antagonist effects of Ro15-4513
(17, 39). A comparison of the structures of Ro15-4513 and
flumazenil shows that these two molecules are identical, except
for one moiety, which is an azido group in Ro15-4513 and a
fluorine in flumazenil (Fig. 2b).
?-CCE Is a Positive GABA Modulator on ?4?3? Receptors. We con-
sistently observed that ?-CCE led to an ‘‘overshoot’’ when we
used it to antagonize the alcohol antagonist effects of Ro15-
4513 on GABA?ethanol-induced currents (see Fig. 2), sug-
gesting that ?-CCE might potentiate alcohol effects on ?4?
6?3? GABAAR. We therefore tested ?-CCE alone and in
combination with 3 mM ethanol for their functional effects on
?4?3? receptors. Fig. 3a shows that ?-CCE not only enhances
alcohol actions, but also increases the activity of ?4?3?
GABAARs in the absence of alcohol. In the same way as
alcohol effects on ?4?3? GABAAR, the ?-CCE-induced en-
hancement of GABA currents is reversed by Ro15-4513 (Fig.
3b). A likely explanation for these findings is that ?-CCE, as
well as Ro15-4513, occupies an overlapping and mutually
exclusive binding site, whereas ?-CCE and ethanol might be
able to bind next to each other in a side-by-side binding pocket,
both microdomains blocked by Ro15-4513 (see Fig. 2b).
Loss of Ro15-4513-Insensitive Ethanol Actions in ?4?3N265M?
GABAAR. The Ro15-4513-insensitive component of ethanol en-
hancement is observed at high alcohol concentrations (?30 mM),
where most recombinant and native GABAAR show ethanol
enhancement that is likely due to alcohol sites determined by
mutations in the second and third transmembrane region of
GABAARs (38). We show here (Fig. 4) that ?4?3N265M? recep-
tors, where the ?3 WT subunit is replaced with the mutated
ment. However, the ?3N265M mutation completely abolished the
Ro15-4513-insensitive ethanol enhancement observed at 100 and
300 mM ethanol (Fig. 4a), and even at 1 M ethanol (data not
show identical ethanol enhancement at alcohol concentrations up
to 30 mM and differ only at the 100- and 300-mM dose (Fig. 4b).
As a consequence of this loss of Ro15-4513-insensitive, high-
concentration alcohol effects, recombinant ?4?3N265M? recep-
tors now have a saturable ethanol response curve with a half-
maximal response of 16 mM [at EC20(300 nM) GABA, Fig. 2b], a
concentration close to the legal blood alcohol limit (17 mM) for
driving in most US states.
Extrasynaptic ? Subunit-Containing Receptors as Targets for Ethanol
Action. GABAARs containing the ? subunit have been shown
to have an extra- or perisynaptic localization (40, 41) and give
rise to tonic (sustained) GABA currents in neurons that
express these GABAAR subunits (11). Recombinant
GABAARs (?4?6??) known to mediate tonic currents, as well
as ? subunit-containing receptor-mediated tonic currents in
neurons, are augmented by low alcohol concentrations
reached during social alcohol consumption (9, 14, 42–44).
Whereas ? subunit-containing GABAARs comprise only be-
tween 5 and 10% of all of GABAARs in the mammalian brain
(45), their constant or tonic activity (in marked contrast to
synaptic receptors that open only for fractions of a second after
synaptic GABA release) more than compensates in total
charge transfer for the low abundance (46), and, therefore,
extrasynaptic receptors are important regulators of neuronal
excitability. The alcohol-induced augmentation of tonic cur-
GABA and potentiated by 30 mM ethanol, and this potentiation was reversed
by 100 nM Ro15-4513. In constant presence of 300 nM GABA, 30 mM ethanol,
and 100 nM Ro15-4513, the BZ site ligands Ro15-1788 (300 nM), ?-CCE (300
whether they reverse Ro15-4513’s ethanol antagonist effects. At the end of
Shown is a representative recording of a total of three experiments. (b)
Chemical structures of the imidazobenzodiazepines Ro15-4513 and Ro15-
1788 show that they differ only at one single position in the molecule. The
clinically used BZ antagonist flumazenil (Ro15-1788) carries a fluorine at the
C7 position of the BZ ring, whereas Ro15-4513 carries the larger azido group.
Ro15-4513 alcohol antagonism is antagonized by flumazenil and
together with ethanol (always in the presence of 300 nM GABA) to oocytes
expressing ?4?3? receptors, and peak GABA?Cl?currents were measured. (b)
www.pnas.org?cgi?doi?10.1073?pnas.0600194103Wallner et al.
rents in neurons and ? subunit-containing GABAARs at
relevant physiologic alcohol doses is expected to lead to a
decrease in neuronal excitability in neurons expressing these
subunits and make these receptors excellent candidates for
mediating acute alcohol effects at intoxicating concentrations.
Our demonstration that the behavioral alcohol antagonist
Ro15-4513 leads to a dose-dependent inhibition of ethanol-
induced current enhancement in recombinant ?4?3?
GABAARs provides strong support for this notion.
?4?3? GABAAR Are Sensitive to Certain BZs and BZ-Site Ligands. The
discovery that the BZ Ro15-4513 reduces alcohol actions on
?4?6?3? receptors is unexpected because it has been thought that
BZ-site ligands (47). In recombinantly expressed functional recep-
tors, ? subunit-containing receptors have been shown to be insen-
sitive to classical BZ agonists like diazepam and flunitrazepam (48,
49). Here, we reveal the activity of Ro15-4513 on ?4?6?3? recep-
tors by their ability to act as an alcohol antagonist (Figs. 1, 2, and
4) whereas the binding of flumazenil and the BZ site ligand ?-CCE
is inferred from their ability to reverse Ro15-4513’s alcohol antag-
onism (Fig. 2a). Consistent with the view that ?4?6?3? GABAAR
are insensitive to classical BZ agonists, we show that flunitrazepam
does not antagonize the alcohol antagonistic actions of Ro15-4513
on recombinant ?4?3? receptors (Fig. 2).
Ethanol?Ro15-4531-Sensitive GABAAR in36Cl?Flux Assays Show Strik-
ing Similarities with Recombinantly Expressed ? Subunit-Containing
GABAAR. Our data on the Ro15-4513 reversal are in agreement
with previous work that showed that Ro15-4513 can abolish
ethanol augmentation of Cl?flux in cerebral cortex synapto-
neurosomal preparations and that the Ro15-4513’s alcohol
antagonism is reversed by flumazenil and ?-CCE (17, 39).
Several lines of evidence support the view that the ethanol-
sensitive Cl?flux through GABAAR in synaptosomal prepara-
tions may be mediated by alcohol-sensitive extrasynaptic recep-
tors: (i) like ? subunit-containing receptors, this Cl?flux seems
highly sensitive to GABA and muscimol (17, 29), and the ability
to carry sustained
desensitization; (ii) both ? subunit-containing receptors, as well
as the36Cl?flux, are strikingly similar in their low-dose response
to physiologically relevant (3–30 mM) ethanol concentrations;
(iii) we show here with recombinant receptors that, as previously
observed in the36Cl?flux assays (17), 30 mM ethanol augmen-
36Cl?flux suggests that they show slow
Why Are GABAAR as Alcohol Targets Controversial?Ourfindingsalso
provide an explanation for the controversial findings concerning
the alcohol sensitivity of GABAARs in native neurons. Whereas
most synaptic physiologists failed to find evidence for low-dose
alcohol effects on GABAergic synaptic transmission (42, 50),
there is abundant evidence that many neurons can respond to
physiological (3–30 mM) ethanol concentrations at conditions
that favor the detection of highly GABA-sensitive nondesensi-
tizing extrasynaptic GABAARs (6, 51). Such conditions are, for
example, the prolonged application of relatively low GABA and
ethanol concentrations, like those resulting from local applica-
tion of GABA?ethanol in the vicinity of neurons, conditions that
synaptic physiologists might have considered nonphysiological.
Consistent with this view and the results presented here, it has
been shown that such low-dose alcohol enhancement of GABA
currents in neurons (51) and the (presumably) resulting changes
in firing frequency (52) can be reversed by the alcohol antagonist
Ro15-4513 (51, 52). The fact that not all neurons express these
alcohol?Ro15-4513-sensitive ? subunit-containing GABAARs is
consistent with the observation that not all neurons are sensitive
to pharmacologically relevant ethanol concentrations (6, 42, 51).
Another complicating factor is that electrophysiological studies
are often performed on neurons and slices harvested from
immature brains that do not yet express ? subunit-containing
extrasynaptic GABAAR (53). Consistent with a slow onset of
expression of ? subunit-containing receptors during develop-
ment and adolescence in rodents, reaching mature levels at
around sexual maturation (54, 55), is the finding that younger
animals are less sensitive to the sedative and motor-impairing
effects of ethanol (56).
Whereas our experimental data explain and extend ?20 yr of
often puzzling observations concerning ethanol actions on
GABAARs, a recent report that fails to reproduce low-dose
ethanol enhancement on ?4?3? receptors, in particular negative
results from the attempted expression of human ?4?3? receptors
in oocytes and mammalian cell lines (57), likely ensures that our
findings and related work (e.g., refs. 14, 17, 29, and 39) will
in the apparent lack of alcohol sensitivity, but also in the peak
currents published by Borghese et al. (57), which are five times
expressed in oocytes reported by Borghese et al. (57) are
marginal, indicating a possible lack of expression of one or more
of their human clones.
In our hands, variable ethanol responses in ?4?3? are likely
due to the tendency of oocytes to express receptors that are
composed of ?? subunits alone. GABAARs composed of ??
alone are insensitive to relevant alcohol concentrations and to
classical BZs but show high sensitivity to etomidate, propofol,
and steroid anesthetics (58, 59). The phenomenon of ??
high alcohol concentrations. (a) A single point mutation (?3N265M in mem-
brane-spanning segment TM2 of the ?3 subunit) abolishes the Ro15-4513-
resistant alcohol enhancement observed at high ethanol concentrations in
coapplications of ethanol and GABA EC20 (300 nM for ?4?3? and
?4?3N265M?, and 30 ?M GABA for ?4?3?2 GABAAR). Currents through
?4?3N265M? GABAAR show a saturable alcohol enhancement and an EC50of
around 16 mM. The complete reversal of even very high dose alcohol effects
is not expected from an ideal competitive relationship of ligands with appar-
ent dissociation constants (10 nM for Ro15-4513 and 16 mM for ethanol). The
on functional receptors remain to be clarified.
A point mutation eliminates Ro15-4513-insensitive alcohol effects at
Wallner et al.PNAS ?
May 30, 2006 ?
vol. 103 ?
no. 22 ?
GABAAR expression in oocytes has been well documented for
?2 subunit-containing receptors and explains the observed vari-
ability in BZ effects in ?1?2?2 receptors expressed in oocytes
(60). The expression of ?? receptors in oocytes, highly sensitive
to GABAAR anesthetics, is also the likely explanation for a
previous challenge (58) to the finding that ? subunit-containing
GABAARs lack anesthetic enhancement (59).
Behavioral Alcohol Effects Antagonized by Ro15-4513. The alcohol
antagonist Ro15-4513 has been reported to prevent many acute
alcohol effects. These effects include increased exploration and
locomotion at very low doses (0.25, 0.5, and 0.75 g?kg in rats)
(61), the anxiolytic effects at low doses (1 g?kg) (17, 39), and
sedative, motor-impairing as well as the amnestic effects at
moderate alcohol doses (2 g?kg) (17, 19, 21, 62). In addition, the
observation that Ro15-4513 reduces alcohol self-administration
(23, 24, 63) suggests that the rewarding effects of ethanol might
be mediated by ethanol?Ro15-4513-sensitive GABAARs. How-
ever, Ro15-4513 does not prevent all ethanol effects: at higher
alcohol doses (?2 g?kg in rats), Ro15-4513 significantly reduces,
but does not prevent the anesthetic (‘‘sleep’’-inducing) effects of
ethanol, and Ro15-4513 does not prevent the hypothermic
effects of ethanol (32, 64). In addition, Ro15-4513 does not
prevent lethal effects at massive alcohol doses (20, 27). This
finding suggests that, at high alcohol doses, Ro15-4513-
insensitive ethanol targets mediate the anesthetic ethanol ef-
fects. Our data on recombinant ?4?3? GABAARs suggest the
possibility that, at such high alcohol concentrations (blood
alcohol levels ?30 mM), Ro15-4513-sensitive recombinant ?4?
6?3? receptors have a high ethanol (?80%) occupancy and are
therefore close to saturated.
Established targets of Ro15-4513-insensitive alcohol actions
are alcohol-sensitive G protein-gated (GIRK) potassium chan-
nels (33) that likely contribute to the analgesic (4) and hypo-
thermic effects of ethanol (34). Other potential alcohol targets
that might contribute to Ro15-4513-insensitive acute alcohol
and voltage- and Ca2?-activated (BK) potassium channels (66,
67). In addition, we show here that the Ro15-4513-insensitive
alcohol action on ?4?3? GABAAR is abolished by a mutation
(?3N265M) in the ?3 subunit. It is therefore possible that
GABAARs (including the abundant synaptic ?2-containing
GABAARs) may contribute to high-dose (?30 mM) Ro15-4513-
resistant, anesthetic ethanol actions.
Potential Mechanisms of Alcohol Antagonism by Ro15-4513. A com-
parison of the structure of the alcohol and BZ antagonist
Ro15-4513 and flumazenil, which differ only at a single moiety,
suggests a possible mechanism of alcohol antagonism. The
larger azido group (at the C7 position of the BZ ring) might
be the group that occupies the alcohol-binding site on the
receptor. Flumazenil and the ?-carbolines ?-CCE and FG7142
likely antagonize Ro15-4513 alcohol antagonist actions by
displacing Ro15-4513 from its binding site. Flumazenil,
?-CCE, and FG7142 do not act as alcohol antagonists by
themselves, because they might fit together with ethanol in the
Ro15-4513-binding pocket. Therefore, we think that the most
parsimonious explanation for Ro15-4513’s alcohol antagonism
is that the unique azido group in Ro15-4513 occupies the
alcohol-binding site. However, there could be other possible
mechanisms: e.g., the azido group in Ro15-4513 may cause
allosteric changes in these receptors. We have used native
immunopurified and recombinant expressed ? subunit-
containing receptors in [3H]Ro15-4513-binding assays to show
further evidence that Ro15-4513 and ethanol have a compet-
itive relationship on ?4?6?? receptors (68).
Alcohol Receptors as Potential Drug Targets.Ourfindingthatalcohol
effects on ?4?3? receptors can be reversed by the BZ Ro15-4513
ligand ?-CCE suggests that these receptors likely contain a modi-
abundant but functionally important GABAAR subtypes, provide
opportunity for new drug development. For example, it might be
agonist effects of Ro15-4513 on certain GABA receptor subtypes.
half-life (?30 min) in vivo. Furthermore, the alcohol antagonist
Ro15-4513 and the clinically used general BZ antagonist flumaze-
nil are highly hydrophobic with likely poor bioavailability when
applied orally or i.p. (flumazenil is administered intravenously in
the clinic as a BZ antidote). Given the high incidence of ethanol
intoxication cases in emergency rooms and the short half-life of
flumazenil, leading to resedation in cases where it is used to
antagonize much longer acting BZs, the identification of ?4?6?3?
GABAARs as ethanol?Ro15-4513 targets may spur the develop-
ment of combined BZ?alcohol antagonists with longer half-life.
Such combined antagonists, without inverse agonist activity and
better solubility, not only might be useful in the clinic, but also may
GABAAR subtypes to certain aspects of acute ethanol actions.
We show that the BZ-site ligand ?-CCE, a ?-carboline, is an
agonist on ?4?3? receptors and like EtOH allosterically en-
hances the GABA response without directly activating these
receptors. The activation of ?4?3? GABAARs by ?-CCE pro-
vides proof of principle that it might be possible to develop
specific alcohol receptor agonists. Ideally, such positive modu-
lators (alcohol mimetics) should be specific for the EtOH?Ro15-
4513 site and lack activity (like the inverse agonist activity of
?-CCE) on classical GABAAR ?1, -2, -3, and -5?2 BZ sites. Such
specific alcohol receptor agonists could be useful to harness
ethanol receptors for therapeutic purposes by mimicking the
anxiolytic, anti-depressive, and mood-enhancing actions of al-
cohol, without the undesired effects like liver toxicity and other
toxic effects of metabolites like acetaldehyde.
Materials and Methods
Electrophysiology. Clones used were as described previously and
were confirmed by sequencing to ensure that they were free of
errors and agreed with the consensus sequences for rat ?4, ?6, ?3,
transcribed after plasmid linearization by using the mMessage
mMachine kit (Ambion, Austin, TX). Transcripts were purified by
LiCl precipitation, and RNA concentration was determined on a
gel and by photometry. Oocytes were coinjected with a mixture of
Currents were measured at room temperature (22°C–24°C) in the
two-electrode voltage clamp configuration at ?80 mV holding
potential with an Axoclamp 2A Axon Instruments (Union City,
CA) amplifier. Two electrode voltage clamp on Xenopus oocytes
mM CaCl2?1 mM MgCl2?5 mM Hepes, pH 7.2). Because of the
slow onset in the expression of highly alcohol-sensitive ? subunit-
containing receptors, oocytes were measured 7–14 days after
injection. Currents were measured either in a tonic current mim-
s) coapplications of 300 nM GABA with drugs (ethanol, BZ-site
ligands), to evoke peak currents, followed by a recovery time of at
least 1 min.
Reagents. Hoffman-LaRoche (Nutley, NJ) kindly provided
Ro15-4513, flumazenil or Ro15-1788 (ethyl 8-fluoro-5,6-
3-carboxylate), diazepam, and flunitrazepam. DMCM was a gift
www.pnas.org?cgi?doi?10.1073?pnas.0600194103Wallner et al.
from Ferrosan (Copenhagen), and FG7142 (N-methyl-?- Download full-text
carboline-3-carboxamide) and ?-CCE were provided by Scher-
ing. Ethanol, GABA, and bicuculline were purchased from
Sigma. Compounds were dissolved in DMSO as a 10-mM stock
solution and were used at the indicated concentrations. DMSO
at final concentrations used did not lead to changes in GABA
receptor currents (data not shown).
We thank Dr. C. Gundersen [University of California, Los Angeles
(UCLA)] and the UCLA Anesthesiology Department for providing
Xenopus oocytes and Qui Vu (UCLA) for help with oocyte injections.
This work was supported by National Institutes of Health (NIH)
Predoctoral Fellowship AA015460 (to H.J.H.), an Alcoholic Beverage
Medical Research Foundation grant (to M.W.), NIH grants NS35985
and AA07680, and funds provided by the State of California for medical
research on alcohol and substance abuse (to R.W.O.).
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