Differential potency of 2,6-dimethylcyclohexanol isomers for positive modulation of GABAA receptor currents

Article abstract of DOI:10.1124/jpet.115.228890

GABA_A receptors meet all of the pharmacological requirements necessary to be considered important targets for the action of general anesthetic agents in the mammalian brain. In the following patch-clamp study, the relative modulatory effects of

Full text of DOI:10.1124/jpet.115.228890

1521-0103/357/3/570–579$25.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics
http://dx.doi.org/10.1124/jpet.115.228890
J Pharmacol Exp Ther 357:570–579, June 2016
Differential Potency of 2,6-Dimethylcyclohexanol Isomers for
Positive Modulation of GABAA Receptor Currents
Luvana Chowdhury, Celine J. Croft, Shikha Goel, Naina Zaman, Angela C.-S. Tai,
Erin M. Walch, Kelly Smith, Alexandra Page, Kevin M. Shea, C. Dennis Hall,
D. Jishkariani, Girinath G. Pillai, and Adam C. Hall
Neuroscience Program, Departments of Biological Sciences (L.C., C.J.C., S.G., N.Z., A.C.-S.T., E.M.W., A.C.H.) and Chemistry
(K.S., A.P., K.M.S.), Smith College, Northampton, Massachusetts; Department of Chemistry, University of Florida, Gainesville,
Florida (C.D.H., D.J., G.G.P.); and Department of Chemistry, University of Tartu, Ravila, Estonia (G.G.P.)
Received September 9, 2015; accepted March 22, 2016
ABSTRACT
GABAA receptors meet all of the pharmacological requirements isomers . . cis,trans-isomer. In molecular modeling studies, the
necessary to be considered important targets for the action of three cyclohexanol isomers bound with the highest binding
general anesthetic agents in the mammalian brain. In the energies to a pocket within transmembrane helices M1 and M2
following patch-clamp study, the relative modulatory effects of of the b3 subunit through hydrogen-bonding interactions with a
2,6-dimethylcyclohexanol diastereomers were investigated on glutamine at the 224 position and a tyrosine at the 220 position.
human GABAA (a1b3g2s) receptor currents stably expressed in The energies for binding to and hydrogen-bond lengths within
human embryonic kidney cells. Cis,cis-, trans,trans-, and cis, this pocket corresponded with the relative potencies of the
trans-isomers were isolated from commercially available 2,6- agents for positive modulation of GABAA receptor currents
dimethylcyclohexanol and were tested for positive modulation (cis,cis . trans,trans . cis,trans-2,6-dimethylcyclohexanol).
of submaximal GABA responses. For example, the addition of In conclusion, the stereochemical configuration within the
30 mM cis,cis-isomer resulted in an approximately 2- to 3-fold dimethylcyclohexanols is an important molecular feature in
enhancement of the EC20 GABA current. Coapplications of conferring positive modulation of GABAA receptor activity and
30 mM 2,6-dimethylcyclohexanol isomers produced a range of for binding to the receptor, a consideration that needs to be
positive enhancements of control GABA responses with a rank taken into account when designing novel anesthetics with en-
order for positive modulation: cis,cis . trans,trans $ mixture of hanced therapeutic indices.
of the action of anesthetics at GABAA receptors have revealed
Introduction
that for select agents, the potentiation of GABA currents
Intravenous sedatives and general anesthetics are some of
correlates with anesthetic potency in vivo (Krasowski et al.,
the most common therapeutic agents used during surgery.
2001; Watt et al., 2008; Hall et al., 2011).
Several of these agents (e.g., propofol and etomidate) are
Given the interest in developing less toxic sedatives and
postulated to sedate patients and render them unconscious
anesthetics, several studies have explored the structure-
through positive modulation of GABAA receptor currents in
activity relationship for agents that enhance GABA-evoked
the central nervous system (Franks and Lieb, 1994; Krasowski
currents and provide anesthesia (e.g., Krasowski et al., 2001;
and Harrison, 1999; Olsen and Li, 2011). GABAA receptors are
Pejo et al., 2014). Previously we demonstrated the potential
membrane-spanning chloride-selective ion channel complexes
for cyclohexanols to act as positive modulators of GABAA
activated through the binding of GABA (Barnard et al., 1998)
receptor currents and as general anesthetics (Hall et al., 2004;
and they are the predominant ionotropic receptor type for
Watt et al., 2008). The structure-activity relationship for a
fast inhibitory neurotransmission in the mammalian central
range of cyclohexanol analogs was further explored; among
nervous system. Their pentameric structure is composed of
those tested, 2,6-dimethylcyclohexanol was determined to be
different subunits (a1–6, b1–4, g1–3, d, «, p, and u) with the
the most potent for both receptor modulation and as a general
anesthetic (Hall et al., 2011).
Stereoselectivity for positive modulation of GABAA recep-
tor currents is not unprecedented, particularly in regard
predominant GABAA receptor combination of a1b2g2 in mam-
malian neurons (McKernan and Whiting, 1996). Investigations
to enantiomers of general anesthetics (e.g., Hall et al., 1994;
Tomlin et al., 1998). Likewise, cyclohexanol-based compounds
(e.g., menthol) have also been shown to exhibit stereoselectivity
of action for these receptors (Corvalán et al., 2009). In the
This work was supported by the Howard Hughes Medical Institute [Grant
52007557 (to C.J.C. and S.G.)], a Smith College Tomlinson Award [(to L.C.)],
the Blakeslee Foundation [(to A.C.H.)], and the European Social Fund [Project
1.2.0401.09-0079, University of Tartu (to G.G.P.)].
dx.doi.org/10.1124/jpet.115.228890.
ABBREVIATIONS: GLIC, gloeobacter ligand-gated ion channel; HEK, human embryonic kidney; NMR, nuclear magnetic resonance; PDB, Protein
Data Bank; TLC, thin-layer chromatography.
570

Modulation of GABAA Currents by 2,6-Dimethylcyclohexanols
571
were maneuvered onto cells to form “gigaohm” seals using a microma-
nipulator (MP-225; Sutter Instrument Company). Junction potentials
were zeroed in the chamber prior to all recordings, the liquid junction
potential was negligible (approximately 2 mV), and cells were routinely
voltage clamped at 250 mV.
following study, we used WSS-1 cells to investigate modulation
of wild-type GABAA receptors (a1b3g2s) by cis,cis-, trans,trans-,
and cis,trans-diastereomers of 2,6-dimethylcyclohexanol
(Fig. 1). In the most stable of the two possible chair confor-
mations, cis,cis-dimethylcyclohexanol has both methyl groups
equatorial and the hydroxyl group axial. The cis,trans-isomer
has the hydroxyl group and one methyl substituent equatorial,
whereas the other methyl is axial. In the trans,trans-
isomer, the hydroxyl group and both methyl groups are all
equatorial, making it the most stable configuration and closest
to planarity. Molecular modeling studies were carried out for
the cis,cis-, trans,trans-, and cis,trans-diastereomers of 2,6-
dimethylcyclohexanol against the b3 subunit of the human
GABAA receptor. The results indicate that stereochemical
configuration within the dimethylcyclohexanols is an impor-
tant molecular feature in conferring positive modulation of
GABAA receptor activity and for binding to the receptor.
Drugs were superfused onto cells using a motor-driven exchange
device (Rapid Solution Changer, RSC-100; Bio-Logic Science Instru-
ments, Claix, France) controlled via Clampex 10 acquisition software
(Molecular Devices/Axon Instruments, Sunnyvale, CA). Flow of
extracellular solutions onto cells was driven by a multichannel
infusion pump (KD Scientific, Holliston, MA). Currents evoked by
GABA with and without the modulators in extracellular solution were
amplified via an Axopatch 200A (Molecular Devices/Axon Instru-
ments), filtered at 1 kHz via a low-pass Bessel filter (Frequency
Devices, Ottawa, IL), and digitized using a Digidata 1440 (Molecular
Devices/Axon Instruments). All currents were measured using
Clampex 10 (Molecular Devices/Axon Instruments) and were further
analyzed using Origin software (OriginLab Corp., Northampton,
MA). Data are expressed as the mean 6 S.E.M. calculated from at
least five individual cells for each data point reported (unless stated
otherwise). Positive modulation of GABA-induced currents was de-
fined as the percentage increase of the control GABA response
(average of pre- and postdrug). Concentration-response data were
fitted with the Hill equation (eq. 1) using Origin software (OriginLab
Corp.):
Materials and Methods
Cell Culture
WSS-1 cells (CRL-2029; American Type Culture Collection, Mana-
ssas, VA) were used for all electrophysiology experiments. WSS-1 cells
are human embryonic kidney (HEK) cells that have been stably
transfected with cDNAs encoding for the rat a1 and g2s subunits of
the GABAA receptor along with expression of an endogenous human
b3 subunit (Wong et al., 1992; Davies et al., 2000) and thus are a
convenient cell line for generating GABA-evoked currents consis-
tently. WSS-1 cells were grown in standard media (90% Dulbecco’s
modified Eagle’s medium and 10% fetal bovine serum, with 100 U/ml
penicillin and 100 mg/ml streptomycin) including 500 mg/ml geneticin
(G-418) to select for cells expressing GABAA receptors (Wong et al.,
1992). Cells were maintained in culture flasks in a humidified
incubator with 5% CO2/95% air at 37°C and passaged on a weekly
basis. During passaging, cells were either plated on poly(L-lysine)
(Trevigen, Gaithersburg, MD)–coated glass coverslips for electrophys-
iological recording or were used to reseed another flask. Cells were
used for up to 30 passages after purchase from ATCC. All culturing
reagents were purchased from Life Technologies (Carlsbad, CA)
unless stated otherwise.
.ꢀ
ꢁ
nH
nH
I 5 Imax: ½agonistꢀ
½agonistꢀnH 1 ½EC50
ꢀ
(1)
where I is the agonist-evoked current at a given concentration, Imax
is the peak current at saturating [agonist], EC50 is the concentration
of agonist that elicits a half-maximal response, and nH is the Hill
coefficient.
Drugs and Reagents
All reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless
stated otherwise. During experiments, GABA and the cyclohexanol
isomers were co-applied to assess the level of current modulation.
Stock solutions (10–100 mM) of the 2,6-dimethylcyclohexanol isomers
in dimethylsulfoxide were diluted daily to the required concentrations
in extracellular medium with dimethylsulfoxide concentrations in
final solutions never exceeding 0.1% (a concentration that had no
effect on GABA-activated currents).
Electrophysiology
Isolation of 2,6-Dimethylcyclohexanol Isomers
Electrophysiological recordings were performed using a standard
2,6-Dimethylcyclohexanol from Acros (Geel, Belgium) was a mixture
whole-cell patch-clamp technique at room temperature. Coverslips were of 45% cis,cis-isomers, 32% trans,trans-isomers, and 23% cis,trans-
transferred to a recording chamber that was continuously superfused isomers (Fig. 1). Thin-layer chromatography (TLC) was performed as
at 3 ml/min with extracellular recording medium containing 140 mM follows: eluant, 10% diethyl ether in petroleum ether; Rf 5 0.1, a
NaCl, 5 mM KCl, 1.2 mM MgCl2, 2.5 mM CaCl2, 11 mM glucose, and mixture of trans,trans- and cis,trans-diastereomers; Rf 5 0.2, cis,cis-
5 mM HEPES (pH 7.4 with NaOH). The electrode solution contained diastereomer; both spots were visualized with vanillin.
140 mM KCl, 2 mM MgCl2, 11 mM EGTA, 0.1 mM Mg21-ATP, and
2,6-Dimethylcyclohexanone was obtained from Sigma-Aldrich as a
10 mM HEPES (pH 7.4 with KOH). Pipettes, fabricated using a mixture of 80% cis-isomers and 20% trans-isomers. TLC was performed
Flaming/Brown micropipette puller (Sutter Instrument Company, as follows: eluant, 5% diethyl ether in petroleum ether; Rf 5 0.23
Novato, CA), typically had resistances in the range of 2–4 MV. Pipettes (trans), 0.42 (cis), visualized with KMnO4.
Fig. 1. Structures of 2,6-dimethylcyclohexanol diastereomers and enantiomers.

572
Chowdhury et al.
2,6-Dimethylcyclohexanone (1.50 g, 11.9 mmol) was applied to a
column of silica gel (150 g) and eluted with 5% ethyl ether in petroleum
anhydrous solution of lithium aluminum hydride in tetrahydrofuran
(1.10 ml, 1.10 mmol). A solution of trans-2,6-dimethyl cyclohexanone
ether to yield cis-2,6-dimethylcyclohexanone (1.01 g, 8.00 mmol), a (128.5 mg, 1.01 mmol) in anhydrous tetrahydrofuran (1 ml) was
mixture of both isomers consisting of 20% cis-2,6-dimethylcyclohex- transferred by cannula from a pear-shaped flask and the mixture was
anone and 80% trans-2,6-dimethylcyclohexanone (115 mg, 0.910 stirred at room temperature for 1 hour.
mmol), and trans-2,6-dimethylcyclohexanone (72.1 mg, 0.570 mmol).
For cis-2,6-dimethylcyclohexanone, TLC was as follows: eluant, 5%
The reaction was quenched with a few drops of water and then
treated with 9% NaOH solution added dropwise. The mixture was
diethyl ether in petroleum ether; and Rf 5 0.42, visualized with filtered through silica gel under a vacuum, rinsed with diethyl ether
KMnO4. 1H nuclear magnetic resonance (NMR) (300 MHz, CDCl3) was (approximately 15 ml), and the filtrate was transferred to a 50-ml
as follows: 1.0 (d, 6 H), 1.2–1.4 (m, 2 H), 1.7–1.9 (m, 2 H), 2.0–2.2 (m, 2 H), separatory funnel. The aqueous layer was extracted with ethyl ether
and 2.3–2.5 (m, 2 H)
(3 ꢁ 10 ml). and the ether layers were combined and dried over MgSO4.
For trans-2,6-dimethylcyclohexanone, TLC was as follows: eluant, 1H NMR on the crude product (42.9 mg, 33%) revealed a mixture
5% diethyl ether in petroleum ether; and Rf 5 0.23, visualized with of cis,trans-2,6-dimethylcyclohexanol (98%) and trans,trans-2,6-
KMnO4. 1H NMR (300 MHz, CDCl3) was as follows: 1.1 (d, 6 H), 1.5–1.6 dimethylcyclohexanol (2%).
(m, 2 H), 1.7–1.8 (s, 2 H), 1.9–2.0 (m, 2 H), and 2.5–2.6 (m, 2 H).
Reduction of cis-2,6-Dimethylcyclohexanone with Lithium
Aluminum Hydride.
cis,trans- and trans,cis-2,6-Dimethylcyclohexanol (Enantio-
mers 3 and 4). TLC was as follows: Rf 5 0.1, eluant 10% diethyl ether
in petroleum ether, visualized with vanillin. 1H NMR (300 MHz,
CDCl3) was as follows: 0.97 (t, 6 H), 1.36–1.55 (m, 6 H), 1.63–1.80
(m, 2 H), 1.90–2.04 (m, 1 H), and 3.28–3.35 (m, 1 H)
A sample of the cis,cis-isomer was also isolated directly from the
commercially available mixture via column chromatography of the
mixture (5 g) on silica gel (300 g) using hexane/ethyl acetate (20:1) to
yield 1.1 g pure cis,cis-isomer (by 1H NMR) as a colorless liquid.
Samples of the cis,cis-isomer from both isolation procedures produced
similar modulation of GABA currents.
An oven-dried 100-ml three-neck flask, equipped with magnetic
stirrer, rubber septum, and gas inlet was filled with N2. Tetrahydro-
furan (anhydrous, 20 ml) was added followed by a 1 M solution of
lithium aluminum hydride in tetrahydrofuran (13.1 ml, 13.1 mmol,
1.10 Eq). cis-2,6-Dimethylcyclohexanone (1.46 g, 11.5 mmol) was then
added dropwise via a syringe and the reaction was stirred at room
temperature for 1 hour.
Molecular Modeling
Molecular docking studies were carried out to define the mode of
interaction between the GABAA receptor propofol and each diastereo-
mer of 2,6-dimethylcyclohexanol. Given previous literature highlight-
ing the role of b subunits in the binding of propofol to GABAA receptors
(Yip et al., 2013) and the availability of a crystal structure [Protein
Data Bank (PDB) identifier 4COF; Miller and Aricescu, 2014], the b3
subunit of the human GABAA receptor was the considered target. This
target was prepared for the docking process by protonating, minimiz-
ing, and examining the missing side chain residues in the protein
using Chimera Software (University of California, San Francisco)
(Pettersen et al., 2004; Goddard et al., 2005). The prepared target was
uploaded to the ProBiS server (http://probis.nih.gov/; National Insti-
tutes of Health, Bethesda, MD) (Carl et al., 2010) for the detection
of binding sites using protein binding site structure similarities. The
ProBiS program aligns and superimposes protein binding sites, and it
enables pairwise alignments and fast database searches for similar
binding sites.
We focused on propofol binding sites highlighted in previous studies
(Nury et al., 2011; Yip et al., 2013; Chiara et al., 2014) and sites based
on the structural similarities of the following proteins (shown by PDB
identifiers): 2M6B (structure of transmembrane domains of human
glycine receptor a1 subunit; Mowrey et al., 2013), 4X5T (a1 glycine
receptor transmembrane structure fused to the extracellular domain
of gloeobacter ligand-gated ion channel(GLIC); Moraga-Cid et al.,
2015), and 3P50 (structure of propofol bound to GLIC; Nury et al.,
The reaction was quenched with 2 ml water added dropwise, then
diluted with 5 ml 15% NaOH solution, followed by another 2 ml water.
The mixture was filtered through silica gel under a vacuum and rinsed
with diethyl ether (approximately 50 ml). The filtrate was transferred
to a 250-ml separatory funnel. The aqueous layer was extracted with
ethyl ether (3 ꢁ 50 ml), and the combined ether layers were dried over
anhydrous MgSO4. 1H NMR on the crude product (1.65 g, 100%)
showed a mixture of cis,cis-2,6-dimethylcyclohexanol (54%) and trans,
trans-2,6-dimethylcyclohexanol (46%). Flash column chromatography
of the crude product using 240 g silica gel and 10:1 petroleum ether to
ethyl ether as eluant gave the cis,cis-isomer (646 mg, 42%), a com-
bination of both isomers (41 mg, 3%), and the trans,trans-isomer
(551 mg, 36%). The total yield was 1.24 g (9.65 mmol, 81%).
cis,cis-2,6-Dimethylcyclohexanol (Isomer 1). TLC was as fol-
lows: Rf 5 0.2, eluant 10% diethyl ether in petroleum ether, visualized
with vanillin. 1H NMR (300 MHz, CDCl3) was as follows: 0.98 (d, 6 H),
1.16 (d, 1 H), 1.28–1.38 (m, 5 H), 1.45–1.59 (m, 2 H), 1.65–1.74 (m, 1 H),
and 3.51–3.56 (m, 1 H)
trans,trans-2,6-Dimethylcyclohexanol (Isomer 2). TLC was
as follows: Rf 5 0.1, eluant 10% diethyl ether in petroleum ether,
visualized with vanillin. 1H NMR (300 MHz, CDCl3) was as follows:
1.07 (d, 6 H), 1.20–1.29 (m, 2 H), 1.30–1.42 (m, 2 H), 1.47 (d, 1 H),
1.56–1.65 (m, 2 H), 1.66–1.76 (m, 2 H), and 2.72 (t of d, 1 H).
Reduction of trans-2,6-Dimethylcyclohexanone with Lith- 2011). Based on these previous studies, we explored intrasubunit
ium Aluminum Hydride.
binding sites within chain A of a single subunit of 4COF (Miller and
Aricescu, 2014). The best binding site with a Z score of 4.21 included
the key amino acid residues Tyr220, Phe221, Gln224, His267, and
Thr271. A second binding site with key amino acid residues Tyr143,
Thr225, Pro228, Ile264, and Leu268 gave a Z score of 4.01. The high
scoring binding sites were then merged because there was consider-
able spatial overlap between the key amino acids. Although it is
recognized that intersubunit sites have been proposed for propofol
binding to GABAA receptors (e.g., Bali and Akabas, 2004), sites
between subunits (e.g., chains A and B) were not considered because
steric clashes were encountered when modeling multiple subunits at
(2)
It should be noted that 3 and 4 shown in eq. 2 are enantiomers and
may (or may not) have equal activity dependent on whether the site is
chiral (not the same activity) or achiral (same activity).
An oven-dried 25-ml two-neck flask, equipped with magnetic these sites.
stirrer, rubber septum, and gas inlet, was filled with dry N2.
Tetrahydrofuran (anhydrous, 1.5 ml) was added together with a 1 M
The protein was loaded to MGL-AutoDock Tools (Scripps Research
Institute, La Jolla, CA) to define the custom binding site grid box for

Modulation of GABAA Currents by 2,6-Dimethylcyclohexanols
573
the docking process. In brief, polar hydrogen atoms and Kollman
charges were assigned to the protein by converting to the PDBQT
format of AutoDock (Morris et al., 2009). The program AutoGrid was
used to generate grid maps for the custom binding site on the protein
and the grid was generated based on selected residues from binding
site analysis. To generate grid maps for different types of ligands (with
possible hydrogen bonding), the grid parameter file was modified to
include O-H bond types from ligands. To generate the custom grid
maps, we defined grid points as x 5 40, y 5 40, and z 5 50 and the grid
center as x 5 10.70, y 5 213.28, and z 5 157.35 with a spacing of 0.38
in the protein three-dimensional structure. The binding site for 2,6-
dimethylcyclohexanols included Tyr143, Tyr220, Phe221, Gln224,
Thr225, Pro228, Ile264, His267, Leu268, and Thr271 amino acids
from the M1 and M2 domains of chain A (Fig. 2).
Propofol, cis,cis-, trans,trans-, and cis,trans-diastereomers of 2,6-
dimethylcyclohexanol were all considered as ligands. The geome-
tries were drawn in MarvinSketch (Chemaxon, Cambridge, MA)
and converted to three-dimensional structures using the MM2
force field. The diastereomeric conformers were frozen for further
quantum chemical optimization using the DFT/B3LYP level of
theory and the 6-31G basis set in HyperChem software (Hypercube,
Inc., Gainesville, FL) (see Table 1 for quantum chemical proper-
ties). The lowest energy conformer was uploaded to MGL Tools to
assign Gasteiger partial charges and for the detection of torsions
to rotate the bonds during the docking procedure. For all ligands,
random initial positions, fixed conformers, and torsions were
parameterized. The number of active torsions and the number of
torsional degrees of freedom were set to default values indicated in
AutoDock. A Lamarckian genetic algorithm was used for minimi-
zation using optimum parameters (from initial docking evalua-
tions) to generate all possible energies to rank the conformers
(Morris et al., 1998). For energy evaluations, we used the following
docking parameters: 250,000 evaluations, 250 genetic algorithm
iterations, and a population size of 150 to generate 250 docked
conformers. For reliable docking results, the root mean square
deviation of the lowest energy conformer and the root mean square
deviation to one another were analyzed to group families of similar
conformations using clustering.
Results
cis,cis- and trans,trans-2,6-Dimethylcyclohexanols Isomers
Are Positive Modulators of GABAA Receptor Currents
We investigated the modulation of submaximal GABA
currents by the three isolated cis,cis-, cis,trans-, and trans,
trans-2,6-dimethylcyclohexanol diastereomers along with the
mixture of the isomers. WSS-1 cells (HEK cells stably ex-
pressing a1b3g2s GABAA receptors) were routinely exposed to
applications of 10 mM GABA that evoked approximate EC20
currents (effective concentration that evoked 20% of maximal
current). Coapplications of 30 mM 2,6-dimethylcyclohexanols
produced potentiations of the GABA responses (Fig. 3). For
example, the addition of 30 mM cis,cis-isomer resulted in
approximately 2- to 3-fold enhancement of the EC20 GABA
current (Fig. 3B). Pre-exposure to cyclohexanols did not
affect the extent of current modulation upon subsequent
coapplication and no direct activation of GABAA receptor
currents was observed by the cyclohexanols even at the highest
concentrations (300 mM) of the isomers tested (data not shown).
Coapplications of 30 mM 2,6-dimethylcyclohexanol isomers
produced a range of positive enhancements of control GABA
responses with the rank order for positive modulation of
GABA EC20 currents: cis,cis . trans,trans $ mixture of
isomers . . cis,trans-isomer (Fig. 3). We confirmed the
relative extent of the potentiations of the GABA receptor
activity in the presence of increasing concentrations of the
isomers (1–300 mM, Fig. 4). For instance, on average with the
addition of 30 mM cis,cis-isomer, the positive modulation of
the current (above control) was 165% 6 27% (n 5 6), whereas
the equivalent for the trans,trans-isomer was 92% 6 24% (n 5 5).
Current modulation by the cis,trans-isomer was negligible
even at the highest concentration tested (at 300 mM, 5% 6 5%,
n 5 6). All of the cyclohexanol effects were fully reversible upon
washout (as previously reported; Hall et al., 2011).
Fig. 2. Binding site and hydrophobicity of the target GABAA b3-subunit protein chain A (PDB identifier 4COF) with cis,cis-2,6-dimethylcyclohexanol.
Binding site is in the adjacent subunit of membrane region (M1 and M2) close to the Cys loop.

574
Chowdhury et al.
TABLE 1
Quantum chemical properties in vacuum
Properties/Ligand
E(RHF) Hatree
Dipole Moment Debye
E (Thermal)
Heat Capacity
Entropy
Zero-Point Energy Correction Hatree
kcal/mol
cal/mol·K
cal/mol·K
cis,cis
trans,trans
cis,trans
2386.97113
2386.97115
2386.96832
1.815
2.169
1.891
161.11
160.99
161.18
35.239
35.556
35.374
91.083
91.592
90.988
0.247
0.247
0.247
The relative potencies for positive modulation of GABA GABAA receptor b-subunits or GLIC channels (Nury et al.,
currents were further supported by recording and plotting the 2011; Yip et al., 2013; Chiara et al., 2014). The binding site
leftward shifts in the GABA concentration-response curves in prediction server ProBiS (Carl et al., 2010) gave the best
the presence of 30 mM 2,6-dimethylcyclohexanol isomers (Fig. 5). binding site score with key amino acid residues Tyr143,
For instance, the EC50 for the control GABA currents (approx- Tyr220, Phe221, Gln224, Thr225, Pro228, Ile264, His267,
imately 21 mM) was shifted to approximately 10 mM by the Thr271, and Leu268.
trans,trans-isomer and to approximately 7 mM in the presence of
This binding site is located between the transmembrane M1
the cis,cis-isomer. The cis,trans-isomer produced a negligible and M2 helices of the b3 subunit (Fig. 2) and includes a
shift in the concentration-response relationship (Fig. 5).
phenylalanine at the 221 position (M1) and a histidine at the
267 position (M2) that were previously photolabeled with an
ortho-propofol diazirine derivative (Yip et al., 2013). In the
same study, replacement of the phenylalanine at 221 by a
tryptophan residue was shown to attenuate propofol’s poten-
tiation of GABA-evoked currents. Interestingly, the three
cyclohexanol isomers were found to bind within this pocket
through hydrogen-bonding interactions with a glutamine at
the 224 position and a tyrosine at the 220 position plus
hydrophobic interactions with the leucine at position 268,
phenylalanine at position 221, and tyrosine at position 220
(Fig. 6; Tables 1–3). By comparison, propofol’s hydrogen
bonding was modeled only through the glutamine residue and
hydrophobic interactions with the leucine at 268, tyrosine
at 220, and threonine at 225 positions (Fig. 7). The ligand
efficiency, van der Waals energy, and electrostatic and H-bond
energy were all considered in the calculation of binding energies
and intermolecular energies for this intrasubunit site. Within
the site the binding energies for the interactions (Table 2) had
a rank order of propofol . cis, cis . trans,trans . cis,trans-2,6-
dimethylcyclohexanol corresponding with the rank order of
potencies for positive modulation of GABAA receptor currents
recorded electrophysiologically [compare with Figs. 3 and 7 from
Davies et al. (2000) and Hall et al. (2011), respectively].
Modeling the Binding of the Isomers to the b3 Subunit of the
GABAA Receptor
Molecular modeling focused on regions of the b3 subunit of
the GABAA receptor (4COF; Miller and Aricescu, 2014) that,
in previous studies, were implicated in propofol binding to
cis-,trans-2,6-Dimethylcyclohexanol as an Inhibitor of
Propofol’s Modulatory Action at GABAA Receptors
Finally, given the lack of modulation observed by cis,trans-
2,6-dimethylcyclohexanol and the modeling of its binding to
the receptor at a site similar to that of propofol’s, we explored
the possibility that this isomer might competitively inhibit
propofol’s modulatory action at the receptor. As expected,
submaximal GABA (3 mM) responses were potently enhanced
by propofol (10 mM; Fig. 8). However, this positive modulation
was only moderately attenuated by the coapplication of 100
mM cis,trans-2,6-dimethylcyclohexanol (Fig. 8). In summary,
positive modulation by propofol was attenuated by 14.1% 6
1.3% (n 5 4) and by 13.4% 6 2.5% (n 5 5) by 100 and 300 mM
cis,trans-2,6-dimethylcyclohexanol, respectively.
Fig. 3. Current recordings illustrating modulation by 2,6-dimethylcyclo-
hexanol isomers of GABAA receptor activity. WSS-1 cells were held at 250
mV and 10 mM GABA was applied for the duration (2 seconds) of the
shaded boxes above each trace. Upper traces indicate current evoked in
the presence of GABA alone. Superimposed lower traces indicate current
evoked in presence of 10 mM GABA plus 30 mM of the 2,6-dimethylcyclo-
hexanol isomer(s) specified. (A) With coapplication of the commercially
available mixture of 2,6-dimethylcyclohexanol isomers. (B) With coappli-
cation of cis,cis-2,6-dimethylcyclohexanol. (C) With coapplication of trans,
trans-2,6-dimethylcyclohexanol. (D) With coapplication of cis,trans-2,6-
dimethylcyclohexanol.
Discussion
In the search for novel anesthetic and sedative compounds,
this study investigated the potential of 2,6-dimethylcyclo-
hexanol stereoisomers as positive modulators of GABAA

Modulation of GABAA Currents by 2,6-Dimethylcyclohexanols
575
Fig. 5. GABA concentration-response curves are shifted to the left with
coapplication of cis,cis- and trans,trans-2,6-dimethylcyclohexanol isomers.
Currents were normalized against the maximum current evoked by any
concentration of agonist with or without coapplication of 2,6-dimethylcy-
clohexanol. Data (n $ 5, mean 6 S.E.M.) were fitted with the Hill equation
(mentioned in the Materials and Methods) in the absence (filled square)
and presence of cis,trans-2,6-dimethylcyclohexanol (open circles), cis,cis-
2,6-dimethylcyclohexanol (open triangles), and trans,trans-2,6-dimethyl-
cyclohexanol (closed inverted triangles).
Fig. 4. cis,cis-2,6-Dimethylcyclohexanol is the most potent of the isomers
for positive modulation of GABAA receptor currents. Relative modulations
of GABA (10 mM) responses were compared by coapplying increasing
concentrations (1–300 mM) of 2,6-dimethylcyclohexanol isomer(s). With
the exception of cis,trans-2,6-dimethylcyclohexanol, the isomers were
effective as positive modulators of GABA currents, with cis,cis-2,6-
dimethylcyclohexanol demonstrating the most potent current enhance-
ment. Data points represent mean 6 S.E.M. modulations for n $ 5 cells.
receptor– mediated currents. In this study, the GABA EC50 the mixture is composed of isomers that may have different
in HEK cells expressing a1b3g2s receptors was determined to modulatory effects on the receptors. Stereoselective action has
be approximately 20 mM with a Hill coefficient of 1.6, which is been previously observed for GABA receptor modulation by an-
reasonably consistent with other reports for similar receptor esthetic agents. For instance, the S(1)-isoflurane isomer was
combinations using the same expression system (e.g., Ueno observed to be more effective in potentiating GABA-induced
et al., 1996). The cyclohexanols in this study all have their currents than the R(2)-isoflurane (Hall et al., 1994) with
aliphatic groups in the 2,6-position relative to the hydroxyl steroselectivity also observed for (1)- and (2)-pentobarbital
group equivalent to the commonly used intravenous anes- (Tomlin et al., 1998). It seemed plausible, therefore, that
thetic, propofol (2,6-di-isopropylphenol). Following on from individual isomers of 2,6-di-methylcyclohexanol would exhibit
studies that demonstrated the efficacy of 2,6-dimethylcyclo- a range of potencies due to differing chemical configurations.
hexanol for positive modulation of GABAA receptor currents 2,6-Di-isopropylphenol (propofol), a potent positive modulator
and for inducing anesthesia (Hall et al., 2011), the major of GABA receptor currents, consists of a hydroxyl group and
electrophysiological findings of our study are as follows. two ortho isopropyl groups in the plane of the benzene ring.
First, the mixture of 2,6-dimethylcyclohexanol isomers By contrast, all three 2,6-di-methylcyclohexanols are chair
(3–300 mM) enhanced GABA currents evoked in HEK cells shaped and are not planar molecules. The trans,trans config-
expressing a1b3g2s receptors. Second, the cis,cis- and trans, uration of 2,6-di-methylcyclohexanol is the closest to planarity
trans-isomers of 2,6-dimethylcyclohexanol positively modu- with the hydroxyl and two methyl groups in equatorial positions
lated GABA currents, with the former being marginally more in the most stable conformer. Thus, we expected that the trans,
potent. Finally, cis,trans-2,6-dimethylcyclohexanol had min- trans-isomer would be the most potent modulator since it might
imal effects on GABA currents.
fit most effectively into an equivalent propofol binding pocket
The modulatory effects of cyclohexanols on GABA recep- within the GABAA receptor. Indeed, previous studies reported
tors have been previously investigated (Hall et al., 2011); how- that cyclohexanols and propofol may share similar sites of
ever, in this instance, they were studied in oocytes expressing action on GABAA receptors (Watt et al., 2008). The cis,cis con-
a1b2g2s receptors (the most prevalent combination found in figurationof 2,6-dimethylcyclohexanol was postulated to be the
the mammalian brain). By comparison, the potentiation of next most potent since the most stable conformation would have
GABA currents by 30 mM 2,6-dimethylcyclohexanol was greater the hydroxyl group axial and the methyl groups equatorial.
in the oocyte studies (approximately 4-fold enhancement; Hall Finally, the cis,trans configuration of 2,6-dimethycyclohexanol
et al., 2011) than in HEK cells expressing a1b3g2s receptor was expected to be the least potent since one methyl must
composition (approximately 1.5-fold enhancement, this study). necessarily be axial thereby reducing planarity. Contrary to
Subunit composition, differences in expression systems and expectations, although the trans,trans-isomer was still an
speed of agonist/modulator application probably contribute to effective modulator, the cis,cis-isomer was the most potent
the discrepancies in the extent of the modulations reported.
diastereomer for positive modulation of GABA responses. As
Although the 2,6-dimethylcyclohexanol mixture of stereoiso- anticipated, the cis,trans-isomer had minimal impact on the
mers enhanced GABA currents at concentrations of 3–300 mM, modulation of GABA receptor currents, suggesting that the

576
Chowdhury et al.
Fig. 6. Three-dimensional interpretations (A–C) and two-dimensional depictions (A’–C’) of molecular docking interactions between the ligands and
GABAA receptor (b3-subunit). cis,cis-2,6-Dimethylcyclohexanol (A), trans,trans-2,6-dimethylcyclohexanol (B), and cis,trans-2,6-dimethylcyclohexanol
with hydrogen-bond interactions (red color), bond distance in Å (red color), and hydrophobic interaction regions in the blue-colored surfaces (C). H-Bond
distances were as follows: O, Gln224 = 1.898 Å (A and A’), 2.014 Å (B and B’), and 2.152 Å (C and C’), respectively; H, Tyr220 = 1.904 Å (A and A’), 1.891 Å
(B and B’), and 2.054 Å (C and C’), respectively. Definitions for the various interacting elements represented in the two-dimensional depiction (A’–C’) are
as follows. First, residues involved in hydrogen-bond, charge, or polar interactions are represented by purple circles. Second, residues involved in van der
Waals interactions are represented by green circles. Third, the solvent accessible surface of an interacting residue is represented by a blue halo around
the residue with the diameter of the halo proportional to the solvent accessible surface. Finally, hydrogen-bond interactions with amino acid side chains
are represented by a blue arrows directed toward the electron donor. Circled residues in the two-dimensional diagrams are indicative of residues within
hydrophobic interaction regions and H-bonding interaction regions, whereas those outside are other relevant binding site residues. Note, in each case,
only residues within 4-Å distance from the ligand are depicted.
23% of this isomer present in the commercially available assay (Hall et al., 2011). 2,6-Dimethylcyclohexanol was one of
mixture acts as pharmacological ballast with regard to GABAA the most potent in this regard with an EC50 of approximately
receptor modulation.
13 mM, with 2,6-di-isopropylcyclohexanol demonstrating
In previous studies, a range of 2,6-substituted cyclohexanols equivalent potency (EC50 of approximately 14 mM). To
with varying sizes of the aliphatic chains were assessed for date, yields of isolated isomers from mixtures of both 2,6-
anesthetic potency and for loss of righting reflex in a tadpole dimethylcyclohexanol and 2,6-di-isopropylcyclohexanol

Modulation of GABAA Currents by 2,6-Dimethylcyclohexanols
577
Fig. 7. Three-dimensional interpretations (left) and two-dimensional depictions (right) of molecular docking interactions between the propofol and
GABAA receptor (b3 subunit). H-Bond distances were as follows: H, Gln224 = 1.874 Å. The green color discs represents van der Waals residue
interactions, and purple represents electrostatic interactions. Circled residues in the two-dimensional diagrams are indicative of residues within
hydrophobic interaction regions and H-bonding interaction regions, whereas those outside are other relevant binding site residues. Hydrogen-bond
interactions with the amino acid main chain is represented by a green arrow directed towards the electron donor Note, in each case, only residues within
4-Å distance from the ligand are depicted.
have been insufficient to enable similar analyses of the relative extensive protein preparation (i.e., minimization to refine
anesthetic potencies of the cis,cis- versus trans,trans-isomers in protein structure). Because this questions the availability of
in vivo tadpole assays.
the proposed site in an assembled receptor, future studies will
Although our electrophysiological studies were conducted require a thorough protein minimization to refine the struc-
on a GABAA receptor consisting of a1, b3, and g2s subunits, the ture and molecular dynamics simulations to address other
closest approximating crystal structure for the molecular potential intra- and intersubunit binding sites.
modeling studies was for a homopentamer of human b3
Our modeling studies revealed several important points
subunits (4COF; Miller and Aricescu, 2014). The binding regarding the binding of the 2,6-dimethylcyclohexanols at
pocket for the cyclohexanol isomers, revealed through our the chosen site. First, the binding energies (Table 2) vary from
modeling studies of a GABAA receptor b3 subunit, was 24.42 through 24.38 to 24.21 kcal/mol for the cis,cis-, trans,
composed primarily of residues from M1 and M2 transmem- trans-, and cis,trans-isomers, respectively. Such small differ-
brane helices. This intrasubunit binding site was predicted by ences, although corresponding to the rank order of potency for
reference to previous propofol binding studies, by similarity receptor modulation, are unlikely to explain changes in current
in the templates used for the homology modeling and by modulation from approximately 300% (300 mM cis,cis) to 0%
identifying key amino acid residues within two sites that (300 mM cis,trans). Binding energy comprises a combination of
produced the highest Z scores. The site included a phenylal- hydrophobic interactions, van der Waal’s forces, and hydrogen
anine at the 221 position and a histidine at the 267 position bonding. Figure 6 shows that the H-bond length to glutamine
(Fig. 7) that have already been implicated in propofol’s 224 (Gln224) increases from 1.898 Å (cis,cis) through 2.014 Ǻ
positive modulation of receptor currents and in propofol’s (trans,trans) to 2.152 Å (cis,trans). Interestingly, these H-bond
binding (Yip et al., 2013). Both active isomers, cis,cis and lengths are inversely related to the extent of current enhance-
trans,trans, were also observed to bind to the receptor subunit ments derived electrophysiologically, suggesting that H bond-
via the histidine 267 and through hydrophobic interactions ing may be an important factor in determining the extent of
with the phenylalanine 221 (Fig. 6, A and B). It should be receptor-positive modulation (Table 3).
noted that Yip et al. (2013) observed interactions of an eq-
It is instructive to compare the modeling data for the
uivalent binding site with the main chain of the neighboring cyclohexanols with those for propofol, which shows a binding
subunit. In our modeling studies, intersubunit sites of action energy of 24.96 kcal/mol. If binding energy was the deter-
were not explored because of steric clashes between subunits mining factor, this only corresponds to an estimated modula-
(e.g., chains A and B) at these sites that were severe even after tion enhancement over cis,cis-2,6-dimethylcyclohexanol of
TABLE 2
Molecular docking results: docked ligand scoring parameters and interacting amino acid residues
Binding
Energy
Predicted Inhibition
Constant
Ligand
Efficiency
Intermolecular
Energy
van der Waal’s
Properties/Ligand
H Bond
Hydrophobic Interactions
Desolvation Energya
kcal/mol
mM
kcal/mol
kcal/mol
cis,cis
24.42
24.38
24.21
24.96
572.09
620.36
813.66
231.96
20.49
20.49
20.47
20.38
24.72
24.67
24.51
25.85
24.51
24.42
24.36
25.71
Tyr220, Gln224 Tyr220, Phe221, Leu268
Tyr220, Gln224 Tyr220, Phe221, Leu268
Tyr220, Gln224 Tyr220, Phe221, Leu268
trans,trans
cis,trans
Propofol
Gln224
Tyr220, Thr225, Leu268, Gln224
avan der Waal’s desolvation energy = van der Waal’s + H bond + desolvation energy.

578
Chowdhury et al.
TABLE 3
Molecular docking results: H-bond distance
Residues/Ligand
Tyr220
Å
Gln224
cis,cis
1.904
1.891
2.054
—
1.898
2.014
2.152
1.874
trans,trans
cis,trans
Propofol
distinct from that predicted to be stabilized by positive
modulators. Moreover, the 4COF structure represents a
modified b3 subunit of the GABAA receptor with extensive
intracellular loop domains removed. Therefore, although
4COF provides an approximation of the relevant structure
for binding studies, interpretation of the molecular modeling
data must be viewed with caution. Furthermore, the struc-
ture of 4COF is that of a homopentameric receptor, whereas
our electrophysiological recordings were derived from recep-
tors with two additional subunits (a1 and g2s), which could
contribute either directly to drug binding or indirectly to
binding site conformations. Given these caveats, other
candidate binding sites may be equally or more relevant to
producing the positive modulations of the receptor activity
observed electrophysiologically.
Fig. 8. Current recording illustrating minimal attenuation by cis,trans-
2,6-dimethylcyclohexanol isomer of the positive modulation of GABAA
receptor activity by propofol. A WSS-1 cell was held at 250 mV and 3 mM
GABA was applied for the duration (2 seconds) of the open boxes above the
trace. During the period of the checkered box a current was evoked by the
presence of 3 mM GABA plus 10 mM propofol. During the filled box, a
current was evoked by the presence of 3 mM GABA plus 10 mM propofol plus
100 mM cis,trans-2,6-dimethylcyclohexanol, resulting in only moderate
inhibition of the propofol-induced positive modulation. Positive modulation
by propofol was attenuated by 14.1% 6 1.3% (n = 4) and by 13.4% 6 2.5%
(n = 5) by 100 and 300 mM cis,trans-2,6-dimethylcyclohexanol, respectively
Finally, we performed competition experiments to deter-
mine whether cis,trans-2,6-dimethylcyclohexanol could act
as a competitive inhibitor of propofol’s action at the receptor
(Fig. 8). In these recordings, we observed only modest inhibi-
tion of the positive modulation induced by propofol. This re-
sult suggests that although cis,trans-cyclohexanol produced
no modulation of GABA responses, its ability to compete for
a propofol binding site is limited, presenting a further caveat
for overinterpretation of the modeling data.
In conclusion, the enhanced activity of cis,cis-2,6-dimethyl-
cyclohexanol presents an interesting lead in the development
of novel anesthetics, given that cyclohexanols in general are
typically well tolerated (Thorup et al., 1983). Further refine-
ment of such agents through isolation of individual isomers
may lead to novel anesthetics with improved therapeutic
indices.
approximately 3-fold, whereas the observed enhancements
using propofol are considerably greater (e.g., compare Fig. 3B
with Fig. 8). According to our modeling, propofol also forms a H
bond with Gln224 with a bond length of 1.874 Ǻ only slightly
shorter than that with cis,cis-2,6-dimethylcyclohexanol. There
is, however, a fundamental difference. The H bond is described
between the hydrogen of the OH group and the basic amide
oxygen of Gln224, possibly due to the acidity of propofol (pKa
of approximately 9). By contrast, the H bond between the
cyclohexanols and Gln224 is between the NH2 group (in
CONH2) of the glutamine and the oxygen atom of the OH
group. Therefore, one may conclude that it is the nature and
lengths of the hydrogen bonds that determines the degree of
receptor-positive modulation, although additional contributions
from the hydrophobic and van der Waal’s interactions must
also contribute to the binding energy.
Acknowledgments
Although we focused on a site selected from predocking
studies with the highest Z scores, many other sites had the
potential to accommodate the isomers, albeit with lesser
scores. Indeed, many other sites have been proposed for
propofol’s interactions and modulation of GABAA receptor
activity, including other sites in M1 (Chang et al., 2003), M2
(Siegwart et al., 2002), and also M3 and M4 helices (Krasowski
et al., 1998; Siegwart et al., 2002; Richardson et al., 2007). For
example, in previous studies, a tyrosine in the 444 position of
b2 subunits proved important for modulation of the GABA
currents by propofol (Richardson et al., 2007) and by menthol,
a monoterpenoid with a neutral cyclohexanol chair structure
(Watt et al., 2008).
The authors thank Harrison Hunter and Salma Bargach for helping
to set up the patch-clamp electrophysiology. They also thank Dr.
Andrew Jenkins (Department of Anesthesiology, Emory University,
Atlanta, GA) for reading of and comments on the manuscript. G.G.
Pillai thanks the University of Tartu Graduate School for Functional
Materials and Technologies.
Authorship Contributions
Participated in research design: Chowdhury, Croft, Goel, Zaman,
Tai, Walch, Smith, Page, Shea, C.D. Hall, Jishkariani, Pillai, A.C.
Hall.
Conducted experiments: Chowdhury, Croft, Goel, Zaman, Tai,
Walch, Smith, Page, Shea, C.D. Hall, Jishkariani, Pillai, A.C. Hall.
Contributed new reagents or analytic tools: Smith, Page, Shea, C.D.
Hall, Jishkariani.
Performed data analysis: Chowdhury, Croft, Goel, Zaman, Tai,
Walch, Smith, Page, Shea, C.D. Hall, Jishkariani, Pillai, A.C. Hall.
Wrote or contributed to the writing of the manuscript: Chowdhury,
Croft, Goel, Zaman, Tai, Walch, Smith, Page, Shea, C.D. Hall,
The drugs investigated in this study were all positive
modulators and, therefore, likely stabilize the “open” state of
the ligand-gated ion channel through an allosteric mecha-
nism. By contrast, the 4COF structure used for our modeling
studies is described by Miller and Aricescu (2014) as repre-
sentative of a “desensitized” state. This state is evidently Jishkariani, Pillai, A.C. Hall.

Modulation of GABAA Currents by 2,6-Dimethylcyclohexanols
579
Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK, and Olson AJ
(1998) Automated docking using a Lamarckian genetic algorithm and an empirical
binding free energy function. J Comput Chem 19:1639–1662.
Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, and Olson
AJ (2009) AutoDock4 and AutoDockTools4: automated docking with selective re-
ceptor flexibility. J Comput Chem 30:2785–2791.
Mowrey DD, Cui T, Jia Y, Ma D, Makhov AM, Zhang P, Tang P, and Xu Y (2013)
Open-channel structures of the human glycine receptor a1 full-length trans-
membrane domain. Structure 21:1897–1904.
Nury H, Van Renterghem C, Weng Y, Tran A, Baaden M, Dufresne V, Changeux JP,
Sonner JM, Delarue M, and Corringer PJ (2011) X-ray structures of general an-
aesthetics bound to a pentameric ligand-gated ion channel. Nature 469:428–431.
Olsen RW and Li GD (2011) GABA(A) receptors as molecular targets of general
anesthetics: identification of binding sites provides clues to allosteric modulation.
Can J Anaesth 58:206–215.
Pejo E, Santer P, Jeffrey S, Gallin H, Husain SS, and Raines DE (2014) Analogues of
etomidate: modifications around etomidate’s chiral carbon and the impact on in
vitro and in vivo pharmacology. Anesthesiology 121:290–301.
Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC,
and Ferrin TE (2004) UCSF Chimera–a visualization system for exploratory re-
search and analysis. J Comput Chem 25:1605–1612.
Richardson JE, Garcia PS, O’Toole KK, Derry JM, Bell SV, and Jenkins A (2007) A
conserved tyrosine in the b2 subunit M4 segment is a determinant of g-amino-
butyric acid type A receptor sensitivity to propofol. Anesthesiology 107:412–418.
Siegwart R, Jurd R, and Rudolph U (2002) Molecular determinants for the action of
general anesthetics at recombinant a(2)b(3)g(2)g-aminobutyric acid(A) receptors.
J Neurochem 80:140–148.
References
Bali M and Akabas MH (2004) Defining the propofol binding site location on the
GABAA receptor. Mol Pharmacol 65:68–76.
Barnard EA, Skolnick P, Olsen RW, Mohler H, Sieghart W, Biggio G, Braestrup C,
Bateson AN, and Langer SZ (1998) International Union of Pharmacology. XV.
Subtypes of g-aminobutyric acidA receptors: classification on the basis of subunit
structure and receptor function. Pharmacol Rev 50:291–313.
Carl N, Konc J, Vehar B, and Janezic D (2010) Protein-protein binding site prediction
by local structural alignment. J Chem Inf Model 50:1906–1913.
Chang CS, Olcese R, and Olsen RW (2003) A single M1 residue in the b2 subunit
alters channel gating of GABAA receptor in anesthetic modulation and direct ac-
tivation. J Biol Chem 278:42821–42828.
Chiara DC, Gill JF, Chen Q, Tillman T, Dailey WP, Eckenhoff RG, Xu Y, Tang P,
and Cohen JB (2014) Photoaffinity labeling the propofol binding site in GLIC.
Biochemistry 53:135–142.
Corvalán NA, Zygadlo JA, and García DA (2009) Stereo-selective activity of menthol
on GABA(A) receptor. Chirality 21:525–530.
Davies PA, Hoffmann EB, Carlisle HJ, Tyndale RF, and Hales TG (2000) The in-
fluence of an endogenous beta3 subunit on recombinant GABA(A) receptor as-
sembly and pharmacology in WSS-1 cells and transiently transfected HEK293
cells. Neuropharmacology 39:611–620.
Franks NP and Lieb WR (1994) Molecular and cellular mechanisms of general an-
aesthesia. Nature 367:607–614.
Goddard TD, Huang CC, and Ferrin TE (2005) Software extensions to UCSF chimera
for interactive visualization of large molecular assemblies. Structure 13:473–482.
Hall AC, Griffith TN, Tsikolia M, Kotey FO, Gill N, Humbert DJ, Watt EE, Yermo-
lina YA, Goel S, and El-Ghendy B, et al. (2011) Cyclohexanol analogues are positive
modulators of GABA(A) receptor currents and act as general anaesthetics in vivo.
Eur J Pharmacol 667:175–181.
Hall AC, Lieb WR, and Franks NP (1994) Stereoselective and non-stereoselective
actions of isoflurane on the GABAA receptor. Br J Pharmacol 112:906–910.
Hall AC, Turcotte CM, Betts BA, Yeung WY, Agyeman AS, and Burk LA (2004)
Modulation of human GABAA and glycine receptor currents by menthol and re-
lated monoterpenoids. Eur J Pharmacol 506:9–16.
Thorup I, Würtzen G, Carstensen J, and Olsen P (1983) Short term toxicity study in
rats dosed with pulegone and menthol. Toxicol Lett 19:207–210.
Tomlin SL, Jenkins A, Lieb WR, and Franks NP (1998) Stereoselective effects of
etomidate optical isomers on gamma-aminobutyric acid type A receptors and ani-
mals. Anesthesiology 88:708–717.
Ueno S, Zorumski C, Bracamontes J, and Steinbach JH (1996) Endogenous subunits
can cause ambiguities in the pharmacology of exogenous g-aminobutyric acidA
receptors expressed in human embryonic kidney 293 cells. Mol Pharmacol 50:
931–938.
Krasowski MD and Harrison NL (1999) General anaesthetic actions on ligand-gated
ion channels. Cell Mol Life Sci 55:1278–1303.
Watt EE, Betts BA, Kotey FO, Humbert DJ, Griffith TN, Kelly EW, Veneskey KC,
Gill N, Rowan KC, and Jenkins A, et al. (2008) Menthol shares general anesthetic
activity and sites of action on the GABA(A) receptor with the intravenous agent,
propofol. Eur J Pharmacol 590:120–126.
Krasowski MD, Jenkins A, Flood P, Kung AY, Hopfinger AJ, and Harrison NL (2001)
General anesthetic potencies of a series of propofol analogs correlate with potency
for potentiation of gamma-aminobutyric acid (GABA) current at the GABA(A) re-
ceptor but not with lipid solubility. J Pharmacol Exp Ther 297:338–351.
Krasowski MD, Koltchine VV, Rick CE, Ye Q, Finn SE, and Harrison NL (1998)
Propofol and other intravenous anesthetics have sites of action on the g-amino-
butyric acid type A receptor distinct from that for isoflurane. Mol Pharmacol 53:
530–538.
McKernan RM and Whiting PJ (1996) Which GABAA-receptor subtypes really occur
in the brain? Trends Neurosci 19:139–143.
Miller PS and Aricescu AR (2014) Crystal structure of a human GABAA receptor.
Nature 512:270–275.
Wong G, Sei Y, and Skolnick P (1992) Stable expression of type I g-aminobutyric
acidA/benzodiazepine receptors in
996–1003.
a transfected cell line. Mol Pharmacol 42:
Yip GMS, Chen Z-W, Edge CJ, Smith EH, Dickinson R, Hohenester E, Townsend RR,
Fuchs K, Sieghart W, and Evers AS, et al. (2013) A propofol binding site on
mammalian GABAA receptors identified by photolabeling. Nat Chem Biol 9:
715–720.
Address correspondence to: Dr. Adam C. Hall, Neuroscience Program,
Department of Biological Sciences, Smith College, Ford Hall 202a, North-
ampton, MA 01063. E-mail: ahall@science.smith.edu
Moraga-Cid G, Sauguet L, Huon C, Malherbe L, Girard-Blanc C, Petres S, Murail S,
Taly A, Baaden M, and Delarue M, et al. (2015) Allosteric and hyperekplexic
mutant phenotypes investigated on an a1 glycine receptor transmembrane struc-
ture. Proc Natl Acad Sci USA 112:2865–2870.