Enantioselectivity of α-Benzyl-α-methyl-γ -butyrolactone-Mediated Modulation of Anticonvulsant Activity and GABA A Receptor Function

Article abstract of DOI:10.1124/jpet.103.063008

Alkyl-substituted butyrolactones have both inhibitory and stimulatory effects on GABA_A receptors. Lactones with small alkyl substitutions at the α-position positively modulate the channel, whereas β-substituted lactones tend to inhibit the GABA

Full text of DOI:10.1124/jpet.103.063008

0022-3565/03/3092-677–683$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics
JPET 309:677–683, 2003
Vol. 309, No. 2
63008/1142230
Printed in U.S.A.
Enantioselectivity of ␣-Benzyl-␣-methyl-␥-butyrolactone-
Mediated Modulation of Anticonvulsant Activity and GABAA
Receptor Function
Eric B. Gonzales, Cathy L. Bell-Horner, Maria Antonette M. de la Cruz,
James A. Ferrendelli, Douglas F. Covey, and Glenn H. Dillon
Department of Pharmacology and Neuroscience, University of North Texas Health Science Center at Fort Worth, Fort Worth,
Texas (E.B.G., C.L.B.H., G.H.D.); Department of Molecular Biology and Pharmacology, Washington University School of
Medicine, St. Louis, Missouri (M.A.M.C., D.F.C.); and Department of Neurology, University of Texas-Houston Medical School,
Houston, Texas (J.A.F.)
Received November 14, 2003; accepted January 20, 2004
ABSTRACT
Alkyl-substituted butyrolactones have both inhibitory and stim- CF-1 mice. The (ϩ)-enantiomer inhibited binding of t-butylbi-
ulatory effects on GABAA receptors. Lactones with small alkyl cyclophosporothionate with a higher affinity than the (Ϫ)-enan-
substitutions at the ␣-position positively modulate the channel, tiomer (IC50 of 0.68 and 1.1 mM, respectively). Whole cell
whereas ␤-substituted lactones tend to inhibit the GABAA re- patch-clamp recordings from recombinant ␣1␤2␥2 receptors
ceptor. These compounds mediate inhibition through the pic- stably expressed in HEK293 cells demonstrated that both com-
rotoxin site of the receptor. A distinct binding site that mediates pounds stimulated GABA-activated current. The maximal stim-
the stimulatory actions of lactones is presumed to exist, al- ulation was approximately 2-fold greater with (ϩ)-␣-BnMeGBL
though no definitive evidence to support this claim exists. In the than that seen with (Ϫ)-␣-BnMeGBL. Both enantiomers of
present study, we used in vivo and in vitro assays to evaluate ␣-BnMeGBL directly gated the GABAA receptor at mM concen-
the effects of the enantiomers of a novel lactone, ␣-benzyl-␣- trations, in a nonstereoselective manner. Our data demonstrate
methyl-␥-butyrolactone (␣-BnMeGBL), on the GABAA receptor. the stimulatory actions of ␣-BnMeGBL on GABAA receptor
R-(Ϫ)-␣-BnMeGBL was 2-fold more potent than the S-(ϩ)-␣- function display enantioselectivity and provide strong evidence
BnMeGBL in blocking pentylenetetrazol-induced seizures in for the existence of a true “lactone site” on the receptor.
The ␥-aminobutyric acidA receptor (GABAAR) is the pre- include the benzodiazepines, steroids, barbiturates, and the
dominant inhibitory receptor in the nervous system (Mac-
donald and Olsen, 1994). The receptor is composed of protein
subunits (␣, ␤, ␥, ␦, ⑀, ␳, ␪, and ␲) (Barnard et al., 1998;
Bonnert et al., 1999) that are arranged in a pentameric
conformation (Barnard et al., 1998). Once activated by the
ligand GABA, the protein changes its conformational struc-
ture from a closed to an open state, allowing ClϪ ions into the
cell. GABAARs are members of the ligand-gated ion channel
superfamily, which include glycine receptors, 5-hydroxytryp-
tamine3 receptors, and the nicotinic acetylcholine receptors
(Ortells and Lunt, 1995). Several classes of compounds bind
to the receptor and influence the channel’s action. These
convulsant picrotoxin (Hevers and Luddens, 1998).
A novel class of compounds that regulate the GABAA receptor
are the ␥-butyrolactones (GBLs) and the related ␥-thiobutyro-
lactones (TBLs). The lactones have either positive or negative
modulatory activity, depending on the substitution incorpo-
rated in the lactone ring. These compounds have been substi-
tuted with alkyl groups (Mathews et al., 1996) and fluorinated
groups (Canney et al., 1998). The presence of relatively small
substituents at the ␣-position generally results in anticonvul-
sant activity and potentiation of GABA-gated ClϪ current,
whereas lactones with larger substituents at this position may
display convulsant activity (Canney et al., 1991; Holland et al.,
1992). Alkyl substitution at the ␤-position results in convulsant
activity and a decrease in GABA-activated current (Klunk et
al., 1982b; Holland et al., 1990b; Yoon et al., 1993), whereas
unsubstituted ␥-butyrolactone has no significant effect on
GABA current (Feigenbaum and Howard, 1996).
This work was supported by National Institutes of Health Grant ES07904
(to G.H.D.) and NS14834 (to D.F.C.).
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
DOI: 10.1124/jpet.103.063008.
ABBREVIATIONS: GABAAR, type A GABA receptor; GBL, ␥-butyrolactone; TBL, ␥-thiobutyrolactone; TBPS, t-butylbicyclophosporothionate;
␣-BnMeGBL, ␣-benzyl-␣-methyl-␥-butyrolactone; HEK, human embryonic kidney; THF, tetrahydrofuran; IR, infrared; PTZ, pentylenetetrazole.
677

678
Gonzales et al.
After the discovery that lactones competitively inhibit test solutions were less than 0.3% (v/v). [35S]TBPS was obtained
binding of the picrotoxin-site ligand [35S]TBPS to the
from PerkinElmer Life Sciences (Boston, MA).
GABAAR (Levine et al., 1985; Holland et al., 1990a), it was
concluded that the effects of lactones are due to binding at
the picrotoxin site. Convulsant (GABA current-inhibiting) or
Synthesis of Racemic ␣-BnMeGBL
General Methods. Unless otherwise noted, the starting materi-
als were obtained from commercial suppliers and used without fur-
anti-convulsant (GABA current-enhancing) activity was pre-
ther purification. Dichloromethane (CH2Cl2) was distilled over cal-
sumed to be due the ability of lactones to act as either
cium hydride. Hexamethylphosphoramide was dried over activated
13ϫ molecular sieves. Tetrahydrofuran (THF) was distilled over
sodium-benzophenone. A Varian model 3700 or 3400 CX gas chro-
matograph equipped with a glass column (0.25-in. i.d., 6-ft length)
packed with 15 SP2401 on 80/100 mesh Supelcoport (Supelco, Belle-
positive or negative modulators, respectively, of the picro-
toxin site (Holland et al., 1990a). Subsequent studies led to
the belief that the inhibitory effects of the lactones are in fact
due to an interaction at the picrotoxin site (Yoon et al., 1993;
Williams et al., 1997) but that the positive modulatory ac- fonte, PA) was used to monitor the progress of the reactions and to
assess the purity of the products. Solvents used for extractions were
dried over either Na2SO4 or MgSO4, filtered, and removed on a
rotary evaporator. Thin layer chromatography was performed on
250-␮m Analtech silica gel plates containing a fluorescent indicator.
Flash chromatography was carried out by using Scientific Adsor-
bents (Atlanta, GA) 40-␮m silica gel. Bulb-to-bulb distillations were
performed on an Aldrich Kugelrohr apparatus. Melting points were
determined with either a Thomas-Hoover capillary or Kofler-type
micro hot stage apparatus and are uncorrected. IR spectra were
obtained with PerkinElmer 1710 FT-IR instrument as thin films on
tions are due to action at a “lactone site” (Holland et al., 1995;
Williams et al., 1997).
Whether a true lactone binding site exists on the GABAAR
is unknown. It is possible, for example, that the GABA cur-
rent-enhancing effects of lactones are secondary to relatively
nonselective alterations of the local physiochemical environ-
ment, which could subsequently influence GABAAR activity.
One approach to provide evidence of a ligand binding site is
demonstration of enantioselective preference. Enantiomers
are nonsuperimposable mirror images of optically active mol- NaCl plate, and NMR spectra (1H NMR and 13C) were recorded in
CDCl3 solutions on a Varian Gemini-300 spectrometer. Chemical
shifts are expressed in parts per million (␦) relative to tetramethyl-
silane as an internal standard. Elemental analyses were performed
at M-H-W Laboratories (Phoenix, AZ).
Racemic ␣-BnMeGBL was prepared by benzylation of ␣-methyl-␥-
butyrolactone. A solution of ␣-methyl-␥-butyrolactone (10.0 g, 100
mmol) in THF (15 ml) was added slowly (ca. 15 min) to a solution of
lithium diisopropylamine [prepared from diisopropylamine (21.0 ml,
150 mmol) and n-butyllithium in hexanes (2.5 M, 57.2 ml, 143
mmol)] in THF (175 ml) at 0°C in a N2 atmosphere, and the mixture
ecules (i.e., compounds with at least one chiral center). They
have identical physiochemical properties, and thus any ef-
fects of them due to alterations of membrane lipids would be
identical (Guyer and Block, 1983; Arnett and Gold, 1986;
Mannock et al., 2003; Westover et al., 2003). Targets that
display an enantioselective preference are thus likely to have
a specific binding site for the parent molecule. GABAARs
have been shown to display enantioselective interactions
with several compounds. These include the volatile anesthet-
ics halothane (Harris et al., 1998) and isoflurane (Hall et al., was stirred for 30 min. The temperature was then decreased to
Ϫ78°C, and a solution of benzyl bromide (26.9 g, 157 mmol) in THF
(10 ml) and hexamethylphosphoramide (15 ml, 86.6 mmol) was
added over a period of 10 min. After 2 h at Ϫ78°C, the reaction
mixture was allowed to warm to room temperature (ca. 3 h), and
stirring was continued overnight (ca. 11 h). The reaction was
quenched by addition of HCl (3 N, 200 ml) at 0°C. The layers were
separated, the aqueous phase was further extracted with diethyl
ether (3 ϫ 75 ml), and the combined organic extract was washed with
100-ml portions of water, saturated NaHCO3, water, and brine.
Removal of the solvent gave 27.7 g of pale yellow-colored viscous
residue, which upon flash chromatography over silica gel (EtOAc/
hexanes, 1:9) followed by bulb-to-bulb distillation [pot temperature
125–130°C (0.4 mm Hg)], afforded the previously described (Jakovac
and Jones, 1979) ␣-BnMeGBL (17.6 g, 92%) as a colorless solid:
melting point 55–56°C (from diethyl ether-pentane at Ϫ5°C); IR 1762
(CϭO), 1604 cmϪ1 (CϭC); 1H NMR ␦ 7.33 to 7.18 (m, 5, PhH), 4.15 to
4.07 (m, 1, diastereotopic H of H-5), 3.78 to 3.71 (m, 1, diastereotopic
H of H-5), 3.04 (d, 1, J ϭ 13.4 Hz, diastereotopic H of CH2Ph), 2.74
(d, 1, J ϭ 13.4 Hz, diastereotopic H of CH2Ph), 2.33 to 2.24 (m, 1,
diastereotopic H of H-4), 1.98 to 1.89 (m, 1, diastereotopic H of H-4),
1.30 (s, 3, CH3); 13C NMR ␦ 181.71, 136.67, 129.93, 128.39, 126.94,
64.98, 43.84, 43.37, 33.27, 23.60. Analysis Calculated for C12H14O2:
C, 75.76; H, 7.42. Found: C, 75.61; H, 7.50.
1994), barbiturates (Rho et al., 1996), and neurosteroids
(Covey et al., 2001).
To more definitively evaluate the possibility that a lactone
binding site exists on the GABAAR, we have synthesized
enantiomers of a novel butyrolactone, ␣-benzyl-␣-methyl-␥-
butyrolactone (␣-BnMeGBL), and studied its actions using in
vitro and in vivo approaches. Our results demonstrate that
␣-BnMeGBL enantioselectively interacts with GABAA recep-
tors and provide the strongest evidence to date in support of
the existence of a lactone binding site on GABAARs.
Materials and Methods
Chemicals
␣-BnMeGBL enantiomers (Fig. 1) and racemic solutions were
made from a 1 M dimethyl sulfoxide stock and diluted in 5 ␮M GABA
in normal saline. The final dimethyl sulfoxide concentrations in the
Separation of Racemic ␣-BnMeGBL into (؉)- and (؊)-Enan-
tiomers. The racemic ␣-BnMeGBL was reacted with S-(Ϫ)-␣-meth-
ylbenzylamine using an established literature procedure to obtain a
65% yield of a mixture of the diastereomeric ring opened ␥-hydroxy-
amides (Bigg and Lesimple, 1992). The diastereomers were sepa-
rated by column chromatography using hexanes/ethyl acetate (1:4)
as solvents. Each pure diastereomer was obtained as white crystals.
Each pure diastereomer was then hydrolyzed with 1 N H2SO4 in
refluxing ethylene glycol to give the ␥-hydroxyacids, which lacton-
ized in situ to give pure (ϩ)- and (Ϫ)-␣-BnMeGBL in 96% yields. The
Fig. 1. Structure of ␣-benzyl-methyl-␥-butyrolactone enantiomers.

Enantioselective Effects of a Novel Lactone
679
(ϩ)-␣-BnMeGBL had [␣]D ϭ (ϩ) 60 and the (Ϫ)-␣-BnMeGBL had tection with 95% confidence (dose-response curve confidence inter-
[␣]D ϭ (Ϫ) 59.
vals at 50% response).
Assignment of R and S Absolute Configurations to the (؉)-
Electrophysiology. The whole-cell patch-clamp technique was
and (؊) Enantiomers. The S absolute configuration for the chiral used to record GABA-induced ClϪ currents. Patch pipettes of boro-
center in the (ϩ) enantiomer was established by the following reac- silicate glass (1B150F; WPI Sarasota, FL) were pulled (Flaming/
tion sequences. The (ϩ)-BnMeGBL was reduced with diisobutylalu-
minum hydride to give 2-benzyl-2-methyl-1,4-butanediol, which had
[␣]D ϭ (ϩ) 4.1. An authentic sample of (S)-n-butyl 2-benzyl-2-methyl-
3-benzoylpropionate was then prepared according to a literature
procedure (Meyers et al., 1990). The benzoyl group of this ketoester
was then oxidized with sodium perborate in refluxing trifluoroacetic
acid to a phenyl ester group, yielding (S)-2-benzyl-2-methylsuccinic
acid phenyl ester, n-butyl ester. Reduction of this diester with lith-
ium aluminum hydride gave (S)-2-benzyl-2-methyl-1,4-butandiol,
which had [␣]D ϭ (ϩ) 3.2. Because the same diol when obtained from
the (ϩ)-␣-BnMeGBL also had a positive optical rotation, (ϩ)-␣-Bn-
MeGBL is established as (S)-(ϩ)-␣-BnMeGBL.
Cloned GABAA Receptors. Human embryonic kidney cells sta-
bly transfected with rat ␣1␤2␥2 GABAA receptors were studied in
this investigation. Cells expressing these receptors were generously
supplied by Pharmacia (Kalamazoo, MI). Preparation and culture of
these cells have been described in detail previously (Hamilton et al.,
1993).
Brown, P-87/PC; Sutter Instrument Co., Novato, CA) to a tip resis-
tance of 1 to 2.5 M⍀ when filled with the following pipette solution:
140 mM CsCl, 10 mM EGTA, 10 mM HEPES, and 4 mM Mg-ATP; pH
7.2. Coverslips containing cultured cells were placed in a small
chamber (ϳ1.5 ml) on the stage of an inverted light microscope
(IMT-2; Olympus, Tokyo, Japan) and superfused continuously (5–8
ml/min) with the following external solution containing 125 mM
NaCl, 5.5 mM KCl, 0.8 mM MgCl2, 3.0 mM CaCl2, 20 mM HEPES,
and 10 mM D-glucose, pH 7.3. GABA-induced ClϪ currents were
obtained using an Axoclamp 200A amplifier (Axon Instruments,
Foster City, CA) equipped with a CV-4 headstage. GABA currents
were low-pass filtered at 5 kHz, monitored on an oscilloscope and a
chart recorder (Gould TA240), and stored on a computer (pClamp
6.0; Axon Instruments) for subsequent analysis. Initial patch char-
acteristics were used as reference throughout the experiment. If a
change in access resistance was observed during the recording pe-
riod, the patch was aborted and the data were not included in the
analysis. All recordings were collected at room temperature with
cells voltage-clamped at Ϫ60 mV.
Radioligand Binding. Competitive radioligand binding studies
were conducted as described previously (Levine et al., 1985). Briefly,
Sprague-Dawley male rats were dissected and their cerebral hemi-
spheres were isolated. Membrane fractions were made and stored at
Ϫ70° until time of experiment. At the time of experiment, fractions
were resuspended with a Polytron and protein concentration was
determined. The assay consisted of 100 ␮l of tissue homogenate with
50 ␮l [35S]TBPS (2 nM) in 1 M NaBr and 50 ␮l of Tris-citrate buffer
(pH 7.5 at 0°C) containing either no additive or varying concentra-
tions of either enantiomer of ␣-BnMeGBL. The samples were incu-
bated at 25°C for 90 min., followed by dilution with 3 ml 0.9% NaCl.
Samples were filtered through Whatman GF/B microfiber filter discs
with slight vacuum applied and washed twice with 3 ml of 0.9%
NaCl. Radioactivity was determined using conventional liquid scin-
tillation counting. Nonspecific binding was characterized with unla-
beled 10 ␮M TBPS and was subtracted to compute specific binding.
␣-BnMeGBL concentration-response data were fitted to the equation
I/Imax ϭ [␣-BnMeGBL]n/([␣-BnMeGBL]n ϩ IC50n), where I is the
degree of inhibition of [35S]TBPS binding at a given concentration of
␣-BnMeGBL, Imax is maximal inhibition observed with ␣-BnMeGBL,
IC50 is the half-maximal effective concentration of ␣-BnMeGBL, and
n is the Hill coefficient.
Experimental protocol. Modulatory effects of ␣-BnMeGBL enanti-
omers were assessed using an EC20 concentration (5 ␮M) of GABA.
GABA with or without ␣-BnMeGBL was prepared in the extracellu-
lar solution and applied (10 s) to each cell by gravity flow using a
Y-shaped tube positioned adjacent to the cell. Control responses were
established by observing two consecutive GABA-induced currents
that varied in amplitude by no more than Ϯ10%. After establishing
the control, effects of lactones at varying concentrations were deter-
mined. Recovery from the lactones was often fully obtained, thus a
complete concentration-response profile could generally be obtained
from a single cell. In experiments where the ability of ␣-BnMeGBL
enantiomers to directly activate the GABAAR was determined, 3 mM
␣-BnMeGBL was used. Concentration-response profiles for the pos-
itive modulatory actions of lactones were generated (Origin 5.0;
OriginLab Corp., Northampton, MA) using the equation I/Imax
ϭ
[␣-BnMeGBL]n/([␣-BnMeGBL]n ϩ EC50n), where I is normalized cur-
rent amplitude at a given concentration of ␣-BnMeGBL, Imax is the
maximum GABA current induced by ␣-BnMeGBL, EC50 is the half-
maximal effective concentration of ␣-BnMeGBL, and n is the Hill
coefficient.
Pentylenetetrazole (PTZ) Convulsant Assay. A PTZ convul-
sant assay was performed using female CF-1 mice (Canney et al.,
1991). All procedures were performed in accordance with the Na-
tional Institutes of Health Guide for Care and Use of Laboratory
Animals. Seizures were induced with PTZ (85 mg/kg) dissolved in
0.9% NaCl. (ϩ)-␣-BnMeGBL, (Ϫ)-␣-BnMeGBL or a racemic mixture
was dissolved in varying concentrations of 30% polyethylene glycol,
and a volume of 0.01 ml/g body weight was administered via intra-
peritoneal injection 30 min before the PTZ injection. Mice were
monitored for seizure activity for up to 30 min after PTZ adminis-
tration. Protection from PTZ-induced seizures was defined as an
absence of convulsions with application of test compound. Ability of
either enantiomer of ␣-BnMeGBL to block PTZ-induced seizures was
recorded at the observed concentration. The experimental data
pooled from two independent experiments for each tested drug were
described as dose-response curves together with 95% confidence
intervals by fitting them into Hill equation using nonlinear curve fit
procedures (R2 Ն 0.949) (SigmaPlot 2000; SPSS Science, Chicago,
IL). The ED50 values, defined as drug concentrations affording 50%
protection, were determined numerically (Calculation Center, Wol-
fram Research, Champaign, IL). The 95% confidence intervals for
ED50 values were determined graphically by reading off the graph
Results
Influence of ␣-BnMeGBL Enantiomers on [35S]TBPS
Binding. The lactone class of compounds has been shown
previously to bind at or near the site of action of convulsants,
particularly TBPS and picrotoxin (Canney et al., 1991). The
binding affinity for this class of compounds to the convul-
sants site ranges from low micromolar to millimolar dissoci-
ation constants (Canney et al., 1991; Holland et al., 1993).
Thus, we investigated the ability of ␣-BnMeGBL isomers to
inhibit [35S]TBPS binding to GABAA receptors in isolated rat
cerebral cortical membranes. (ϩ)-␣-BnMeGBL displaced
[
35S]TBPS with an IC50 of 0.68 Ϯ 0.01 mM. The ability of
(Ϫ)-␣-BnMeGBL to displace [35S]TBPS was less (IC50
ϭ
1.1 Ϯ 0.01 mM) that that seen with the (ϩ)-enantiomer.
Compared with ␣-IMGBL, the (ϩ)-isomer of ␣-BnMeGBL
displayed higher affinity (Holland et al., 1993). Holland et al.
(1993) demonstrated that alkyl-substituted ␥-butyrolactones
do not compete for the TBPS site. These lactones act at a
the minimum and maximum drug concentrations offering 50% pro- distinct, allosteric site that increases the dissociation rate of

680
Gonzales et al.
TABLE 1
35
Effects of ␣-benzyl-␣-methyl-␥-butyrolactone on seizure activity, ͓S͔TBPS displacement, and GABA-gated ClϪ current
35
Compound
Anticonvulsant Potencya
͓S͔TBPS Displacementb
GABA Current Potentiationc
%
(ϩ)-␣-BnMeGBL
(Ϫ)-␣-BnMeGBL
(Ϯ)-␣-BnMeGBL
65 (53–84)
158 (97–250)
65 (50–90)
0.68 Ϯ 0.01
1.1 Ϯ 0.01
0.92 Ϯ 0.01
149 Ϯ 32
80 Ϯ 8.7
ND
ND, not determined.
a ED50 (milligrams per kilogram) against PTZ-induced seizures, 95% confidence interval in parentheses, n ϭ 6 to 12 for each compound. Doses tested were 0, 25, 50, 100,
and 200 mg/kg for (ϩ)-BnMeGBL; 0, 25, 50, 100, 200, and 300 mg/kg for (Ϫ)-BnMeGBL; and 20, 25, 40, 50, 80, 100, 200, and 250 mg/kg for (Ϯ)-BnMeGBL.
35
b Mean IC50 to inhibit ͓S͔TBPS binding in cerebral cortex of rat; errors are S.E.M. for duplicate experiments run in triplicate.
c Maximal stimulation above control of GABA (5 ␮M) current recorded from HEK293 cells expressing rat ␣1␤2␥2 GABAA receptors; n ϭ 5.
the radioligand. ␣-BnMeGBL may interact with the receptor
in a similar manner to inhibit TBPS binding.
Effects of ␣-BnMeGBL Enantiomers on GABA-Acti-
vated Cl؊ Current. If the anticonvulsant effects of ␣-Bn-
Anticonvulsant Activity of ␣-BnMeGBL Enanti- MeGBL are due to their interaction with GABAARs, the
omers. ␥-Butyrolactones have been characterized as convul- above-mentioned results suggest ␣-BnMeGBL should also
sant or anticonvulsant, depending in large part on the loca- modulate GABA-gated currents in an enantioselective man-
tion of the substituent on the lactone ring (Klunk et al., ner. We thus evaluated the actions of (ϩ)-␣-BnMeGBL and
1982a,b; Canney et al., 1991). Whether an enantioselective (Ϫ)-␣-BnMeGBL on GABA-activated currents recorded from
effect of the anticonvulsant actions of ␥-butyrolactones exists human embryonic kidney 293 cells stably expressing ␣1␤2␥2
has never been studied. We thus tested the hypothesis that GABAARs. (ϩ)-␣-BnMeGBL facilitated GABA current in a
␣-BnMeGBL would protect against PTZ-induced convulsions, concentration-dependent manner (Fig. 2A). Maximal en-
and whether this protection might be enantioselective. Both hancement of GABA current was roughly 2.5-fold of the con-
enantiomers of ␣-BnMeGBL and the racemic mixture inhib- trol and occurred at 2 mM (ϩ)-␣-BnMeGBL. The EC50 value
ited PTZ-induced convulsions. A clear enantioselective effect and Hill coefficient were estimated to be 0.5 Ϯ 0.08 mM and
was observed, because the potency of the (ϩ)-␣-BnMeGBL 1.3 Ϯ 0.25, respectively. At the highest concentrations (at
was more than 2-fold greater than that observed with (Ϫ)-␣- and above 1 mM), enhancement of current decay became
BnMeGBL (Table 1).
apparent.
Fig. 2. Enantioselective modulation of GABA-activated ClϪ current by ␣-BnMeGBL. Aa, typical response of GABA-activated currents to increasing
concentrations of (ϩ)-␣-BnMeGBL. Currents were recorded from rat ␣1␤2␥2 receptors expressed in HEK293 cells. (ϩ)-␣-BnMeGBL caused current
enhancement up to a concentration of 2 mM and then facilitated current decay. Ab, mean results illustrating the effects of (ϩ)-␣-BnMeGBL. Current
amplitude was maximally increased roughly 2.5-fold compared with the control. Concentration-response analysis yielded an EC50 value of 0.5 Ϯ 0.08
mM and a Hill coefficient of 1.3 Ϯ 0.25. The data point recorded at the highest concentration of (ϩ)-␣-BnMeGBL was not included in the curve fitting
analysis due to the onset of current inhibition. Ba, typical response of GABA-activated currents to increasing concentrations of (Ϫ)-␣-BnMeGBL.
Whereas (Ϫ)-␣-BnMeGBL also facilitated GABA-gated current, the magnitude of enhancement was approximately 50% of that observed with the (ϩ)
enantiomer. (Ϫ)-␣-BnMeGBL also displayed a biphasic effect, because current decay was enhanced at concentrations above 1 mM. Bb, mean results
illustrating the effects of (Ϫ)-␣-BnMeGBL. n ϭ a minimum of four cells tested at each concentration for both enantiomers. Scale bars represent current
amplitude in picoamperes and time in seconds.

Enantioselective Effects of a Novel Lactone
681
(Ϫ)-␣-BnMeGBL also enhanced GABA-gated current in
␣1␤2␥2 receptors (Fig. 2B). Although the apparent affinity of
the negative enantiomer (EC50 ϭ 0.22 Ϯ 0.02 ␮M) was higher
than that of (ϩ)-␣-BnMeGBL, it had roughly 50% of the
efficacy observed with the (ϩ)-enantiomer. As seen with the
(ϩ)-enantiomer, high concentrations of (Ϫ)-␣-BnMeGBL elic-
ited enhancement of current decay (Fig. 2B, traces at 2 and 3
mM). It should be noted that the presence of both inhibitory
and direct activating effects of lactones at high concentra-
tions (below) make accurate determination of EC50 and Hill
coefficient values difficult. Nevertheless, the estimated val-
ues do provide some useful comparative value. The enantio-
selectivity seen with both anticonvulsant actions and GABA
current-enhancing actions strongly supports the existence of
a lactone binding site on the GABAA receptor. Table 1 sum-
marizes the data for binding, convulsant, and patch-clamp
experiments.
␣-BnMeGBL Directly Activates the GABAA Receptor.
Barbiturates and other anesthetics, in addition to allosteri-
cally modulating the GABAAR, can also directly activate the
channel (Rho et al., 1996; Krasowski et al., 1998). The ability
of ␥-butyrolactone compounds to directly activate the
GABAAR has not been fully explored. We thus evaluated
whether enantiomers of ␣-BnMeGBL could directly open
␣1␤2␥2 GABAARs. As shown in Fig. 3, high concentrations (3
mM) of each enantiomer resulted in GABAAR activation. The
direct activating effect was relatively modest, i.e., about 30%
of the amplitude of the response seen with application of 5
␮M GABA (GABA EC20). Due to solubility considerations, we
did not evaluate direct activating effects of ␣-BnMeGBL at
higher concentrations. At a concentration of 3 mM, however,
where a clear enantioselective effect was seen for modulatory
effects of GABAAR activity, no enantioselective effect was
observed for direct activating effects of ␣-BnMeGBL.
Fig. 3. Direct activation of GABAA receptors by ␣-BnMeGBL. A, initial
trace is response to application of 5 ␮M GABA. Subsequent traces dem-
onstrate the response to each enantiomer when applied in the absence of
GABA. Application of either enantiomer of ␣-BnMeGBL (3 mM) resulted
in opening of GABAA receptors. B, mean results from above-mentioned
experiments (n ϭ 3 cells for each enantiomer). The direct channel acti-
vating effects were the same for both enantiomers, and the magnitude of
resulting currents was modest compared with the response to 5 ␮M
GABA (EC20 concentration).
Discussion
Despite extensive study, definitive evidence for the exis-
tence of a lactone binding site on the GABAAR has proven to
be elusive. Whereas early studies led to the conclusion that
both positive modulatory (GABA current-enhancing) and
negative modulatory (GABA current-inhibiting) actions of
the lactone compounds were due to an interaction at the
picrotoxin site of the receptor (Holland et al., 1991), subse-
quent work led to the suggestion that positive modulatory
actions of lactones resulted from modulation of a novel site
(Yoon et al., 1993; Holland et al., 1995). The fact that both
GABA current stimulatory and inhibitory effects of lactones
are not due to differential modulation of a single site is best
supported by the results of Williams et al. (1997). They
showed that the ability of convulsant lactones to inhibit
GABA-activated current was abolished by a mutation known
to also abolish sensitivity to picrotoxin. However, the GABA
current-enhancing effects of lactones could not be abolished
by this mutation. Although the results of Williams et al.
(1997) are consistent with the suggestion that a lactone bind-
ing site is present on the GABAAR, alternative explanations
Effects of ␣-BnMeGBL Are Similar to Other ␣-Sub-
stituted Lactones. The actions of the novel butyrolactone
␣-BnMeGBL were evaluated using both in vivo (anticonvul-
sant) and in vitro (whole-cell patch-clamp recording, radioli-
gand binding) assays. Its ability to protect against pentyle-
netetrazole-induced seizures and to enhance GABA-
activated ClϪ currents is comparable with that seen with
other ␣-substituted lactones (Canney et al., 1991). ␣-Bn-
MeGBL also inhibited binding of the picrotoxin site ligand
[
35S]TBPS. Like with other ␣-substituted lactones, displace-
ment of [35S]TBPS requires much higher concentrations than
necessary with ␤-substituted lactones (Canney et al., 1991).
At higher concentrations of ␣-BnMeGBL (above 1 mM),
inhibition of GABA-activated current became apparent. This
effect, which has also been observed for other ␣-substituted
lactones (Williams et al., 1997), can be explained by several
possibilities. Our radioligand binding studies demonstrate
are not precluded. Thus, the conclusion that a true lactone that ␣-BnMeGBL inhibits [35S]TBPS with IC50 values near 1
site exists on the receptor requires additional verification. mM. Thus, an obvious possibility is that the inhibition could
Results of the present studies provide additional evidence in reflect effects due to binding at the convulsant site predom-
support of this conclusion.
inating over effects due to binding at the positive modulatory

682
Gonzales et al.
lactone site. Alternatively, as noted by Williams et al. (1997), of barbiturates and ␣-BnMeGBL occurs through a similar
the decrease in GABA current induced by ␣-BnMeGBL may domain of the receptor remains to be determined.
be due to its facilitation of entry into the desensitized state.
The results of the present study provide further evidence
Finally, the existence of another low affinity inhibitory site that positive modulatory actions of butyrolactones and thio-
for lactones cannot be ruled out. Williams et al. (1997) found butyrolactones are due to interaction with a distinct protein
that GABAA receptors expressing a mutation that rendered binding site. Although we presume the binding site is located
them insensitive to the inhibitory effects of ␮M ␤-EMTBL on the GABAA receptor itself, the possibility that lactones
were still inhibited by higher concentrations (millimolar) of may be binding to an associated protein that regulates
␤-EMTBL.
GABAA receptor function cannot be dismissed. Distinguish-
Enantioselectivity of ␣-BnMeGBL. Although in the ing between these two possibilities will require additional
present studies we have characterized a novel anticonvulsant studies.
lactone, the major goal of this research was to evaluate
whether enantiomers of this lactone had differential anticon-
vulsant and GABAAR-modulating properties. Because enan-
tioselective preference is typically considered to be evidence
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Address correspondence to: Dr. Glenn H. Dillon, Department of Pharma-
cology and Neuroscience, University of North Texas Health Science Center at
Fort Worth, 3500 Camp Bowie Blvd., Fort Worth, TX 76107. E-mail:
gdillon@hsc.unt.edu