Antagonism of Inhalant and Volatile Anesthetic Enhancement of
Glycine Receptor Function*
Michael J.
Beckstead
§¶,
Rachel
Phelan§, and
S. John
Mihic§
From the
Department of Physiology and Pharmacology,
Wake Forest University School of Medicine,
Winston-Salem, North Carolina 27157 and § Section of
Neurobiology,
Waggoner Center for Alcohol and Addiction
Research, Institute for Neuroscience, and the Institute for Cellular
and Molecular Biology, University of Texas, Austin, Texas 78712
Received for publication, December 22, 2000, and in revised form, May 4, 2001
 |
ABSTRACT |
Recent studies suggest that alcohols, volatile
anesthetics, and inhaled drugs of abuse, which enhance
-aminobutyric
acid, type A, and glycine receptor-activated ion channel function, may share common or overlapping molecular sites of action on these receptors. To investigate this possibility, these compounds were applied singly and in combination to wild-type glycine
1 receptors expressed in Xenopus
laevis oocytes. Data obtained from concentration-response curves
of the volatile anesthetic enflurane constructed in the presence and
absence of ethanol, chloroform, or toluene were consistent with
competition for a common binding pocket on these receptors. A mutant
glycine receptor, insensitive to the enhancing effects of ethanol but
not anesthetics or inhalants, demonstrated antagonism of anesthetic and
inhalant effects on this receptor. Although ethanol (25-200
mM) had no effect on its own in this receptor, it was able
to inhibit reversibly the enhancing effect of enflurane, toluene, and
chloroform in a concentration-dependent manner. These data
suggest the existence of overlapping molecular sites of action for
ethanol, inhalants, and volatile anesthetics on glycine receptors and illustrate the feasibility of pharmacological antagonism of the
effects of volatile anesthetics.
 |
INTRODUCTION |
Ethanol, volatile anesthetics, and inhaled drugs of abuse are
central nervous system depressants. Although it was once thought that
they produced their effects in vivo by the nonspecific
disordering of cell membranes, it is now generally accepted that these
compounds instead exert their actions on specific proteins (1-3).
Among these protein targets are the glycine receptors
(Gly-Rs),1 responsible for
the majority of inhibitory neurotransmission in the brain stem and
spinal cord. Gly-Rs are members of a superfamily of ligand-gated ion
channels that also include the serotonin-3,
-aminobutyric acid, type
A (GABAA), and nicotinic acetylcholine receptors (4). These
receptor complexes are composed of five protein subunits surrounding a
central ion pore. Each subunit has four transmembrane spanning domains
and distinct binding sites for a variety of ligands (5). Specific
single amino acid mutations alter the actions of allosteric modulators
on Gly-Rs (6, 7), and chemically diverse compounds, such as
n-alcohols, anesthetics, and inhalants, may even share a
common binding site on these ligand-gated ion channels (3, 7-8).
Mutations affecting alcohol and volatile anesthetic actions on
receptors may hinder the physical interactions of these compounds with
their binding sites or may instead interfere with the abilities of
alcohols and anesthetics to transduce their signals to modulate channel
function after the compounds have bound.
A conventional investigation of binding sites on receptors would
include radioligand competition studies. Unfortunately the low
potencies (µM to mM) of alcohols, inhalants,
and volatile anesthetics, coupled with the current lack of an
anesthetic site antagonist, make these studies unfeasible (9).
Recently, an indirect experimental approach by Mascia et al.
(7) addressed the anesthetic-binding site/transduction site issue by
substituting cysteine for single amino acids believed to line the
anesthetic-binding pocket on homomeric
1 Gly-Rs: the
amino acids serine 267 and alanine 288. Propanethiol or propyl
methanethiosulfonate covalently cross-linked to the receptor at the
substituted cysteine at Ser-267 irreversibly enhanced Gly-R currents
and prevented further enhancement by the anesthetics enflurane or
isoflurane (7). In the present study, we demonstrate a reversible
binding site interaction through the simultaneous administration of two
positive allosteric modulators on wild-type and mutant
1
Gly-Rs. Specifically, we tested the hypothesis that ethanol (EtOH), the
volatile anesthetics enflurane (ENF) and chloroform
(CHCl3), and the inhaled drugs of abuse toluene (TOL) and
1,1,1-trichloroethane (TCE) share common or overlapping binding sites
on these receptors. Some of this work has been presented previously in
abstract form (10).
 |
EXPERIMENTAL PROCEDURES |
Materials--
Penicillin, streptomycin, gentamicin,
3-aminobenzoic acid ethyl ester, GABA, glycine, collagenase, ENF,
CHCl3, EtOH, TOL, and TCE were purchased from Sigma. All
other chemicals used were of reagent grade. Frogs of the species
Xenopus laevis were obtained from Xenopus Express
(Homosassa, FL).
Oocyte Isolation and cDNA Nuclear Injection--
The oocyte
isolation and cDNA injection procedures were performed as described
previously (3). Oocytes were injected with cDNAs (1.5 ng/30 nl) of
wild-type or mutated human glycine receptor
1-subunits
subcloned into either the pCIS2 or the pBKCMV vector, which had been
modified by removal of the lac promoter and the lacZ ATG (6). Oocytes usually expressed receptors in 1-2
days, and electrophysiological measurements were made 1-7 days after cDNA injection.
Oocyte Electrophysiological Recording--
Animal poles of
oocytes were impaled with two high resistance (0.5-10 megohms) glass
electrodes filled with 3 M KCl. Oocytes were
voltage-clamped at
70 mV using a Warner Instruments OC-725C oocyte
clamp (Hamden, CT). The concentration of agonist producing 10% of a
maximal effect (EC10) was determined for each oocyte and
subsequently used in experiments testing effects of allosteric modulators. Washout periods (5-18 min) were allowed to ensure complete
recovery of receptors from desensitization between drug applications.
During testing, oocytes were preincubated with modulator (EtOH, ENF,
CHCl3, TCE, or TOL) for 60 s before application of the
glycine/modulator solution for an additional 30 s. Oocytes were
perfused with modified Barth's saline at a rate of 2 ml/min using a
Masterflex USA peristalsis pump (Cole Parmer Instrument Co., Vernon
Hills, IL) that connected drug-containing vials to the perfusion
chamber through 18-gauge polyethylene tubing. Changes in the clamping
current were recorded on a strip-chart recorder (Cole Parmer Co.), and
the peak currents were measured and used in data analysis. Oocyte bath
concentrations of modulators were determined by gas chromatography as
described previously by Eger et al. (11).
Statistics--
Statistics were performed on data obtained from
oocytes, by one-way or two-way analysis of variance and appropriate
post hoc tests. Within-subject designs were employed whenever feasible. Data are presented as mean ± S.E.; n values refer to
the number of different oocytes from which data were obtained.
Statistical significance was defined as p < 0.05 on
paired t tests, Tukey's or Dunnett's post hoc test. All
groups of data were collected using oocytes obtained from at least two
different frogs. The theoretical curves presented in Fig. 9 were
created using the following biphasic sigmoidal Equation 1,
|
(Eq. 1)
|
where Y is the current; X is the
concentration of drug; A and D are maximal
possible currents; B and E are EC50
values, and C and F are slope constants.
A = 100, B = 1.3, C = 0.6, and D = 0 for the agonist-only curve.
A = 100, B = 2.3, C = 0.6, and D = 0 for the addition of the competitive
antagonist. The inhibitory component of the curves was created with the
addition of the second term with the following parameters:
D = 100, E = 3, and F = 0.3.
 |
RESULTS |
EtOH, TCE, ENF, and CHCl3 were applied singly and in
combination to Xenopus oocytes expressing wild-type
1 glycine receptors, and the resulting currents were
analyzed for possible interactions between compounds (Figs.
1 and 2).
Each of four compounds enhanced the currents elicited by
EC10 concentrations of glycine applied alone. The
co-application of a second modulator produced no additional effect,
except when ENF and CHCl3 were co-applied
(p < 0.01 versus ENF alone,
p < 0.01 versus CHCl3 alone,
Tukey's test; Fig. 2B).

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Fig. 1.
Co-application of 1,1,1-trichloroethane
minimally affects the enhancing effects of enflurane, chloroform, and
ethanol on Gly-Rs. TCE (0.84 mM) had no effect in the
absence of glycine but enhanced the current produced by a 10% maximal
concentration of glycine. Enhancement of Gly-R function was also
produced by 1.0 mM ENF (A), 1.5 mM
CHCl3 (B), and 200 mM EtOH
(C). When co-applied with each of the other allosteric
modulators, TCE produced little to no further enhancement compared with
that produced by the other modulator alone. Representative tracings are
illustrated above each graph. Compounds were pre-applied to
oocytes for 60 s before being co-applied with EC10
glycine for a further 30 s. Reversibility of all effects was
determined by reapplication of EC10 glycine, which was 95 µM for these particular tracings (not shown). Data are
presented as the mean ± S.E. of six oocytes.
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Fig. 2.
Effects of co-applying combinations of
ethanol, enflurane, and chloroform on Gly-R, compared with the effects
produced by the applications of single compounds. EtOH (200 mM) was unable to enhance glycinergic currents when it was
co-applied with 1.0 mM ENF (A) or 1.5 mM CHCl3 (C). However, when 1.0 mM ENF and 1.5 mM CHCl3
(B) were co-applied, there was slightly greater enhancement
than that produced by either compound alone. Compounds were pre-applied
to oocytes for 60 s before being co-applied with EC10
glycine for a further 30 s. Representative current tracings are
provided above each graph. Data are presented as the
mean ± S.E. of six oocytes.
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ENF concentration-response curves were next constructed in the
absence or presence of EtOH (Fig.
3A), TOL (Fig. 3B),
or CHCl3 (Fig. 3C). Single concentrations of
EtOH, TOL, and CHCl3 each enhanced glycine receptor
function, but these enhancing effects were progressively decreased as
the ENF concentration was increased. Consistent with the data shown in
Fig. 2B, 1.0 mM CHCl3 maintained a
significant effect in the presence of all concentrations of ENF,
whereas the enhancing effects produced by EtOH or TOL vanished when
they were co-applied with even moderate concentrations of ENF. To
determine if CHCl3 could be acting at two distinct sites on
glycine receptors, with only one site shared with EtOH, TOL, TCE, and
ENF, we replaced an amino acid believed to compose part of the putative
alcohol and anesthetic-binding site (serine 267) with the large
phenylalanine residue (S267F). This substitution completely eliminated
chloroform enhancement of currents elicited by an EC10
concentration of glycine (
0.1 ± 4.7% enhancement, n = 6, versus 265 ± 15.1%,
n = 10 in wild-type
1 Gly-R, data not
shown), thus suggesting that a second separate site for chloroform does
not exist.

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Fig. 3.
Ethanol, toluene, and chloroform enhancement
of wild-type Gly-R function decreases in the presence of increasing
concentrations of enflurane. Concentration-response curves for the
volatile anesthetic ENF (0.0-1.74 mM bath concentration)
were generated in the absence and presence of 100 mM EtOH
(A), 0.42 mM TOL (B), or 1.5 mM CHCl3 (C). All three of these
compounds enhanced Gly-R function in the absence of ENF, but this
enhancement decreased as the ENF concentration was raised. Unlike EtOH
and TOL, CHCl3 produced a statistically significant
enhancement of Gly-R function even in the presence of 1.74 mM ENF. TOL and EtOH had non-significant effects in the
presence of the high concentrations of ENF. Compounds were pre-applied
to oocytes for 60 s before being co-applied with EC10
glycine for a further 30 s. Data are presented as the mean ± S.E. of 13-16 oocytes.
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Alcohols and volatile anesthetics produce leftward shifts of glycine
concentration-response curves, and as a result, percent enhancement of
receptor function by alcohols and anesthetics decreases when higher
agonist concentrations are used. Alcohols and anesthetics have minimal
effects on peak current amplitudes when maximally effective glycine
concentrations are used. We tested whether the apparent competition
observed between ENF and the other compounds could be due to the
current produced by ENF already being maximal, thus preventing any
further enhancement by a second modulator. First we identified the
concentration of glycine (ECEQ) that closely approximated
the size of the current elicited by EC10 glycine + 1.74 mM ENF. We then tested the ability of 100 mM
EtOH to enhance currents of that size, whether elicited by a low
concentration of glycine in the presence of ENF or by a higher
ECEQ concentration of glycine in the absence of ENF. Fig.
4 illustrates that 100 mM
EtOH potentiated currents elicited by ECEQ glycine but not currents elicited by EC10 glycine in the presence of 1.74 mM ENF. Similar results were obtained with 0.56 mM TCE in the presence of 1.0 mM ENF (data not
shown).

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Fig. 4.
Ethanol enhances currents produced by a high
concentration of glycine but not a low concentration of glycine plus
enflurane. The concentration of glycine that produced a 10%
maximal response (EC10) was co-applied with 1.74 mM ENF. EtOH (100 mM) was unable to enhance
these currents. However, 100 mM EtOH enhanced currents of
identical size (p = 0.007, paired t test)
elicited by the application of a higher concentration of glycine
(ECEQ) in the absence of enflurane. Representative current
tracings are provided above the graph. Data are presented as
the mean ± S.E. of five oocytes.
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We next tested to determine if competition for a modulator-binding site
could be observed in a Gly-R mutated at a residue at or near the
putative modulator-binding pocket. When the serine residue at amino
acid 267 is mutated to an isoleucine (S267I), mutant Gly-Rs are created
that retain their sensitivities to ENF and CHCl3 but are
insensitive to EtOH (6). We tested the hypothesis that EtOH was still
binding to its putative binding site, despite having no efficacy on
S267I
1 Gly-Rs. This could be demonstrated by its
antagonism of the effects of other modulators that still had enhancing
effects on S267I Gly-Rs (Fig. 5). EtOH
(200 mM), while having essentially no effect on its own,
was able to antagonize the potentiation produced by 0.42 mM
TOL (p < 0.001, n = 5, paired t test), 0.25 mM CHCl3
(p < 0.001, n = 6), and 0.5 mM ENF (p = 0.002, n = 6).
EtOH antagonized the enhancing effects of 0.25 mM
CHCl3 in a concentration-dependent manner
(F (4,16) = 37.1, p < 0.0001], even
at EtOH concentrations as low as 25 mM (Fig. 6). No significant enhancing or
inhibiting effects of 25-200 mM EtOH were noted on S267I
1 Gly-R when it was co-applied with EC10
glycine in the absence of CHCl3. Ethanol (200 mM) antagonism of the CHCl3 enhancement of
function on S267I
1 Gly-Rs was reversible as illustrated
in the representative tracings shown in Fig.
7A. In addition to minimally
affecting the peak amplitude of the S267I
1 Gly-R,
ethanol also did not appear to affect glycine-induced steady-state
currents (Fig. 7B).

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Fig. 5.
EtOH acts as an anesthetic site antagonist on
S267I 1 Gly-Rs. TOL (0.42 mM), CHCl3 (0.25 mM), and ENF (0.5 mM) each enhanced currents elicited by EC10
glycine. These enhancing effects were antagonized when these compounds
were applied to oocytes in the presence of 200 mM EtOH.
Compounds were pre-applied to oocytes for 60 s before being
co-applied with EC10 glycine for a further 30 s.
Statistics are provided in the text. Data are presented as the
mean ± S.E. of 5-6 oocytes.
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Fig. 6.
Ethanol antagonizes chloroform enhancement of
Gly-R currents in a concentration-dependent manner.
Representative tracings illustrate that ethanol antagonism of
chloroform enhancement of S267I 1 Gly-R function occurs
in a concentration-dependent manner. A presents
data as the mean ± S.E. of five oocytes. All concentrations of
ethanol tested antagonized the enhancing effects of chloroform on S267I
1 receptors. Compounds were pre-applied to oocytes for
60 s before being co-applied with EC10 glycine for a
further 30 s.
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Fig. 7.
Ethanol antagonism of chloroform enhancement
of S267I 1 Gly-R function is
reversible. Top panel, ethanol blockade of a glycine + CHCl3 current is fully reversible after allowing for a
700-s washout period. Bottom panel, 3-min applications of
low and high concentrations of glycine in the absence and presence of
100 mM EtOH demonstrate minimal EtOH effects on peak
height, desensitization rates, and steady-state currents in S267I
1 Gly-R.
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EtOH antagonized the enhancement of receptor function produced by a
range of CHCl3 concentrations. Co-application of ethanol with CHCl3 shifted the CHCl3
concentration-response curve down and to the right (Fig.
8A). A similar shift was
observed for the anesthetic ENF (Fig. 8B), although EtOH had
no effect when co-administered with exceedingly high ENF concentrations
(~8 times the concentration required to anesthetize humans (12)). If
EtOH was competing with CHCl3 and ENF for common sites on
the S267I
1 Gly-R, we should have expected to see
rightward shifts in the CHCl3 and ENF
concentration-response curves, with no effects of EtOH at the highest
chloroform and enflurane concentrations tested. That this was not seen
is almost certainly due to an inhibitory action that occurs at higher
concentrations of volatile anesthetics, at an anesthetic site separate
from that at S267I. Evidence favoring this hypothesis was obtained
using the S267F
1 Gly-R mutant. This receptor shows no
enhancing effects of low concentrations of enflurane but demonstrates
inhibition at higher concentrations (Fig. 8C).

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Fig. 8.
Ethanol effects on chloroform and enflurane
concentration-response curves in S267I
1 Gly-Rs. Ethanol antagonizes the
enhancement of Gly-R function produced by a range of CHCl3
(A) and ENF (B) concentrations. Inhibition of
Gly-R function is observed when higher concentrations of anesthetics
are applied. We tested whether inhibition of receptor function at these
higher concentrations was independent of the enhancing effects observed
at lower concentrations. A Gly-R bearing a phenylalanine residue at
position 267 (S267F) was resistant to the enhancing effects of ENF.
This S267F Gly-R still displayed the inhibition of receptor function by
higher concentrations of ENF (C). Data are presented as the
mean ± S.E. of 4-8 oocytes.
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 |
DISCUSSION |
The serine residue at position 267 of the
1-subunit, proposed to compose part of the alcohol and
anesthetic-binding pocket in Gly-Rs, is located near the extracellular
terminus of the second of four membrane-spanning regions on the
protein. A series of single amino acid mutations made at Ser-267
1 Gly-R alter the enhancing effects of ethanol (6),
volatile anesthetics (8, 13), and inhaled drugs of abuse (3) on
GABAA and glycine receptors. An excellent linear
correlation between the effects of the ether ENF and the halogenated
alkane TCE across a panel of Ser267 mutant receptors is consistent with
a common binding site despite the dissimilarities in their chemical
structures (3). Mutations in the glycine
1-subunit at
positions 267 and 288 (in TM3) change alcohol cut off, defined as the
largest chain n-alcohol capable of enhancing receptor
function, which could be interpreted as a change in the size of the
alcohol-binding pocket (14). GABAA
2
1 (S265I)-receptors (the equivalent
amino acid as serine 267 in the
1 glycine receptor) lose
sensitivity to isoflurane but not its isomer ENF (8). Finally,
irreversible enhancement of Gly-R function is observed as a
consequence of cross-linking propyl methanethiosulfonate to S267C
mutant Gly-Rs; this also blocks further enhancement by enflurane and
isoflurane (7). A substantial body of data thus implicates this region of the glycine and GABAA receptors as being critical for
mediating the functional effects of alcohols and volatile anesthetics.
Molecular sites for propofol and other intravenous anesthetics
are located elsewhere on the protein (15), as are the sites for
benzodiazepines on the GABAA receptors (16).
As with any ligand-receptor interaction, the effect that an allosteric
modulator has on a ligand-gated ion channel depends on both the
compound and the receptor. Compounds that bind at a receptor site may
be classified as agonists, antagonists, and inverse agonists, depending
on their efficacies, which can be positive, zero, or negative,
respectively. Modulation of receptor function can be altered by either
changing the structure of the drug or by changing a relevant portion of
the receptor. For example, after residue 142 on the GABAA
receptor
2-subunit is conservatively mutated,
benzodiazepine receptor antagonists and inverse agonists function as
partial agonists (17). Mutation of
1 Gly-R
Ser-267 to isoleucine (S267I) results in a receptor that demonstrates responses to glycine similar to those of wild-type receptors and retains sensitivity to the enhancing effects of enflurane but not
ethanol (6). By mutating
1 Gly-R Ser-267 to
isoleucine, we altered modulator efficacy, changing EtOH to an
antagonist capable of antagonizing the enhancing effects of ENF, TOL,
and CHCl3. At the concentrations tested, ethanol does not
significantly decrease currents evoked by glycine in S267I
1 Gly-R, strongly suggesting that EtOH is binding to a
modulator site common to anesthetics and inhalants, but possesses
minimal efficacy, rather than binding elsewhere to favor the
stabilization of a closed conformation of the chloride channel.
The ethanol effects on ENF and CHCl3 concentration-response
curves (Fig. 8, A and B) do not initially suggest
a competitive interaction between EtOH and the volatile anesthetics in
the S267I mutant receptors. EtOH produces a rightward shift at low
concentrations of ENF and CHCl3, but the curves fail to
reach the same maximum, which would be expected if the interaction were
competitive. Despite the shapes of these concentration-response curves,
we believe competitive antagonism may be occurring at a common
alcohol/anesthetic-binding site. There are two possible explanations
for the shapes of the observed concentration-response curves. First,
EtOH could be binding to a site distinct from that of ENF and
CHCl3 and decreasing the affinities of ENF and
CHCl3 to bind. Although the shapes of the curves favor this
possibility at low concentrations of ENF and CHCl3, it is
difficult to reconcile the effects seen at the high end of the ENF
curve. The second possibility is that there are two separate sites for
ENF on these receptors, the first a pocket at Ser-267 where EtOH can
compete, and the second a low affinity inhibitory site. This second
site has long been postulated based on the non-sigmoidal nature of
volatile anesthetic concentration-response curves, including decreasing
enhancing effects at high anesthetic concentrations as well as rebound
currents that are observed upon washout of drug (18). We created a
mutant (S267F
1 Gly-R) that illustrates the
dissociation between the enhancing effects of volatile anesthetics and
their inhibiting effects (Fig. 8C). S267F
1
Gly-R show no signs of the enhancing effects of low concentrations of
enflurane but do exhibit inhibition of Gly-R function at higher concentrations of ENF. A hypothetical composite ENF
concentration-response curve is presented in Fig.
9, illustrating the potential
contribution from a second inhibitory binding site for ENF, based on
data presented here and observed by others (18). At low
anesthetic concentrations the enhancing effects predominate, but when
higher concentrations are achieved, inhibition predominates. In the
S267I receptors, EtOH antagonism of ENF binding to the higher affinity
enhancing site but not the lower affinity inhibitory site could make
competitive antagonism appear to be non-competitive.

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Fig. 9.
The downward shift of the enflurane and
chloroform concentration-response curve in S267I mutant Gly-Rs does not
necessarily indicate non-competitive antagonism. Hypothetical
sigmoidal anesthetic concentration-response curves in the absence
(A) or presence (B) of EtOH as might be expected
if EtOH and anesthetic were competing for binding to the same site in
S267I 1 Gly-R. Dashed lines C and
D illustrate the contributions of a second, independent,
inhibitory site when its effects are combined with those of the
enhancing site.
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Regardless of the mechanism responsible, our observations suggest the
possibility that compounds acting as ethanol and volatile anesthetic
receptor antagonists may exist. Currently, no known compound
competitively antagonizes anesthetics from their sites of action on
wild-type ligand-gated ion channels, although saturable and
displaceable binding of volatile anesthetics has been demonstrated on
bovine serum albumin (19), on Ca2+-ATPase (20), and in rat
brain synaptosomes (21). The ethanol antagonism of anesthetic effects
we demonstrate in S267I
1 Gly-R is a promising
indication that receptor antagonists might be designed for alcohol-,
inhalant-, and anesthetic-binding sites on wild-type GABAA
and glycine receptors. Mascia et al. (7) demonstrated that
the Cys-267
1 Gly-R residue could be irreversibly
labeled with an anesthetic compound resulting in persistent enhancement of receptor function. The current study extends this work to show that
compounds that are not covalently bound to this alcohol/anesthetic site
can also be used to demonstrate competition for binding to this site.
In summary, ethanol, volatile anesthetics, and inhaled drugs of abuse
appear to compete for a fixed number of binding sites as evidenced by
sub-additivity of enhancement of ligand-evoked currents of recombinant
Gly-Rs expressed in X. laevis oocytes. EtOH antagonized the
enhancement produced by several of these compounds on a mutated
receptor previously shown to be insensitive to EtOH. Finally, our
results suggest that it may be feasible to consider the possibility
that compounds could be designed specifically to antagonize alcohol,
inhalant, and volatile anesthetic effects on ligand-gated ion channels.
Such discoveries could lead to improved treatments for acute alcohol,
anesthetic, or inhalant overdose, or even new pharmacotherapeutic
approaches for alcoholism.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Peter Schofield for providing
the
1 Gly-R cDNA used in this study. We also thank
Drs. Adron Harris and Susan Bergeson for helpful discussions.
 |
FOOTNOTES |
*
This work was supported by National Institute on Alcohol
Abuse and Alcoholism Grant AA11525 (to S. J. M.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Section of
Neurobiology, 2500 Speedway, MBB 1.148, University of Texas, Austin, TX
78712-1095. Tel.: 512-232-7173; Fax: 512-232-2525; E-mail: beckstead@
mail.utexas.edu.
Published, JBC Papers in Press, May 9, 2001, DOI 10.1074/jbc.M011627200
 |
ABBREVIATIONS |
The abbreviations used are:
Gly-R, glycine
receptor;
GABAA,
-aminobutyric acid, type A;
EtOH, ethanol;
ENF, enflurane;
CHCl3, chloroform;
TOL, toluene;
TCE, 1,1,1-trichloroethane;
ECX, the concentration of agonist
(glycine) that produces X% of a maximal current.
 |
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