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INTRODUCTION |
Depression of central nervous system function by ethanol is a
complex phenomenon (1, 2), which may involve several neurotransmitter systems. Ligand-gated ion channels play an important role in the regulation of neuronal excitability and are sensitive to
pharmacologically relevant concentrations of ethanol. Several such
channels are thought to be involved in the behavioral effects of
ethanol, for example the
NMDA1 and non-NMDA subtypes
of glutamate receptors (3-5) and the GABAA receptors (for
review, see Ref. 6). Recent data also suggest that the related glycine
receptor (Gly-R) may also be an important site of alcohol action.
Gly-Rs constitute the major inhibitory neurotransmitter receptor system
in the brainstem and spinal cord but are also found in significant
numbers in higher brain regions such as the olfactory bulb, midbrain,
cerebellum, and cerebral cortex (7). Evidence has recently been
obtained to implicate the Gly-R in some of the behavioral effects of
ethanol. Intracerebroventricular administration of glycine augments
ethanol-induced loss of righting reflex in the mouse (8), and this
effect is blocked by the glycine antagonist strychnine. Enhancement of
Gly-R function would be consistent with the behavioral actions of
ethanol in vivo. Electrophysiological studies have shown
that ethanol enhances Gly-R function in spinal cord neurons from mouse
and chick (9, 10), and increases glycine-mediated Cl
uptake into rat brain synaptoneurosomes (11). Recently, Mascia et
al. (12, 13) demonstrated ethanol enhancement of the function of
Gly-R of defined composition expressed in Xenopus oocytes, with concentrations of ethanol as low as 10 mM being
effective in potentiating the actions of low concentrations of glycine. Gly-Rs are members of the "superfamily" of related ligand-gated ion
channels (14-16). These receptors are pentameric complexes, with the
five subunits arranged around a central pore through which permeant
ions pass. The composition of most native neuronal glycine receptors is
believed to be
1
1
1
, but receptors can also be
efficiently assembled from
1 subunits alone. Each subunit has four
putative transmembrane (TM) domains, with the second (TM2) believed to
form the lining of the pore. We have recently demonstrated that ethanol
enhancement of Gly-R function involves a critical domain encompassing
TM2 and TM3 of the
subunit, and that a specific point mutation
within TM2 (Ser-267
Ile) could completely remove ethanol
enhancement of Gly-R function (17). In the present study, we extended
our original observations on the critical TM2/TM3 domain by studying in
detail alcohol modulation of a complementary pair of receptor chimeras
created by domain exchange between Gly-R
1 and the GABA
1 subunit,
another member of the ligand-gated chloride channel "subfamily,"
which can readily form homomeric receptors. We also studied an
extensive series of single amino acid replacements at Ser-267 in the
Gly-R
1 subunit, and have investigated the effects of each mutation
on allosteric modulation by ethanol.
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EXPERIMENTAL PROCEDURES |
Chimeric Receptors and Site-directed Mutagenesis--
A pair of
chimeric receptors, denoted C1 and C2, were first constructed by
methods described previously (17). The C1 chimera contains mostly
Gly-R
1, except the large intracellular loop and TM4, which are from
GABA
1. C2 is the converse chimera, consisting mostly of GABA
1,
except for the large intracellular loop and TM4 from Gly-R
1. Using
site-directed mutagenesis methods described below, a unique
BssHII restriction site was then introduced into the
cDNA sequence encoding the conserved amino acid sequence "PAR" at the beginning of TM2 in both C1 and C2 chimeras. Utilizing a unique
SspI restriction site existing on the vector pCIS2, the BssHII/SspI fragments were exchanged, resulting
in chimeric receptors C6 and C7 (Fig. 1).
All chimeric receptors were confirmed by double-stranded sequencing
(Sequenase 2.0, U.S. Biochemical Corp.). To create the point mutant
series at Gly-R
1 Ser-267, mutations were introduced into the
cDNA encoding the human Gly-R
1 subunit at bases 883-885, with
simultaneous loss of a SacI restriction site.
Oligonucleotides 24-30 bases in length were obtained from Operon
Technologies (Alameda, CA), 5
-phosphorylated using polynucleotide
kinase, and used to create mutations using the unique site elimination
method (USE kit; Pharmacia Biotech; Ref. 18). SspI digestion
was then used to select in favor of mutants, and clones were screened
for the appearance of the desired mutation by digestion with
SacI. All restriction enzymes and polynucleotide kinase were
obtained from New England Biolabs (Beverly, MA). In addition, a few
mutations were created using a Pfu-based polymerase chain
reaction method (QuikChange; Stratagene). The sequences of both 5
- and
3
-cDNA termini and the sequences through the mutation site were
confirmed by sequencing.

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Fig. 1.
Chimeric glycine/GABA receptors C6 and
C7. The chimera "C6" consists of the GABA 1 subunit
(depicted by the thick lines), with 45 amino acid residues,
roughly including TM2 and TM3, replaced by the analogous regions from
the Gly-R 1 subunit (thin lines). The chimera "C7"
consists of the Gly-R 1 subunit with 45 amino acid residues
(thin lines), roughly including TM2 and TM3, replaced by the
analogous regions from the GABA 1 subunit (thick
lines).
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Oocyte Expression and Electrophysiology--
The methods used
for oocyte preparation and cDNA nuclear injection, and the
electrophysiological assay of receptor function using
Xenopus oocytes have been described previously (19). Drugs were applied for 0.5-3 min (depending on the GABA or glycine
concentration), by which time the peak current response was obtained. A
5-min washout period was allowed between applications when low GABA or
glycine concentrations were used, increasing to 10 min for higher
concentrations. Modulatory drugs were always tested against an
EC10 concentration of glycine or GABA, i.e. a
concentration of agonist giving 10% of the maximal response obtainable
in that egg. Data were always obtained from 4-13 oocytes taken from at least two different frogs.
HEK 293 Cell Culture, Transfection, and
Electrophysiology--
Wild-type or mutant glycine receptor cDNAs
were also expressed by transfecting human embryonic kidney (HEK) 293 cells as described previously (21). Recordings from HEK 293 cells were
made using the whole-cell patch-clamp technique, as described in
previous publications (21). Patch pipettes contained (in
mM): 145 N-methyl-D-glucamine hydrochloride, 5 K2ATP, 1.1 EGTA, 2 MgCl2, 5 HEPES/KOH, 0.1 CaCl2 (pH 7.2). Pipette resistance was 4-5
megohms. The extracellular medium contained (in mM): 145 NaCl, 3 KCl, 1.5 CaCl2, 1 MgCl2, 6 D-glucose, 10 HEPES/NaOH (pH 7.4). HEK 293 cells were
voltage clamped at
60 mV. In addition to the continuous slow bath
perfusion, the extracellular saline, glycine, and the alcohols were
rapidly applied to the cell by local perfusion using a motor-driven
solution exchange device (Bio Logic Rapid Solution Changer RSC-100;
Molecular Kinetics, Pullman, WA) (21). Numerical data are presented
throughout as mean ± S.E. ATP was from Calbiochem; ethanol was
from AAPER Alcohol & Chemical Co.; butanol (HPLC-grade) was from Sigma;
strychnine, bicuculline, and picrotoxin were from RBI (Natick, MA); all
other chemicals were from Sigma.
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RESULTS |
Alcohol Pharmacology of Wild-type Glycine Receptors--
Ethanol
potentiation of Gly-R
1 receptor function has previously been
described in receptors expressed in Xenopus oocytes (12,
17). In the present study, we extend these observations to the longer
chain alcohol, n-butanol, and to Gly-R
1 expressed in the
HEK 293 cell line. Fig. 2A
illustrates the potentiation of glycine responses by ethanol (100-200
mM) in a cell expressing wild-type Gly-R
1.
Concentration-response analysis revealed a leftward shift of the entire
glycine dose-response curve in the presence of 100 mM
ethanol, with no significant change in the maximal current amplitude
elicited by glycine (Fig. 2C), indicating that the alcohol
causes an increase in the apparent affinity of the receptor for
glycine. A similar potentiating action and a leftward shift of the
glycine concentration-response curve was seen with butanol (Fig. 2,
B and D), although, as expected, butanol was more
potent than ethanol.

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Fig. 2.
Alcohol pharmacology of wild-type glycine
1 receptors. A, enhancement of submaximal glycine
responses by ethanol in a HEK 293 cell expressing wild-type Gly-R 1.
The current traces shown illlustrate control responses to a low (30 µM) concentration of glycine, after which 30 µM glycine is reapplied in the presence of 100-200
mM ethanol. Bars over current traces indicate
glycine applications for 4 s and ethanol applications for 6 s
(2-s pretreatment before glycine application). B, a similar
experiment, illustrating enhancement of submaximal glycine responses by
10-20 mM butanol in a HEK 293 cell expressing wild-type
Gly-R 1. C, concentration-response curves for glycine
obtained in the presence and absence of 100 mM ethanol in
293 cells expressing wild-type Gly-R 1. Concentration-response data
for glycine were normalized to the maximal current in each cell, and
fitted according to the logistic equation of the form: I = Imax × {[A]n/(EC50n + [A]n}, where I is current,
Imax is the maximal current recorded in a given
cell, [A] is the glycine concentration, n is
the Hill coefficient, and EC50 the concentration of glycine
eliciting 50% of the maximal current. Each point represents the
mean ± S.E. mean of the normalized current from six individual
experiments. The best fit values of the glycine EC50 values
and Hill coefficients (in parentheses) are as follows: control, 83 µM (n = 2.7); ethanol, 67 µM (n = 2.1). D,
concentration-response curves for glycine obtained in the presence and
absence of 20 mM butanol in 293 cells expressing Gly-R 1.
The best fit values of the glycine EC50 values and Hill
coefficients from six experiments are as follows: control, 83 µM (n = 2.7); butanol, 41 µM (n = 1.8).
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Pharmacology of Chimeric Glycine/GABA
Receptors: Gating by
Agonists--
A complementary pair of chimeric receptors was tested,
in which a 45-amino acid domain had been exchanged between Gly-R
1 (the function of which is enhanced by ethanol, as shown above) and
GABA-R
1 (the function of which is inhibited by ethanol; Ref. 22).
The first chimera tested was denoted "C7" (nomenclature from Ref.
17), consisting almost entirely of Gly-R
1, with the 45-amino acid
TM2-TM3 domain provided by GABA
1 (Fig. 1). This chimera expressed
relatively poorly in HEK 293 cells, relative to wild-type Gly-R
1,
but could be gated by glycine. Interestingly, although the TM2 domain
was contributed by GABA
1, the kinetics of both activation and
deactivation were rapid, resembling the gating behavior of the
wild-type Gly-R
1 (Fig. 3A),
with an EC50 of 293 µM and a Hill coefficient
of 1.3 (Fig. 3C). The converse chimera, denoted C6 (see Fig.
1), consisting almost entirely of GABA
1, with the 45 amino acid
domain (comprising TM2, TM3, and the intervening short extracellular
loop) provided by Gly-R
1 (17), expressed quite efficiently in HEK
293 cells (Fig. 3B), and was gated by GABA with an
EC50 of 1.3 µM and a Hill coefficient of 2.3 (Fig. 3D). Again, although the TM2 domain was contributed entirely by Gly-R
1, the kinetics of both activation and deactivation were extremely slow, strongly resembling the gating behavior of the
wild-type GABA-R
1 (Fig. 3D).

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Fig. 3.
Pharmacology of chimeric glycine/GABA receptors: gating by agonists. A, gating of the chimera C7
by 20-1000 µM glycine. Note that both activation and
deactivation of this receptor are very rapid. B, gating of
the chimera C6 by 0.2-10 µM GABA. Note that both
activation and deactivation of this receptor are extremely slow.
C, concentration-response curve for glycine activation of the chimera C7. Each point represents the mean ± S.E. of
the normalized current from 11 experiments. Glycine EC50,
293 µM; n = 1.3. D, concentration-response curve for GABA activation of the chimera C6.
Each point represents the mean ± S.E. of the normalized current from four experiments. GABA EC50, 1.3 µM;
n = 2.3.
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Chimeric Glycine/GABA
Receptors: Regulation by
Alcohols--
The regulation of these chimeric receptors by alcohols
was examined in detail and compared with those of the corresponding wild-type receptors. GABA
1 subunit function is inhibited by ethanol
in Xenopus oocytes (22); in the present study, we expressed this subunit in HEK 293 cells and also observed
concentration-dependent inhibition by both ethanol and
butanol (Fig. 4A). We then
studied the chimera C6, in which GABA responses were potentiated by
both ethanol and butanol (Fig. 4B). The
concentration-dependence and efficacy of these enhancing effects were
similar to those exhibited by the wild-type Gly-R
1 (cf.
Fig. 2). Chimera C7 showed very weak inhibition of glycine currents by
both ethanol and butanol (Fig. 4C). The efficacy of these
effects were smaller than those seen in the wild-type GABA
1
(cf. Fig. 4A).

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Fig. 4.
Pharmacology of chimeric glycine/
receptors: regulation by alcohols. A, inhibition of
submaximal GABA responses by 50-200 mM ethanol in a HEK
293 cell expressing the wild-type GABA 1 subunit. The current traces
shown illlustrate control responses to 2 µM GABA, after
which 2 µM GABA is reapplied in the presence of 100 and
200 mM ethanol. Bars over current traces
indicate glycine applications for 2 s and alcohol applications.
Left-hand side shows the concentration-response curves for
the inhibition of GABA currents by ethanol (filled symbols)
and by butanol (open symbols). B, enhancement of
submaximal (0.5 µM) GABA responses by 100-200
mM ethanol in a HEK 293 cell expressing the chimera C6.
Left-hand side shows the concentration-response curves for the enhancement of GABA currents by ethanol (filled symbols)
and by butanol (open symbols). C, weak inhibition
of submaximal (100 µM) glycine responses by 100-200
mM ethanol in a HEK 293 cell expressing the chimera C7.
Left-hand side shows the concentration-response curves for
the inhibition by ethanol (filled symbols) and butanol (open symbols).
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These findings strongly confirmed and extended our original findings
from experiments in Xenopus oocytes that suggested the pre-eminent importance of the TM2 and TM3 domains in the regulation of
the Gly-R by alcohols (17). We next examined the effects of ethanol and
butanol on the mutant S267I receptor in HEK 293 cells. The mutant S267I
receptor expressed well in HEK cells, at levels comparable to those of
the wild-type Gly-R
1. Gating by glycine was efficient, with normal
rapid activation and deactivation kinetics, an EC50 of 57 µM and a Hill coefficient of 2.2 (Fig. 5A). Desensitization at high
glycine concentrations appeared to be reduced in Gly-R(S267I) relative
to wild-type Gly-R
1.2 In
HEK 293 cells expressing the mutant Gly-R
1(S267I) subunit, up to 200 mM ethanol had no effect on the response to submaximal glycine (Fig. 5B, cf. Fig. 2A);
similar observations were made with 20 mM butanol (Fig.
5C, cf. Fig. 2B). Neither ethanol (100 mM) nor butanol (20 mM) had any significant
effect on the concentration-response curve for glycine in the S267I
mutant (Table I).

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Fig. 5.
Alcohol pharmacology of mutant S267I glycine
receptors. A, glycine responses in the S267I receptor mutant
expressed in an individual HEK 293 cell. B, lack of effect
of ethanol (100-200 mM) on submaximal glycine responses in
a cell expressing Gly-R 1(S267I). The current traces shown
illlustrate control responses to a low (20 µM)
concentration of glycine, after which 20 µM glycine is reapplied in the presence of 100-200 mM ethanol.
C, a similar experiment, illustrating the lack of effect on
submaximal glycine responses of 10-20 mM butanol in a cell
expressing Gly-R 1(S267I).
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Table I
EC50 values and Hill coefficients for certain Gly-R Ser-267
mutants
Concentration-response curves for glycine were obtained for the
wild-type Gly-R 1 and several mutant receptors.
Concentration-response curves for glycine were also obtained in the
presence of 100 mM ethanol in cells expressing the
wild-type Gly-R 1 and Gly-R 1(S267I). The best fit values of the
EC50 and Hill coefficients are given here.
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Gating of Other Serine 267 Mutants by Glycine--
The results
obtained with the S267I mutant strongly suggested an important role for
Ser-267 in the regulation of Gly-R
1 by alcohols. Because the side
chain of isoleucine is larger and more hydrophobic than that of serine,
we were curious to determine which amino acid residues at this position
allowed for receptor function, and also to determine whether size,
hydrophilicity or some other physical parameter of these residues
dictated the abilities of the alcohols to potentiate Gly-R
1
function. We therefore mutated Ser-267 to the remaining 18 amino acid
residues commonly found in mammalian proteins. Due to the large number
of mutants, we chose to express these in Xenopus oocytes, in
which rapid sampling and screening of multiple constructs was
facilitated by the use of a robotic system (23). A few selected mutants
were also examined in HEK 293 cells. All of the mutant receptors
expressed successfully in the oocytes. Replacement of Ser-267 with His
also produced a functional Gly-R in HEK 293 cells, which was gated by
glycine with an EC50 of 251 µM and a Hill
coefficient of 1.5 (Table I). Replacement of Ser-267 with Tyr also
produced a functional receptor gated by glycine, with an
EC50 of 226 µM, Hill slope of 1.3 (Table I).
Even the introduction of charged residues at position 267 produced
functional Gly-R in Xenopus oocytes.
Modulation by Alcohols of Multiple Mutants at Ser-267--
We
studied the regulation of each of these mutant receptors by ethanol in
the Xenopus oocyte preparation. Each was tested by applying
200 mM ethanol and the results expressed in terms of
potentiation of the response to a test concentration of glycine. In
each case, we standardized the experiment by using an EC10 concentration of glycine, i.e. a concentration of glycine
that gave 10% of the maximal response obtained in that mutant
receptor, the maximal response being usually assessed as the response
to 10 mM glycine. Some mutant receptors demonstrated
enhancement of glycine by ethanol; for example, the S267A and S267N
mutants showed potentiation of glycine. Another group of mutants,
including S267I and S267V, demonstrated almost no detectable effects of 200 mM ethanol. Unexpectedly, and perhaps most
interestingly, another group of mutants, including S267Y, S267H, and
S267F, showed quite strong inhibitory effects of
ethanol. The results are summarized in the histogram in Fig.
6, in which the amino acids substituted at residue
267 are arranged in descending order of potentiation of
Gly-R function by ethanol, from Ser-267, which gave the largest degree
of potentiation, down to Tyr-267, which showed the largest degree of
inhibition of Gly-R function by ethanol.

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Fig. 6.
Modulation by alcohols of multiple mutants at
Ser-267. The modulation of glycine responses in 19 mutants at
Ser-267, all expressed in Xenopus oocytes, and all tested
for modulation by 200 mM ethanol of the response to a
EC10 test concentration of glycine. Each point represents
the mean ± S.E. of the normalized modulation from 4-13
individual oocytes.
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Ethanol Modulation of Mutants at Ser-267 Is Inversely Correlated
with Molecular Volume of the Amino Acid Substituent--
To study
further the nature of the hypothetical interaction between the alcohols
and the amino acid residues at position 267 in Gly-R
1, we
investigated the relationship between some physical properties of the
amino acid side chains and the alcohol response of the resultant mutant
receptors. We found a significant inverse correlation between the
extent of alcohol modulation and the volume of the amino acid side
chain (Fig. 7), but no correlation
between ethanol responsiveness and the polarity, hydrophobicity, or
hydrophilicity of the amino acid residues.

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Fig. 7.
Modulation by ethanol of Gly-R Ser-267 X mutants is inversely correlated with molecular volume of
the amino acid substituent, X. Correlation of amino
acid side chain physical properties with ethanol potentiation. Nineteen
Gly-R mutants at Ser-267 and the wild-type Gly-R 1 receptor were all
tested for the effects of 200 mM ethanol on Gly-R function.
The observed effects of ethanol were correlated with the
hydropathicities (32), hydrophilicities (33), polarities (34), and
volumes (24) of the 20 amino acids. Only in the case of amino acid side
chain volume was the slope of the linear regression (dashed)
significantly different from zero (t = 5.46, p < 0.0005), with an r2 value
of 0.62. No significant effects were noted for hydropathicity (t = 0.59, p > 0.56, r2 = 0.002), hydrophilicity (t = 1.76, p > 0.09, r2 = 0.15), or
polarity (t = 0.24, p > 0.81, r2 = 0.003). Statistical comparisons were done
using a t test, as described in Ref. 35.
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DISCUSSION |
Earlier studies showed that ethanol and other alcohols enhance the
action of agonist on homomeric Gly-R consisting only of
1 or
2
subunits (12, 13), but inhibit the function of GABA-R composed of
1
subunits (22). This raises the question of whether these two opposite
actions of alcohols are due to their actions on homologous regions of
these two receptors, with different functional consequences, or whether
they are due to drug interactions with distinct, non-conserved
sequences. Our initial studies with chimeric and mutant receptors
suggested that the 45-amino acid domain (comprising TM2, TM3, and the
intervening short extracellular loop) accounted for both enhancing and
inhibitory actions of the alcohols (17), and this finding is confirmed
and extended in the present study. The testing of "mini-chimeras"
demonstrated that a 45-amino acid sequence in the Gly-R
1 and GABA
1 subunits appears to be the prime determinant of both the enhancing
and inhibitory actions of alcohols on this subfamily of receptors. One
caveat here is that the alcohols do not show their full efficacy as
inhibitory modulators at the C7 chimera, when compared with their
effects on the wild-type GABA
1 receptor (Fig. 4), suggesting that
for the inhibitory actions of the alcohols, there may well be an
additional contribution to this effect from receptor structures outside
TM2 and TM3.
Another interesting issue with respect to the minichimeras is that the
receptor gating kinetics are apparently not a property of TM2, since
the 45 amino acid domain (comprising TM2, TM3, and the intervening
short extracellular loop) from GABA-R
1 could be inserted into a
Gly-R skeleton to create C7 without significantly slowing channel
opening or closing, while the minichimera C6 retains the extremely slow
gating behavior of the parent GABA
1 receptor (Fig. 3). These
observations suggest that the rate-limiting steps for the opening of
these channels might be in the binding of agonist, or might otherwise
be controlled in other parts of the molecule not represented within
TM2, TM3, or the TM2-TM3 loop.
In our earlier study, we showed that mutation of Ser-267 to Ile
abolished the action of ethanol (17), and we therefore selected the
Ser-267 residue for extensive mutagenesis. The resulting 19 mutant
receptors with different amino acid residues at
267 showed a
remarkable gradation of ethanol actions, ranging from 88% potentiation (Ser-267) to 55% inhibition (Tyr-267) of glycine-induced currents. The
nature of this distribution (Fig. 6) suggested that a continuous property of the amino acids might determine the action of ethanol. Correlation analysis (Fig. 7) demonstrated that the molecular volume
(24) of the substituent at
267 was associated with more than 60% of
the variation in ethanol activity among the mutant receptors. On the
other hand, there was no relationship between ethanol action and
hydrophilicity, hydrophobicity, or charge (Fig. 7). These data suggest
that the physical size of the residue at
267 is a key regulator of
the effects of alcohols on the Gly-R. It is believed that Ser-270 in
the GABAA-R
1 subunit (a homolog of Ser-267 in Gly-R
1)
does not form part of the channel lining, but instead is likely to be
sequestered within the interior of the protein (25). Our observations
on Ser-267 mutants are consistent with the idea that the binding of
small molecules such as alcohols to the receptor protein could alter
the subtle thermodynamic equilibria associated with channel gating in
the Gly-R. However, it appears unlikely that Ser-267 itself is involved
in the primary alcohol binding site, although this may be in the
vicinity of residue 267.
An obvious question to be considered here is how mutation of a single
amino acid residue can alter the modulatory action of ethanol on Gly-R
function so dramatically, from enhancement of receptor function
(wild-type, S267G), through no effect (S267I, S267V), to inhibition
(S267F, S267Y)? One possible explanation is based on the idea that, in
the wild type glycine
1 receptor, ethanol preferentially binds to
and thereby stabilizes the open state of the receptor;
conversely, in the wild-type GABA
1 receptor, ethanol would bind to
and stabilize the closed state of the receptor. Selective
binding to active or inactive states of receptors has been proposed as
the basis of the actions of agonists, antagonists, and "inverse
agonists" on many types of receptors (26). Such an action of alcohols
would be analogous to the preferential binding of local anesthetics to
the inactivated state of the voltage-activated sodium channels
(e.g. Refs. 27 and 28). According to such a model, a lack of
detectable alcohol effect on a mutant Gly-R such as S267V would be
explained by the drug binding with equal affinity to open and closed
states, so that an equilibrium between enhancement and inhibition of
receptor function is achieved. Applying this model to our data, the
effect of mutations would be to change the relative affinity for
ethanol binding to the open state of the receptor relative to the
closed state, with the specific requirement that larger amino acid
residues at 267 must favor the binding of ethanol to the
closed state. These mutants are thus inhibited by ethanol.
Such a mechanism does not, of course, require that ethanol affect
channel function by binding in the vicinity of Ser-267. Ser-267 may
therefore act as a "transduction" site to determine the action of
ethanol, which arises from a different binding site located elsewhere
on the Gly-R
1 polypeptide.
Without detailed information on receptor structure (although molecular
models have been proposed; see Ref. 29), and in the absence of direct
drug binding studies, it is difficult to distinguish between these two
possibilities. In this respect, the lack of effect of long chain
alcohols may be a useful tool. The so-called "cut-off" phenomenon
is thought to be due to the exclusion of larger alcohols from an
amphiphilic binding cavity or "pocket," and the cut-off may serve
as an indicator of the molecular size of such cavities within a given
protein (30, 31). Studies of the Gly-R suggest a distinct cut-off at
around dodecanol (13); cut-off analysis on the chimeras and mutants
studied here should help determine whether Ser-267 controls the
size of such a binding pocket in Gly-R. In addition,
single-channel kinetic studies of several of these mutant Gly-R would
help to evaluate the hypothesis that ethanol preferentially stabilizes
open or closed states of the channel operated by glycine.
We are indebted to Prof. Heinrich Betz, Garry
Cutting, and Peter Schofield, who supplied cDNAs used in this
study, and to Dr. Gareth Tibbs for helpful discussion.