ABSTRACT
The glycine receptor is a member of the superfamily of ligand-gated ion
channels, of which many are important targets for alcohol and anesthetic
actions. In the present study, I examined the sensitivity of cloned alpha1 and
alpha2 homomeric glycine receptors expressed in Xenopus oocytes to ethanol,
butanol, and halothane. Ethanol (50-200 mM) significantly potentiated
responses seen with low (<EC20) glycine concentrations in both alpha1 and
alpha2 subunit containing oocytes. Butanol (2.5-40 mM) potentiated glycine
receptor function to a similar degree in oocytes expressing alpha1 or alpha2
subunits. Enhancement of glycine responses by butanol ranged from ~ 20
to1000%. The volatile anesthetic, halothane, increased glycine mediated
currents in oocytes expressing either subunit. Increases of approximately 40
to 500% were observed with concentrations of halothane ranging from 0.16 to 10
mM. Significant increases in glycine receptor function were observed below 1
minimum alveolar concentration for anesthesia (MAC). The ability of both
alcohols and halothane to enhance receptor function declined as glycine
concentrations were increased, suggesting that these drugs enhance the potency
and not the efficacy of the receptor. Since glycine receptor subtypes are
sensitive to pharmacologically relevant concentrations of alcohols and
anesthetics, they are good candidates for participating in the mechanism(s) of
action of these drugs.
INTRODUCTION
Glycine receptors are the predominant inhibitory
neurotransmitter receptors in brain stem and spinal cord (reviewed in Betz,
1991). Purified glycine receptors contain two homologous polypeptides, alpha
and beta. To date, four alpha and one beta subunits have been identified
through molecular cloning techniques. The ligand-binding site of the glycine
receptor has been localized to the alpha subunits, which form homo-oligomeric
receptors in heterologous expression systems, such as the Xenopus laevis
oocyte (Grenningloh et al., 1990a). In addition to being an integral subunit
of the native glycine receptor, the beta subunit appears to be the site
through which gephyrin, a putative cytoskeletal docking protein, binds to
localize the receptor in postsynaptic membranes of neurons (Kirsch and Betz,
1995).
Glycine receptors in adult brain and spinal cord are accepted as being
composed of alpha and beta subunits, given their insensitivity to picrotoxin,
which the beta subunit confers (Pribilla et al., 1992). In contrast, glycine
receptors in developing rat brain are presumed to be homomeric alpha2
subunit receptors due to their sensitivity to picrotoxin (Akaike and Kaneda,
1989; Mori-Okamoto and Tatsuno, 1985) and the predominance of the alpha2
subunit in the early stages of development (Betz, 1991). Glycine receptors are
members of the superfamily of ligand-gated ion channels, of which the
nicotinic receptor is the prototype. Many of these receptors are targets for
alcohol and anesthetic modulation (Harris et al., 1995; Franks and Lieb, 1994;
Sanna and Harris, 1993) and because of their roles in regulating synaptic
transmission, ligand-gated ion channels are prime candidates for producing the
mechanism of action for both classes of drugs. Very little work has been done
to characterize the sensitivity of glycine receptors to alcohols or
anesthetics. In general, ethanol has been reported to enhance glycine-induced
Cl- currents in chick spinal cord neurons (Celentano et al., 1988), rat brain
synaptoneurosomes (Engblom and Akerman, 1991), and cultured mouse neurons (Aguayo
and Pancetti, 1994). The volatile anesthetics, halothane and enflurane,
stimulate glycine-induced currents in dissociated neurons (Wakamori et al.,
1991), while isoflurane enhances alpha2 glycine receptors expressed
in HEK293 cells (Harrison et al., 1993).
While alcohols and anesthetics share many
neurochemical features, recent work has indicated that protein domains may
confer sensitivity to these drugs, and furthermore, that these sites in ligand-gated
ion channels may, in fact, be distinct. For instance, ethanol and halothane
both enhance 5-hydroxytryptamine3 receptor function, but halothane potentiates
receptor function in the presence of saturating concentrations of ethanol (Machu
and Harris, 1994). Subunit requirements observed for stimulation of GABAa
receptor function by subanesthetic concentrations of ethanol (Wafford et al.,
1991) are not shared by enflurane (Mihic et al., 1994), isoflurane (Harrison
et al., 1993) or the intravenous anesthetic, propofol (Jones et al., 1995).
Furthermore, members of the kainate/AMPA (GluR 1-3) and kainate (GluR6) family
of receptors are all inhibited by ethanol (Dildy-Mayfield and Harris, 1995),
while GluR1-3 are inhibited and GluR6 is stimulated by volatile anesthetics
(Harris et al., 1995), respectively.
The sensitivities of cloned glycine receptors to alcohols and anesthetics
have not been studied. As a first step, I have examined the actions of
ethanol, butanol, and halothane on glycine mediated Cl- currents in oocytes
expressing alpha1 or alpha2 glycine receptors. The
characterization of these compounds on defined channels will provide the
framework for further study of primary and secondary structural requirements
for drug action.
MATERIALS AND METHODS
Xenopus laevis female frogs were obtained from Xenopus I (Ann Arbor, MI).
Glycine was purchased from Bio-Rad (Hercules, CA) and strychnine was obtained
from Sigma (St. Louis, MO). Halothane was purchased from Ayerst (New York,
NY). The bath concentration of halothane, 25% of its original bottle
concentration, was determined by gas chromatography as previously described by
Lin et al. (1992). The alpha1 and alpha2 glycine
receptor cDNAs were provided by Dr. Heinrich Betz.
Isolation of Xenopus oocytes. X. laevis frogs were kept in tanks in
dechlorinated tap water at 19o C on a 12-hour light/dark cycle and
were fed a diet of mealworms and beef heart 3 times per week. Frogs were
anesthetized by immersion in ice water for 45 min and were kept on ice during
surgery. After surgical removal, ovarian lobes were placed in modified Barth's
solution (MBS) containing (in mM) NaCl 88, KCl 1, NaHCO3
2.4, HEPES 10, MgSO4 0.82, Ca(NO3)2
0.33, CaCl2 0.91, (pH 7.5).
Before dissection, ovarian lobes were placed in hypertonic isolation media
containing (in mM) NaCl 108, KCl 2, EDTA 1, and HEPES 10 (pH 7.5). Stage V and
VI oocytes were dissected with fine surgical forceps as previously described (Dildy-Mayfield
and Harris, 1992) and placed in MBS. Oocytes were treated with collagenase
Type 1A (0.5 mg/ml) in (mM) NaCl 83, KCl 2, MgCl2 1 and HEPES 10 (pH 7.5) to
remove the follicular cell layer, and rinsed in MBS.
Microinjection of oocytes with Glycine Receptor cDNA. Glycine receptor
cDNAs were subcloned in the pCis vector which expresses well when injected
into the oocyte nucleus. The "blind" method for nuclear injection
described by Colman (1984) was used to inject 50 pg - 5 ng of glycine receptor
cDNA dissolved in 30 nl of diethyl pyrocarbonate (DEPC)-treated water per
oocyte. Oocytes were maintained at 18oC in sterile filtered
incubation media (MBS containing 10 mg/l streptomycin, 50 mg/l gentamicin,
10,000 U penicillin/l, 0.5 mM theophylline, and 2 mM sodium pyruvate).
Incubation media was changed daily. Oocytes were recorded from day 1 through 7
following injection.
Electrophysiological Recording of Glycine Receptor Responses. Oocytes were
perfused (2 ml/min) in a 100 u1 volume recording chamber with MBS via a roller
pump (Cole Parmer Instrument, Co., Chicago, IL). Oocytes were impaled with 2
glass electrodes (1.2 mm outside diameter and 1 - 10 megohm resistance) filled
with 3 M KCl. Oocytes were voltage clamped to -70 mV using a Warner
Instruments Model OC-725A (Hamden, CT) oocyte clamp. Clamping currents were
plotted on a strip chart recorder (Cole Parmer Instrument Co., Chicago, IL).
Glycine was dissolved in MBS and applied for 30 sec. Alcohols and
anesthetics were coapplied with glycine. Alcohols were preapplied for 1 min
prior to glycine plus alcohol application.
Data Analysis Prism Software (San Diego, CA) was used to determine EC50s
and Hill coefficients. Prism or Instat (San Diego, CA) Software was used to
perform one- or two- way analysis of variance (ANOVA) and Student's t-tests.
One-way ANOVA was used, in some cases, to evaluate the effects of drug
concentration on a construct. Two-way ANOVA was used to simultaneously examine
the effects of drug concentration and receptor construct. The unpaired
Student's t-test was used to examine any differences in the effects of a
single concentration of drug on the two receptor constructs.
Calculation of MAC values. Concentrations of anesthetics in the oocyte
perfusion chamber were converted to concentrations at 37o C with
the equation reported by Franks and Lieb (1993):
(Concentration at 22o C)/(Concentration at 37o C) =
exp [(-4.08(37-22))/(273.15+22)]
Concentrations at 37o C were converted to MAC by the equation:
Molar concentration at 37o C = MAC x (saline/gas partition
coefficient) x ((273/310)/22.4)
RESULTS
Currents produced by glycine in oocytes expressing alpha1 or
alpha2 (Fig.1)
receptors were almost completely blocked by strychnine (500 nM). Recovery was
essentially complete after several min of washout. Glycine
concentration-response curves were generated for the alpha1 and
alpha2 (Fig.2)
receptor constructs. An EC50 of 85 uM and a Hill coefficient of 2.5 were
calculated for oocytes expressing the alpha1 receptor. Glycine was
less potent in oocytes expressing the alpha2 receptor, with an EC50
of 142 uM and a Hill coefficient of 2.29. Hill coefficients and EC50s fall
within the range of that previously reported for recombinant glycine receptors
(Taleb and Betz, 1994; Bormann et al., 1993; Cascio et al., 1993; Langosch et
al., 1990).
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| Figure 1: Glycine mediated inward
currents in representative oocytes expressing alpha1or alpha2 subunits
of the receptor are almost completely blocked by the selective glycine
antagonist, strychnine. After 10 min of washout, recovery of responses
is observed. |
|
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| Figure 2: A concentration response curve
was generated for alpha1 or alpha2 subunit expressing oocytes. Oocytes
were perfused with glycine (5-1000uM) for 30 sec. Values are expressed
as a percent of the maximal glycine (500 uM) response (n=5-15). An
EC50 of 85 uM and a Hill coefficient of 2.5 were calculated for alpha1
subunit receptors, and an EC50 of 142 uM and a Hill coefficient of
2.29 were calculated for alpha2 subunit receptors, respectively. |
Ethanol stimulated glycine receptor function in alpha1 and alpha2
receptor containing oocytes (Fig.
3). Ethanol stimulation was concentration dependent, but did not plateau
over the range of concentrations tested (Fig.4).
In either alpha1 or alpha2 receptor containing oocytes,
ethanol concentrations less than 50 mM produced potentiation of receptor
function in approximately half the oocytes tested, but the enhancement was
small (<15%, data not shown). Concentrations of ethanol higher than 200 mM
tend to produce oscillations in the baseline, which may be due to release of
intracellular Ca2+ stores (Wafford et al., 1989). Therefore, higher ethanol
concentrations were not tested. There were no significant differences between
the two constructs in their sensitivity to ethanol (p=0.9474), but there was a
significant effect of ethanol concentration (p<0.0001). Approximately 15%
of oocytes expressing either receptor subtype were insensitive to ethanol
(50-200 mM); these data were not included in Fig.4.
This lack of sensitivity was not oocyte batch dependent, nor was it a function
of the level of expression of the receptor in a given oocyte. When tested,
these ethanol "insensitive" oocytes were modulated by higher chain
alcohols and halothane. Finally, the ability of ethanol (100 mM) to enhance
glycine mediated currents in either receptor construct was attenuated by
increasing glycine concentrations (>EC20, data not shown).
|
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| Figure 3: Electrophysiological recordings
of inward currents produced by glycine in oocytes containing alpha1 or
alpha2 receptors in the absence and presence of ethanol. Oocytes were
perfused with ethanol (100 mM) for 1 min prior to the application of
ethanol (100 mM) plus glycine (alpha1: 25 uM) or glycine (alpha2:
50uM) for 30 sec |
|
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| Figure 4: Ethanol produces a
concentration dependent stimulation of alpha1and alpha2 receptors.
Oocytes were bath perfused with approximately equieffective
concentrations (EC4) of glycine, 15uM (alpha1) and 50 uM (alpha2) for
30 sec. Oocytes were perfused with ethanol (50-200 mM) for 1 min prior
to exposure to glycine plus ethanol. Values are expressed as a percent
above the respective control glycine response (n=4-11). |
Figure
5 contains representative tracings of butanol stimulation of glycine
receptor responses. Butanol also enhanced glycine receptor function in a
concentration dependent manner (Fig.6),
but to a greater degree than ethanol. In addition, the difference in potency
between ethanol and butanol is consistent with the Meyer-Overton hypothesis
(Meyer,1901; Overton, 1896), which predicts that lipophilicity is correlated
with anesthetic potency. The enhancement of glycine mediated currents did not
reach a maximum within the range of butanol concentrations tested, and there
were no significant differences between the two constructs in their
sensitivity to butanol (p=0.8886). There was a significant effect of butanol
concentration (p<0.0001). The ability of butanol (10 mM) to enhance glycine
receptor responses was tested as a function of increasing concentrations of
glycine (Fig.7).
In either alpha1 or alpha2 receptor expressing oocytes,
butanol stimulation of function was reduced with increasing concentrations of
glycine (p<0.0001, alpha1 and alpha2). At maximal
glycine concentrations, no enhancement of function was seen, suggesting that
butanol enhances the potency of glycine, but not its efficacy, in both
receptor constructs.
|
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| Figure 5: Electrophysiological recordings
of inward currents produced by glycine in oocytes containing alpha1
and alpha2 receptors in the absence and presence of butanol. Oocytes
were perfused with butanol (5 mM) for 1 min prior to the application
of butanol (5 mM) plus glycine (alpha1: 25 uM) or glycine (alpha2: 50
uM) for 30 sec. |
|
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| Figure 6: Butanol produced a
concentration dependent stimulation of alpha1 and alpha2 receptors.
Oocytes were perfused with approximately equieffective concentrations
(EC4) of glycine, 15uM (alpha1) and 50 uM (alpha2) for 30 sec. Oocytes
were perfused with butanol (2.5-40 mM) for 1 min prior to exposure to
glycine plus butanol. Values are expressed as a percent potentiation
above the respective control glycine response (n=4-9). |
|
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| Figure 7: Butanol increases the potency,
but not the efficacy of glycine receptors. Oocytes (alpha1: upper
panel, alpha2: lower panel) were perfused with glycine (15-500 uM) for
30 sec. Oocytes were perfused with butanol (10mM) for 1 min prior to
application of glycine plus butanol for 30 sec. Values are expressed
as a percent potentiation above the respective control glycine
response (n=5-8). |
Inward currents produced by glycine in the absence and presence of
halothane in oocytes expressing alpha1 and alpha2
receptors are depicted in Fig
8. Halothane enhanced glycine mediated currents in a concentration
dependent manner (Fig.9).
Potentiation was seen with the lower concentrations (0.16 and 0.32 mM) tested,
which correspond to that used clinically. Like ethanol and butanol,
potentiation of receptor function did not reach a maximum over the range of
concentrations tested. Concentrations in excess of 2.5 mM (10 MAC) were not
tested because they are not pharmacologically relevant. While potentiation
varied with increasing halothane concentration (p< 0.0001), there was no
significant difference between the two constructs in their sensitivity to
halothane (p=0.0533). However, there was a definite tendency for the oocytes
expressing the alpha2 subunit to be more sensitive to halothane,
which most likely may be explained by the fact that 50 uM glycine was used to
test both constructs. This concentration of glycine represents an EC62 for the
alpha1 receptor and an EC4 for the alpha2 receptor,
respectively. Figure
10 shows that there is a distinct glycine concentration dependency for
halothane potentiation. When equieffective concentrations of glycine at the
two constructs ( alpha1: 15 uM and alpha2: 50 uM) are
compared with respect to potentiation by 0.25 mM halothane, there is no
significant difference (Student's unpaired, two tailed t-test, p=0.1). The
finding that halothane's ability to enhance glycine responses declines
markedly with increasing concentrations of glycine (p<0.0001, alpha1
and alpha2) suggests that this anesthetic enhances glycine potency,
but not efficacy, at alpha1 and alpha2 receptors.
|
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| Figure 8: Electrophysiological recordings
of inward currents produced by glycine in oocytes containing alpha1
and alpha2 receptors in the absence and presence of halothane. Oocytes
were perfused with glycine (alpha1: 25 uM) or glycine (alpha2: 50 uM)
with or without halothane (0.32 mM) for 30 sec. |
|
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| Figure 9: Halothane produced a
concentration dependent stimulation of alpha1 and alpha2 receptors.
Oocytes were perfused with glycine (50 uM) for 30 sec. Oocytes were
then perfused with glycine plus halothane (0.16-2.5 mM) for 30 sec.
Values are expressed as a percent potentiation above the respective
control glycine response (n=4-5). |
|
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| Figure 10: Halothane mediated stimulation
of glycine receptors is dependent on glycine concentration. Oocytes
(alpha1: upper panel; alpha2: lower panel) were treated with glycine
(10-500 uM) for 30 sec. Oocytes were then treated with halothane (0.25
mM) plus glycine for 30 sec. Values are expressed as a percent
potentiation above the respective control glycine response (n=4-5). |
DISCUSSION
In recent years, the hypothesis that alcohols and anesthetics produce
central nervous system depression through the disordering of membrane lipids
has fallen into disfavor. While lipophilicity, in general, is a good indicator
of alcohol/anesthetic potency (Meyer, 1901; Overton, 1896), it is clear that
membrane disordering can be produced by compounds that do not produce
intoxication or anesthesia (Buck et al., 1989). Furthermore, Franks and Lieb
(1984) showed that anesthetics have potent actions on purified proteins in the
absence of any lipids. Their groundbreaking work with firefly luciferase gave
rise to the hypothesis that alcohols and anesthetics may act through
partitioning into hydrophobic domains of proteins. Because of their roles in
mediating synaptic transmission, ligand-gated ion channels are prime protein
candidates for mediating the behavioral sequelae associated with
intoxicant-anesthetic drugs.
Numerous studies have been conducted on most of the members of the
superfamily of ligand-gated ion channels, as well as the glutamate family of
receptors, with respect to their modulation by alcohols (reviewed in Sanna and
Harris, 1993) and anesthetics (reviewed by Harris et al., 1995). However,
scant attention has been given to the strychnine sensitive glycine receptor,
which is important in regulating motor and sensory functions (reviewed by
Betz, 1991). While sparsely present in higher brain structures, the glycine
receptor is plentiful in brainstem and spinal cord, two areas of the central
nervous system which have been largely overlooked in recent years with regard
to the pharmacology of alcohols and anesthetics. Two lines of evidence
indicate that anesthesia may be produced by subcortical structures. In goats,
the MAC for halothane is ~ 2.5 fold greater when midbrain and forebrain
structures alone are administered drug than when the entire body is perfused (Antognini
and Schwartz, 1993). MAC is unaltered in rats by decerebration (Rampil et al.,
1993). While many of the behaviors associated with ethanol intoxication may
likely be due to impairment of higher brain structures, motor incoordination
could be, in part, attributable to ethanol actions at brain stem and spinal
cord. Recent findings have shown that glycine administered
intracerebroventricularly enhances the depressant properties of a hypnotic
dose of ethanol as measured by loss of righting reflex (Williams et al.,
1995). This enhanced depressant effect was antagonized by low doses of
strychnine, suggesting that it occurred through the inhibitory glycine
receptor and not through the glycine sensitive site of the NMDA receptor.
The stimulation of alpha1 and alpha2 glycine
receptors by ethanol and butanol that I report here represents the first
demonstration that the cloned glycine receptor is modulated by alcohols.
Previous work by other investigators in spinal cord neurons (Celanto et al.,
1988), rat brain synaptoneurosomes (Engleblom and Akerman, 1991), and cultured
mouse neurons (Aguayo and Pancetti, 1994) has shown that native glycine
receptors are stimulated by ethanol. Aguayo and Pancetti (1994) found that
ethanol enhancement of glycine receptors in spinal neurons was not saturable,
even at high ethanol concentrations (400 mM), which is similar to my finding
of a lack of saturation with 200 mM ethanol. Similarly, I did not see
saturation with butanol over the range of concentrations tested. To be
consistent with a "hydrophobic pocket" hypothesis in which there are
a finite number of sensitive site(s) in the receptor, there theoretically must
be a concentration high enough to saturate the sites. However, in many cases,
this concentration may not be water soluble, as we have observed with long
chain alcohols (Mascia et al., submitted). The observation that butanol is
more potent than ethanol is consistent with the hydrophobic pocket hypothesis,
given that butanol, a more lipophilic alcohol than ethanol, would be predicted
to partition into alcohol sensitive sites with higher affinity. In agreement
with the other investigators cited above, I observed that ethanol had actions
on glycine receptors in the intoxicating range (25-50 mM), as well as the
anesthetic range (>150 mM), suggesting that glycine receptors could
participate in the mechanism of action of both ethanol induced intoxication
and anesthesia.
My finding that the function of alpha1 and alpha2
subunits of the glycine receptor is enhanced by the volatile anesthetic,
halothane, is consistent with the hypothesis that general anesthesia may be
entirely, or in part, mediated by an increase in inhibitory neurotransmission,
as has been reported for the GABAa receptor (reviewed by Harris et
al., 1995). Only one other group has investigated glycine receptor subunit
subtypes' sensitivities to anesthetics. Harrison and coworkers (1993) reported
that alpha2 glycine receptors expressed in HEK293 cells were
stimulated by 0.82 mM isoflurane. Most evidence that glycine receptors are
targets of anesthetics has been obtained in nervous tissue preparations.
Wakamori and coworkers (1991) examined the actions of halothane and enflurane
on glycine induced currents in neurons dissociated from ratnucleus tractus
solitarius. Their studies showed that both volatile anesthetics enhanced
glycine receptor function in a concentration dependent manner. Furthermore,
they reported that enflurane and halothane decreased the Kd for glycine,
without having any effect on the maximum glycine response. These results are
mirrored by mine, since I observed an increase in glycine potency, but not
efficacy with both alpha1 and alpha2 glycine receptors.
I also observed that halothane increased glycine receptor responses in a
concentration dependent manner. My results, as well as that of Harrison et al.
(1993) and Wakamori et al. (1991), suggest that these volatile anesthetics act
at glycine receptors in a pharmacologically relevant range. Wakamori et al.
(1991) reported an increase in glycine mediated currents with halothane
concentrations as low as ~ 0.5 MAC and plateauing at ~ 4 MAC. I observed
potentiation of both alpha1 and alpha2 receptors with
halothane concentrations as low as 0.25 MAC, but I did not observe saturation
at the highest concentration (10 MAC) tested. These results all suggest that
glycine receptors are potentiated by concentrations of halothane within the
range of that used clinically (0.75-1.25 MAC), thus underscoring the
possibility that the glycine receptor plays a role in the mechanism of action
of these anesthetics.
A large number of classes of compounds produce anesthesia, some of which
are fairly selective to one type of ligand-gated ion channel . For instance,
the steroid alphaxalone primarily modulates GABAa receptors (Hill-Venning
et al., 1993), and the barbiturates are much more potent at GABAa
receptors than at other receptors. In the glutamate family of receptors, NMDA
receptors are selectively inhibited by the dissociative anesthetic, ketamine
(Macdonald et al., 1989; Macdonald et al., 1987). These observations suggest
that, in some cases, specific drugs may act at one receptor type to produce
anesthesia. However, other classes of anesthetics, such as the volatile
anesthetics, are less discriminating and may produce anesthesia by interacting
at multiple ligand-gated ion channels. These observations are consistent with
the idea that anesthesia may be the sum total of stimulation of inhibitory
neurotransmission and/or depression of excitatory neurotransmission that is
specific to the respective class of anesthetic in question. Thus, the fact
that glycine receptors are not responsive to barbiturates (Hales and Lambert,
1991) or alphaxalone (Hill-Vening et al., 1993), for instance, does not
preclude their participation in mediating anesthesia produced by other classes
of compounds such as the volatile anesthetics.
Stimulation by alcohols and anesthetics of homomeric alpha1 and
alpha2 subunits of the glycine receptor expressed in oocytes
reveals that a minimal requirement for such action in a glycine receptor is an
alpha subunit. Glycine receptors present in adult brain or spinal cord are
generally accepted to be composed of alpha and beta subunits. The premise for
this notion is that native glycine receptors are insensitive to picrotoxin,
whereas alpha subunit homomers are picrotoxin sensitive; the beta subunit
confers picrotoxin insensitivity (Pribilla et al., 1992). However, in
developing rat brain, alpha2 subunits predominate (Betz, 1991), and
these are presumed to be homomeric receptors, given the picrotoxin sensitivity
of glycine receptors in developing rat brain (Akaike and Kaneda, 1989;
Mori-Okamoto and Tatsuno, 1985). The results of the present study, taken
together with previous work in nervous tissue preparations (Aguayo and
Pancetti, 1994; Engleblom and Akerman, 1991; Wakamori et al., 1991; Celento et
al., 1988), suggest that the presence of the beta subunit does not alter the
sensitivity of glycine receptors to alcohols or anesthetics. Beta subunits are
not very efficiently assembled with alpha subunits in expression systems, such
as the Xenopus oocyte (Grenningloh et al., 1990b), and therefore were not
tested. Thus the contribution, if any, of beta subunits to alcohol or
anesthetic action glycine receptors is unknown. Beta subunits are more widely
distributed in the CNS and in greater numbers than are alpha subunits (Betz,
1991), Beta subunit homomers in expression systems are weakly responsive to
glycine in comparison with alpha subunits or native alpha plus beta subunits (Grenningloh
et al., 1990b); therefore a direct contribution of beta subunit homomeric
receptors, if they do occur in vivo, to glycine mediated neuronal inhibition
via an increase in Cl- influx is doubtful. However, beta subunits appear to
perform an important function in binding the glycine receptor to gephyrin, a
cytoskeletal anchoring protein (Kirsch and Betz, 1995).
The nature of the site or sites in which alcohols and anesthetics act in
glycine receptors, as well as other ligand-gated ion channels, remains
unknown. Subunit specific requirements in recombinant GABAa (Wafford
et al., 1991) and kainate receptors (Harris et al., 1995) for
alcohol/anesthetic modulation suggest a protein site of action. However, these
sites may not be the same, given the lack of subunit specificity in the GABAa
receptor for potentiation by anesthetics (Jones et al., 1995; Mihic et al.,
1994) versus the required presence of the PKC site of the gamma2L subunit of
the GABAa receptor for ethanol potentiation (Wafford et al., 1991).
The requirement of the gamma2L insert of the GABAa receptor for
ethanol action remains controversial (reviewed by Macdonald, 1995). In any
case, the ethanol induced stimulation that I report in the alpha1
glycine receptor is not analogous to the GABAa receptor, since I
did not use the alpha1 splice variant that contains a consensus
sequence for PKC. However, other sites for protein kinase dependent
phosphorylation in the glycine receptor have been described (Ruiz- Gomez et
al., 1991), and these should be investigated for their potential role in the
modulation of glycine receptor function by ethanol. My results with the 5-HT3
receptor suggest separate sites for ethanol and halothane, since halothane
potentiates receptor function in the presence of a saturating concentration of
ethanol (Machu and Harris, 1994). Similar studies were not done with the
glycine receptor subtypes since saturating concentrations of either ethanol or
halothane were not obtained. Thus, it remains unknown whether alcohols and
anesthetics share a common site of action in glycine receptors.
In summary, I have demonstrated that cloned glycine receptors are targets
for alcohol and anesthetic modulation. My work extends previous studies done
in native nervous tissue preparations by showing that not only is an alpha
subunit sufficient for modulation by these drugs, but also that there is no
difference between the two alpha subunit subtypes tested. Glycine receptor
subunits, like the 5-HT3 receptor, form homomeric receptors, which make them
ideal candidates for testing amino acid requirements that confer sensitivity
to alcohols or anesthetics.
ACKNOWLEDGMENTS
Supported by funds from NIH grant AA10561.
REFERENCES
Aguayo, L.G. and Pancetti, F.C. (1994) Ethanol modulation of the gamma-aminobutyric
acidA- and glycine-activated Cl- current in cultured mouse neurons. J.
Pharmacol. Exp. Ther., 270: 61-69.
Akaike, N. and Kaneda, M. (1989) Glycine-gated currents in acutely isolated
rat hypothalamic neurons . J. Neurophysiol. 62, 1400-1409.
Antognini, J.F., and Schwartz, K. (1993) Exaggerated anesthetic
requirements in the preferentially anesthetized brain. Anesthesiol. 79,
1244-1249.
Betz, H. (1991) Glycine receptors: heterogeneous and widespread
distribution in the mammalian brain. Trends Neurosci. 14, 458-461.
Bormann, J., Rundstrom, N., Betz, H., and Langosch, D. (1993) Residues
within transmembrane segment M2 determine chloride conductance of glycine
receptor homo- and hetero-oligomers. EMBO J. 12, 3729-3737.
Buck, K.J., Allan, A.M., and Harris, R.A. (1989) Fluidization of brain
membranes by A2C does not produce anesthesia and does not augment muscimol-stimulated
36Cl- influx. Eur. J. Pharmacol. 160, 359-367.
Cascio, M., Schoppa, N.E., Grodzicki, R.L., Sigworth, F.J., and Fox, R.O.
(1993) Functional expression of a homomeric human alpha1 glycine
receptor in baculovirus-infected insect cells. J. Biol. Chem. 268,
22135-22142.
Celentano, J.J., Gibbs, T.T., and Farb, D.H. (1988) Ethanol potentiates
GABA- and glycine- induced currents in chick spinal cord neurons. Brain Res.
455, 377-380.
Colman, A. (1984) Expression of exogenous DNA in Xenopus oocytes. In
Transcription and Translation: A Practical Approach., E.B. Hanes and S.J.
Higgins, eds., Oxford Press, Washington, D.C., pp. 49-69.
Dildy-Mayfield, J.E. and Harris, R.A. (1992) Comparison of ethanol
sensitivity of rat brain kainate,
DLa-amino-3-hydroxy-5-methyl-4-isoxaloneproprionicacid and N-methyl-D-aspartate
receptors expressed in Xenopus oocytes. J. Pharmacol. Exp. Ther. 262, 487-494.
Dildy-Mayfield, J.E. and Harris, R.A. (1995) Ethanol inhibits kainate
responses of glutamate receptors expressed in Xenopus oocytes: role of calcium
and protein kinase C. J. Neurosci. 15, 3162-3171.
Engblom, A.C. and Akerman, K.E.O. (1991) Effect of ethanol on gamma amino
butyric acidA and glycine receptor- coupled Cl- fluxes in rat brain
synaptoneurosomes. J. Neurochem. 57, 384-390.
Franks, N.P. and Lieb, W.R. (1984) Do general anaesthetics act by
competitive binding to specific receptors? Nature 300, 487-493.
Franks, N.P. and Lieb, W.R. (1993) Selective actions of volatile general
anesthetics as molecular and cellular levels. Br. J. Anaesth. 71, 65-76.
Franks, N.P. and Lieb, W.R. (1994) Molecular and cellular mechanisms of
general anesthesia. Nature 367, 607-614.
Grenningloh, G., Schmieden, V., Schofield, P.R., Seeburg, P., Siddique, T.,
Mohanda, T., Becker, C.-M., and Betz, H. (1990a) Apha subunit variants of the
human glycine receptor: primary structures, functional expression and
chromosomal localization of the corresponding genes. EMBO J. 9, 771-776.
Grenningloh, G., Pribilla, I., Prior, P., Multhaup, G., Beyreuther, K.,
Taleb, O., and Betz, H. (1990b) Cloning and expression of the 58 kD beta
subunit of the inhibitory glycine receptor. Neuron 4, 963-970.
Hales, T.G., and Lambert, J.J. (1991) The actions of propofol on inhibitory
amino acid receptors of bovine adrenal medullary chromaffin cells and rodent
central neurons. Br. J. Pharmacol. 104, 619-628.
Harris, R.A., Mihic, S.J., Dildy-Mayfield, J.E., and Machu, T.K. (1995)
Actions of anesthetics on ligand-gated ion channels: role of receptor subunit
composition. FASEB J. 9, 1454-1462.
Harrison, N.L., Kugler, J.L., Jones, M.V., Greenblatt, E.P., and Pritchett,
D.B. (1993) Positive modulation of human gamma-aminobutyric acid type A and
glycine receptors by the inhalation anesthetic isoflurane. Mol. Pharmacol. 44,
628-632.
Hill-Venning, C., Peters, J.A., and Lambert, J.J. (1993) The interaction of
steroids with inhibitory and excitatory amino acid receptors. Clin.
Neuropharmacol. 15 (Suppl. 1), 683A- 684A.
Jones, M.V., Harrison, N.L., Pritchett, D.B., and Hales, T.G. (1995)
Modulation of the GABAa receptor by propofol is independent of the
gamma subunit. J. Pharmacol. Exp. Ther. 274, 962-968.
Kirsch, J. and Betz, H. (1995) The postsynatpic localization of the glycine
receptor-associated protein gephyrin is regulated by the cytoskeleton. J.
Neurosci. 15, 4148-4156.
Langosch, D., Becker, C.-M., and Betz, H. (1990) The inhibitory glycine
receptor: a ligand- gated ion chloride ion channel of the central nervous
system. Eur. J. Biochem. 194, 1-8.
Lin, L.-H., Chen, L.L., Zirrolli, J.A., and Harris, R.A. (1992) General
anesthetics potentiate gamma amino butyric acid actions on GABAa
receptors expressed in Xenopus oocytes: lack of involvement of intracellular
calcium. J. Pharmacol. Exp. Ther. 263, 569-578.
MacDonald, J.F., Miljkovic, Z., and Pennefeather, P. (1987) Use-dependent
block of excitatory amino acid currents in cultured neurons by ketamine. J.
Neurophysiol. 158, 251-261.
MacDonald, J.F., Mody, I., and Salter, M.W. (1989) Regulation of
N-methyl-D-aspartate receptors revealed by intracellular dialysis of murine
neurons in culture. J. Physiol. 414, 17-34.
Macdonald, R.L. (1995) Ethanol, GABA type A receptors, and protein kinase
C. Proc. Natl. Acad. Sci. 92, 3633-3635.
Machu, T.K. and Harris, R.A. (1994) Alcohols and anesthetics enhance the
function of 5- Hydroxytryptamine3 receptors expressed in Xenopus laevis
oocytes. J. Pharmacol. Exp. Ther. 271, 898-905.
Mascia, M.P., Machu, T.K., and Harris, R.A. Enhancement of glycine receptor
function by long chain alcohols and anaesthetics. Br. J. Pharmacol.,
submitted.
Meyer, H. (1901) Zur theorie der alkolnarkose: Der einfluss wechselnder
temperatur sur wirkungsstarke und theilungscoefficient dar narcotica. Naunyn
Schiedebergs Arch. Pharmacol. 46, 388-346.
Mihic, S.J., Whiting, P.J., and Harris, R.A. (1994) Anaesthetic
concentrations of alcohols potentiate GABAa receptor mediated
currents: lack of subunit specificity. Eur. J. Pharmacol. 268, 209-214.
Mori-Okamoto, J. and Tatsuno, J. (1985) Development of sensitivity to GABA
and glycine in cultured cerebellar neurons. Dev. Brain Res. 20, 249-258.
Overton, E. (1896) Ueber die osmotischen eigenschaften der zelle in ihrer
bedeutung fur die toxikologie und pharmakologie. Z. Phys. Chem. 22, 189-209.
Pribilla, I., Takagi, T., Langosch, D., Bormann, J., and Betz, H. (1992)
The atypical M2 segement of the beta subunit confers picrotoxin resistance to
inhibitory glycine receptor channels. EMBO J. 11, 4305-4311.
Rampil, I.J., Mason, P., and Singh, H. (1993) Anesthetic potency (MAC) is
independent of forebrain structures in the rat. Anesthesiol. 78, 707-712.
Ruiz-Gomez, A., Vaello, M.L., Valdivieso, F., and Mayor, F., Jr. (1991)
Phosphorylation of the 48-kDa subunit of the glycine receptor by protein
kinase C. J. Biol. Chem. 266, 559-566.
Sanna, E. and Harris, R.A. (1993) Neuronal ion channels. In: Recent
Developments in Alcoholism. Volume 11: Ten Years of Progress, Marc Galanter,
ed., Plenum Press, New York, pp. 169-186.
Taleb, O. and Betz, H. (1994) Expression of the human glycine alpha1
subunit in Xenopus oocytes: apparent affinities of agonists increase at high
receptor density. EMBO J. 13, 1318- 1324, 1994.
Wafford, K.A., Burnett,D., Leidenheimer, N.J., Burt, D.R., Wang, J.B.,
Kofuji, P. Dunwiddie, T.V., Harris, R.A., and Sikela, J.M. (1991) Ethanol
sensitivity of the GABAa receptor requires eight amino acids
contained in the gamma2L subunit of the receptor complex. Neuron 7, 27-33.
Wafford, K.A., Dunwiddie, T.V., and Harris, R.A. (1989) Calcium-dependent
Cl- currents elicited by injection of ethanol. Brain Res. 505, 212-219.
Wakamori, M., Ikemoto, Y., and Akaike, N. (1991) Effects of two volatile
anesthetics on the excitatory and inhibitory amino acid responses in
dissociated CNS neurons of the rat. J. Neurophysiol. 66, 2014-2021.
Williams, K.L., Ferko, A.P., Barbieri, E.J., and DiGregorio, G.J. (1995)
Glycine enhances the central depressant properties of ethanol in mice.
Pharmacol., Biochem., and Behav. 50, 199-205.
