Molecular mechanisms of anesthetic action on
neurotransmitter receptors are poorly understood. The major excitatory
neurotransmitter in the central nervous system is glutamate, and recent
studies found that volatile anesthetics inhibit the function of the
-amino-3-hydroxyisoxazolepropionic acid subtype of glutamate
receptors (e.g. glutamate receptor 3 (GluR3)), but enhance
kainate (GluR6) receptor function. We used this dissimilar pharmacology
to identify sites of anesthetic action on the kainate GluR6 receptor by
constructing chimeric GluR3/GluR6 receptors. Results with chimeric
receptors implicated a transmembrane region (TM4) of GluR6 in the
action of halothane. Site-directed mutagenesis subsequently showed that
a specific amino acid, glycine 819 in TM4, is important for enhancement
of receptor function by halothane (0.2-2 mM). Mutations of
Gly-819 also markedly decreased the response to isoflurane (0.2-2
mM), enflurane (0.2-2 mM), and 1-chloro-1,2,2-trifluorocyclobutane (0.2-2 mM). The
nonanesthetics 1,2-dichlorohexafluorocyclobutane and
2,3-dichlorooctafluorobutane had no effect on the functions of either
wild-type GluR6 or receptors mutated at Gly-819. Ethanol and
pentobarbital inhibited the function of both wild-type and mutant
receptors. These results suggest that a specific amino acid, Gly-819,
is critical for the action of volatile anesthetics, but not of ethanol
or pentobarbital, on the GluR6 receptor.
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INTRODUCTION |
There has been a shift of research focus in the field of
mechanisms of anesthetic action from nonspecific lipid perturbation to
specific protein sites of action (1). Anesthetics inhibit the activity
of the lipid-free enzyme luciferase (2) and produce stereoselective
actions on ion channel function (3, 4), suggesting receptor specificity
in anesthetic action. Recently, Mihic et al. (5) used
site-directed mutation of glycine and GABAA 1
receptors to identify specific amino acids in transmembrane domains 2 and 3 that are critical for the allosteric modulation of these receptors by alcohols and volatile anesthetics. We employed a similar
strategy in this study.
The neurotransmitter glutamate plays a major role in synaptic
excitation in the central nervous system and is critical for information storage in memory and learning (6). There has been an
explosion in understanding the biochemistry and molecular
biology of all the glutamate receptor types (N-methyl
D-aspartate, AMPA (
-amino-3-hydroxyisoxazolepropionic acid), kainate, and
metabotropic) (7). Molecular cloning studies have identified many
different glutamate receptors (GluRs), with GluR1-4 (AMPA), GluR5-7
(kainate), and KA1-2 (kainate-binding proteins) representing three
distinct families of non-N-methyl D-aspartate
glutamate receptors based on sequence homology and pharmacological
properties (7). Kainate receptors are widely distributed throughout the
central nervous system (8). GluR6 is found in high levels in most
regions of the hippocampus and also in the granule cells of the
cerebellum, and an understanding of its roles in brain function is
emerging. Previous studies have suggested a presynaptic function on
unmyelinated moves (C-fibers) in the spinal cord (9) and on excitatory
synapses within the CA3 region of the hippocampus (10). Recent studies have found that GABA and glutamate release is inhibited by presynaptic kainate receptors in rat hippocampus (11, 12). A role for kainate
receptors in synaptic transmission in the CA3 region of the hippocampus
was demonstrated in other recent studies (13, 14). In addition, a
genetic linkage study suggests that GluR6 is associated with the
pathogenesis of Huntington's disease (15).
With regard to anesthesia, AMPA receptor antagonists decrease the
minimum alveolar concentration (MAC) for halothane or isoflurane (16-19), but there are no reports of the effects of kainate receptor antagonists on MAC. There are few studies of the effects of volatile anesthetics on AMPA and kainate receptors, but Dildy-Mayfield et
al. (20) found that enflurane, halothane, and isoflurane (at
concentrations achieved during anesthesia) inhibited the function of
GluR3 receptors, but enhanced GluR6 receptor function.
One goal of this study was to investigate the mechanisms of anesthetic
action on GluR6 receptors. For this purpose, we constructed GluR3/GluR6
chimeras and used these to determine a region of GluR6 that is
essential for anesthetic action. We then constructed a series of mutant
GluR6 receptors to define specific sites of anesthetic action in
GluR6.
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EXPERIMENTAL PROCEDURES |
Materials--
cDNAs for rat GluR3(flop), GluR5(Q), and
GluR6(R) subunits were kindly provided by the laboratory of Dr. Steven
Heinemann (6, 21). Adult Xenopus laevis female frogs were
purchased from Xenopus I (Ann Arbor, MI). Dimethyl
sulfoxide, kainate, concanavalin A type IV, collagenase type 1A, and
pentobarbital were purchased from Sigma. Ethanol was purchased from
Aaper Alcohol and Chemical Co. (Shelbyville, KY). Halothane was from
Halocarbons Laboratories (River Edge, NJ). Enflurane and isoflurane
were from Ohmeda Pharmaceutical Products (Liberty Corner, NJ).
1-Chloro-1,2,2-trifluorocyclobutane (F3),
1,2-dichlorohexafluorocyclobutane (F6), and 2,3-chlorooctafluorobutane (F8) were obtained from PCR Inc. (Gainesville, FL). The Ultracomp Escherichia coli transformation kit was from Stratagene (La
Jolla, CA). A QIAGEN plasmid kit was used for purification of plasmid cDNA. GluR3, GluR5, and GluR6 cRNAs were prepared using the mCAP mRNA capping kit (Stratagene). The QuikChange site-directed
mutagenesis kit (Stratagene) was used for preparation of the single
amino acid mutations.
GluR cRNA Preparation--
The cDNAs for the GluRs were
inserted into the pGEM-HE vector. The cDNAs were linearized with
NheI, phenol/chloroform-extracted, and ethanol-precipitated
with sodium acetate, and cRNA was prepared using the Stratagene mCAP
mRNA capping kit. The cRNAs were extracted using phenol/chloroform
and precipitated with ethanol and sodium acetate.
To study the effects of anesthetics on GluR3/GluR6 chimeras, we used
some GluR3/GluR6 chimeras (R6(R3S2), R6TM1R3, R3TM4R6) that were
described by Stern-Bach et al. (21) and made additional GluR3/GluR6 chimeras. Splice sites for composition of chimeric receptor
subunits are as follows: C1,
R6(1-816)/R3(815-838)/R6(841-908); C2,
R6(1-811)/R3(809-838)/R6(841-908); C3,
R6(1-635)/R3(627-813)/R6(816-908); C4,
R6(1-635)/R3(627-838)/R6(841-908) (21); C5, R6(1-562)/R3(549-888) (21); and C6, R3(1-838)/R6(841-908) (21). The original names of the
C4, C5, and C6 chimeras were R6(R3S2), R6TM1R3, and R3TM4R6, respectively (21).
Site-directed mutagenesis was performed using the QuikChange
site-directed mutagenesis kit. All point mutations were verified by
double-stranded DNA sequencing.
Whole Cell Voltage Clamp of Injected Oocytes--
Isolation and
microinjection of Xenopus oocytes were performed as
described by Dildy-Mayfield et al. (22). Xenopus
oocytes were injected with 50 ng of cRNA coding for the GluRs. Oocytes were placed in a 100-µl recording chamber and perfused with modified Barth's solution containing 88 mM NaCl, 1 mM
KCl, 2.4 mM NaHCO3, 10 mM HEPES,
0.82 mM MgSO4, 0.33 mM
Ca(NO3)2, and 0.91 mM
CaCl2, pH 7.5, at a rate of 1.8 ml/min at room temperature.
Recording and clamping electrodes (1-5 megaohms) were pulled from
1.2-mm outside diameter capillary tubing and filled with 3 M KCl. A recording electrode was impaled into the animal
pole, and once the resting membrane potential stabilized, a clamping
electrode was inserted, and the resting membrane potential was allowed
to restabilize. A Warner OC 725-B oocyte clamp was used for
voltage-clamping each oocyte at
70 or
90 mV. For oocytes expressing
wild-type GluR5 or GluR6, chimeras from GluR6, or mutants of GluR6, 10 µM concanavalin A in modified Barth's solution was
preapplied for 2 min to prevent desensitization (23-25). The
anesthetics (halothane, isoflurane, enflurane, and F3), nonanesthetics
(F6 and F8) (26), ethanol, and pentobarbital were preapplied for 2 min
to allow for complete equilibration in the bath. Solutions of volatile
compounds were prepared immediately before use. The anesthetic,
nonanesthetic, and ethanol concentrations in the figures represent bath
concentrations, measured as described previously (20, 27).
In most experiments, we used maximally effective concentrations of
kainate to study anesthetic modulation; specifically, 1, 10, or 400 µM was used, depending upon the sensitivity of the particular receptor (see figure legends for details). However, we used
a submaximal concentration of kainate (100 nM) for Fig. 3
to obtain large enhancement effects of anesthetics because
Dildy-Mayfield et al. (20) showed that the potentiation by
halothane is greater at low concentrations of kainate than at maximally
effective concentrations.
Data Analysis--
Results are expressed as percentages of
control responses due to variability in oocyte expression. The control
responses were measured before and after each drug application to take
into account possible shifts in the control currents as recording
proceeded. The n values refer to the number of oocytes
studied. Each experiment was carried out with oocytes from at least two
different frogs. Statistical analyses were performed using t
tests with Dunnett's correction or analysis of variance for repeated
measures. EC50 values for concentration-response curves
were calculated using GraphPad Inplot software.
Molecular Modeling--
The amino acid sequence of TM4 for GluR6
was modeled as an
-helix using the Biopolymer module of Insight II
(MSI, San Diego, CA). The backbone atoms of the helix were tethered
with a force constant of 10 kcal/Å, and side chain packing was
optimized with Discover 97 (MSI) using the CFF91 force field. The
effect of the G819A mutation was modeled by replacing Gly-819 with
alanine and then re-optimizing the side chain packing while using a
force constant of 10 kcal/Å to tether the backbone atoms. A molecule of halothane was modeled with the Builder module of Insight II and
positioned near the pocket formed by Gly-819 in GluR6. The CFF91 force
field was assigned to both TM4 (which was tethered as described above)
and halothane. The halothane molecule was then docked onto TM4 using
Discover 97. The difference in molecular volume caused by the mutation
of glycine to alanine as well as the molecular volume of halothane were
calculated with Spartan (Wavefunction, Inc., San Diego, CA).
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RESULTS |
Effects of Halothane on Chimeric GluR3/GluR6 Receptors--
We
studied effects of 2 mM halothane on chimeras between GluR3
and GluR6 (Fig. 1). The enhancement
produced by halothane on wild-type GluR6 was eliminated in C1 and C2 by
replacement of TM4 and some of the sequence N-terminal to TM4 with the
same region from GluR3. Results with these chimeras indicated that TM4
was important for halothane potentiation. We also replaced the N- and
C-terminal regions of GluR3 with GluR6 (C5 and C6). These chimeras were
similar to GluR3, and halothane inhibited the kainate-induced current,
indicating that neither the C- nor N-terminal region was the site of
enhancement in GluR6 or the site of inhibition in GluR3. Likewise, 2 mM halothane inhibited the kainate-induced current by 47%
on C4, suggesting that the N terminus, TM1, TM2, and C terminus were
not very important in either enhancement or inhibition by halothane. It
is notable that the inhibitory effect of halothane on C4 was converted
to a stimulatory effect in C3 by changing the TM4 region.

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Fig. 1.
Halothane actions on chimeras between GluR3
and GluR6 subunits. The effects of 2 mM halothane on
kainate-induced responses were tested in oocytes expressing GluR3/GluR6
chimeras. Construction of chimeric receptor subunits is described under
"Experimental Procedures." GluR6 (wild-type), C1, C2, and C3 were
stimulated by 1 µM kainate. We used 10 µM
for C4 and C5. GluR3 (wild-type), C5, and C6 were stimulated by 400 µM kainate. Halothane was preapplied for 2 min to allow
for complete equilibration in the bath. Values represent percent change
in kainate response produced by halothane and are the means ± S.E. from 6 to 12 oocytes.
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To further define the site of halothane enhancement in TM4 of GluR6, we
made a series of mutants of C1 (Fig. 2).
We replaced the 4 amino acids just before TM4 (T2) and in the middle of
TM4 (T1 and T3). Halothane had little effect on T2 and T3; however, it
enhanced the function of T1 by 72 ± 6%. These results indicate that the sequence GGIFI in TM4 is critical for the enhancing effects of
halothane on GluR6.

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Fig. 2.
Halothane actions on mutants derived from
C1. The effects of 2 mM halothane on kainate (1 µM) responses were tested in oocytes expressing mutants
derived from C1 (see Fig. 1). Values represent percent change in
kainate response produced by halothane and are the means ± S.E.
from four to eight oocytes.
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Effects of Single Amino Acid Mutations in TM4--
Next, we made
single amino acid mutations of the GGIFI sequence as well as of the
flanking amino acids, selecting residues that were different between
GluR3 and GluR6 and changing the GluR6 residue to GluR3 (Fig.
3). Halothane enhanced the M1 (G814S), M2
(V815L), and M3 (Q816S) mutant receptor function to an extent similar
to that seen with the wild-type receptor (158 ± 12%). However,
halothane enhancement was reduced in the M4 (I818V), M5 (G819A), M6
(I821V), and M7 (I823Y) mutant receptors to 80 ± 13, 34 ± 10, 92 ± 24, and 68 ± 9% of control, respectively. The
reduction was greatest in GluR6(G819A), and these results suggested
that position 819 was particularly important for halothane action, but
the amino acids at positions 818, 821, and 823 may also have a role in
the enhancement. It is important to note that mutations in the TM4
region did not alter the EC50 for kainate responses (Table
I).

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Fig. 3.
Halothane actions on mutants derived from
GluR6 subunits. The effects of 2 mM halothane on
kainate (100 nM) responses were tested in oocytes
expressing wild-type and mutant GluR6 receptors. Values represent
percent change in kainate response produced by halothane and are the
means ± S.E. from four to eight oocytes. Statistical analyses
were performed using an unpaired t test and Dunnett's
correction. * and ***, p < 0.05 and p < 0.001 versus wild-type GluR6, respectively.
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Table I
EC50 values for kainate activation of wild-type and
mutant GluRs and GluR chimeras
Oocytes were voltage-clamped at 70 mV; kainate was applied for
20 s, and peak current was measured. For oocytes expressing GluR6,
chimeras from GluR6, and mutants of GluR6, 1 µM
concanavalin A was pre-applied for 2 min to prevent desensitization.
Values are the means ± S.E. from four to nine oocytes. Data for
C4, C5, and C6 are from Stern-Bach et al. (21).
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Effects of Anesthetics, Ethanol, and Nonanesthetics on Gly-819
Mutants--
We compared the effects of halothane, isoflurane,
enflurane, ethanol, the novel halogenated compound F3 (26), and the
nonanesthetics F6 and F8 (26) on wild-type GluR6 and mutant
GluR6(G819A) receptors. Halothane, isoflurane, enflurane, and F3
enhanced the actions of kainate on the wild-type GluR6 receptor in a
concentration-dependent manner,
as reported earlier (20). However, these
compounds had no significant effects on GluR6(G819A) (Figs. 4 and
5A). The nonanesthetics F6 and
F8 had little effect on either wild-type GluR6 or GluR6(G816) (Fig. 5,
B and C). At concentrations corresponding to
anesthesia (MAC), halothane (0.25 mM) produced 30%
enhancement of wild-type GluR6 function, enflurane (0.6 mM)
produced 45% enhancement, isoflurane (0.28 mM) produced
20% enhancement, and F3 (0.8 mM) produced 60% enhancement
(Fig. 4).

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Fig. 4.
Concentration-response curves for the effects
of halothane (0-2 mM) (A), enflurane
(0-2 mM) (B), and isoflurane (0-2
mM) (C) on the kainate-induced currents
for the wild-type GluR6 and GluR6(G819A) receptors. Anesthetics
were preapplied for 2 min before being coapplied with kainate (1 µM) for 20 s. Values are the means ± S.E. from
six to eight oocytes.
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Fig. 5.
Concentration-response curves for the effects
of F3 (0-2 mM) (A), F6 (0-35.6
µM) (B), and F8 (0-17.6
µM) (C) on the kainate-induced currents
for the wild-type GluR6 and GluR6(G819A) receptors. F3, F6, and F8
were preapplied for 2 min before being coapplied with kainate (1 µM) for 20 s. Values are the means ± S.E. from
six to eight oocytes.
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We also measured the effects of ethanol and pentobarbital on
GluR6(G819A) function. Ethanol inhibited the kainate-induced currents
in a concentration-dependent manner with both wild-type GluR6 and GluR6(G819A) (Fig. 6). The
pentobarbital inhibition was similar for wild-type GluR6 and
GluR6(G819A). On wild-type GluR6, pentobarbital inhibited the
kainate-induced currents by 27 ± 4 and 40 ± 5% at 100 and
200 µM, respectively. Pentobarbital (100 and 200 µM) also inhibited, by 23 ± 4 and 35 ± 3%,
respectively, the kainate-induced currents in oocytes expressing
GluR6(G819A) (Fig. 6).

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Fig. 6.
A, concentration-response curves for the
effect of ethanol on the kainate-induced currents for the wild-type
GluR6 and mutant GluR6(G819A) receptors. Drugs were preapplied for 2 min before being coapplied with kainate (1 µM) for
20 s. Values are the means ± S.E. from six to eight oocytes.
Analysis of variance for repeated measures showed a significant effect
of ethanol concentration (F = 33, p < 0.001), but did not show a significant effect of mutation
(F = 1.82, p > 0.05) or an interaction
between concentration and mutation (F = 0.67, p > 0.05). B, pentobarbital inhibition of
the kainate-induced currents for the wild-type GluR6 and mutant
GluR6(G819A) receptors. Pentobarbital was preapplied for 2 min before
being coapplied with kainate (1 µM) for 20 s. Values
are the means ± S.E. from six to eight oocytes.
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Next, we studied whether substitution of other amino acids at positions
819 would also abolish the action of volatile anesthetics. We
made GluR6(G819C), GluR6(G819F), GluR6 (G819H), GluR6(G819L), GluR6(G819Q), GluR6(G819S), and GluR6(G819W) and measured the effects
of volatile anesthetics on these mutants (Fig.
7). All of the mutations reduced (or
abolished) the potentiation of kainate action by volatile anesthetics.
The action of halothane was affected the least by the G819F mutation,
but this was still significantly different from halothane's action on
wild-type GluR6 (p = 0.03). It should be noted that the
modest enhancement produced by halothane (and the even smaller
enhancement produced by isoflurane) on the mutant receptors may be due
to the relatively high concentrations used for these drugs in Fig. 7.
Halothane and isoflurane were tested at concentrations eight and seven
times greater than the anesthetic (MAC) concentrations, whereas
enflurane and F3 were tested at concentrations corresponding to three
and one times MAC, respectively. In contrast to their effects on
the actions of the volatile anesthetics, none of the mutations affected
the action of ethanol or pentobarbital (Fig.
8).

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Fig. 7.
Effects of halothane (2 mM),
enflurane (2 mM), isoflurane (2 mM), and F3
(0.8 mM) on the kainate-induced currents for the wild-type
GluR6, GluR6(G819A), GluR6(G819C), GluR6(G819F) GluR6(G819H)
GluR6(G819L) GluR6(G819Q) GluR6(G819S), and GluR6(G819W)
receptors. Anesthetics or ethanol was preapplied for 2 min before
being coapplied with kainate (1 µM) for 20 s. Values
are the means ± S.E. from 4 to 20 oocytes. All results with the
GluR6 mutants are significantly different from those with wild-type
GluR6.
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Fig. 8.
Effects of ethanol (200 mM) and
pentobarbital (100 µM) on the kainate-induced currents
for the wild-type GluR6, GluR6(G819A), GluR6(G819C), GluR6(G819F),
GluR6(G819H), GluR6(G819L), GluR6(G819Q), GluR6(G819S), and
GluR6(G819W) receptors. Anesthetics or ethanol was preapplied for
2 min before being coapplied with kainate (1 µM) for
20 s. Values are the means ± S.E. from 4-20 oocytes.
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Effects of Ethanol and Anesthetics on GluR5--
Because the GluR5
subunit is homologous in the Gly-819 region to GluR6 but not to GluR3
(GluR6 and GluR5 have identical amino acid sequence from Ala-810 to
Ala-836) (6), we tested the effects of ethanol and anesthetics on the
function of homomeric GluR5 receptors. Modulation of GluR5 was quite
similar to that of GluR6, with volatile anesthetics enhancing function
and ethanol and pentobarbital inhibiting function (Fig.
9 and Table I). Comparison of drug action
on GluR3, GluR5, and GluR6 supported the idea that Gly-819 in GluR6 is
a critical determinant of the effects of volatile anesthetics on
AMPA/kainate receptors. Again, it should be noted that halothane and
isoflurane were tested at eight and seven times MAC and enflurane and
F3 at three and one times MAC, respectively, in Fig. 9.

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Fig. 9.
Effects of halothane (2 mM),
isoflurane (2 mM), and enflurane (2 mM)
(A) and of ethanol (200 mM), pentobarbital
(100 µM), and F3 (0.8 mM) (B) on
the kainate-induced currents for GluR5. The drug concentrations
were the same as tested on GluR6 in Figs. 7 and 8. Anesthetics or
ethanol was preapplied for 2 min before being coapplied with kainate
(100 µM) for 20 s. Values are the means ± S.E.
from four to eight oocytes. Hal., halothane;
Iso., isoflurane; Enf., enflurane;
Pento., pentobarbital.
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Molecular Modeling of the G819A Mutation--
The model of TM4
shows that Gly-819 provides a small cavity that may not be filled by
side chains of adjoining helices (Fig. 10A). This cavity can be
filled by interaction with a halothane molecule (Fig. 10B).
The halothane molecule not only occupies the space provided by the
small glycine side chain, it has favorable interactions with adjoining
amino acids. Substitution of alanine for Gly-819 reduces the cavity at
that position, as shown in the profile of the
-helix in Fig.
10C. The difference in molecular volume caused by the
mutation of glycine to alanine is 99.2-78.9 = 20.3 Å3. Mutation of glycine to tryptophan decreases the
molecular volume by 224.0-78.9 = 145.1 Å3. These
decreases in volume can be compared with the 121.7-Å3
volume of halothane itself.

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Fig. 10.
The amino acid sequence of TM4 for GluR6 was
modeled as an -helix, with the pore of the ion channel toward the
left (A). A molecule of halothane was modeled,
positioned near the pocket formed by Gly-819 in GluR6, and then docked
onto TM4 using Discover 97 (see "Experimental Procedures" for
details) (B). The effect of the G819A mutation was modeled
by replacing Gly-819 with alanine and then re-optimizing the side chain
packing (C).
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DISCUSSION |
In this study, we identified specific amino acids in the TM4
region of GluR6 that determine actions of volatile anesthetic action on
this receptor. In particular, several mutations of Gly-819 markedly
decreased halothane, isoflurane, enflurane, and F3 enhancement. Recently, Mihic et al. (5) used mutagenesis to implicate
several residues of the glycine
1-receptor subunit and
of the GABAA
- or
-subunits as important sites for
ethanol and enflurane actions on these receptors, suggesting that
alcohol and volatile anesthetics act on a specific region of these
proteins. Our results are consistent with findings in the
GABAA and glycine receptors in that specific mutations
markedly affect anesthetic action on all these receptors.
Glutamate receptors have been proposed to contain a large extracellular
N-terminal domain, four hydrophobic domains, and an intracellular
C-terminal domain (6, 21, 28, 29). It has been hypothesized that TM2 is
inserted into the ion channel pore, based on comparison of the amino
acid sequence between the N-methyl D-aspartate
receptor and K+ channels (28). These models indicate that
the region of TM4 implicated in anesthetic action on GluR6 in our study
is close to the extracellular membrane surface. The site of enflurane
action on GABAA and glycine receptors also appears to be
near the extracellular surface (5). The binding site for local
anesthetics recently established on Na+ channels is also
near the membrane surface, but in this case, it is the cytoplasmic
surface (30).
Our results raise the question of why anesthetics had little effect on
the kainate-induced currents for the GluR6 Gly-819 mutants. Previously,
it was reported that domains in the extracellular loop between TM3 and
TM4 are part of the kainate-binding site based on chimeric receptors
(21). In the present study, mutations in the TM4 region did not alter
the EC50 for kainate responses, suggesting that this
Gly-819 site is not close to the kainate-binding site. Halothane
(0.2-2 mM), in contrast to its affect on wild-type GluR6,
did not enhance the kainate-induced currents for GluR6(G819A); however,
it is interesting to note that the hydrophilicity, hydropathicity, and
polarity of alanine are similar to those of glycine (31-33). In
addition, a variety of amino acid substitutions differing in physical
properties all reduced anesthetic action. These results suggest that
the hydrophilicity, hydropathicity, and polarity of amino acids at the
site of action of anesthetics are not important factors for their
effects. However, mutation of glycine to alanine may produce sufficient
increase in volume to prevent halothane from binding within a cavity
formed (in part) by the Gly-819 region of TM4. Modeling of TM4 shows
that Gly-819 resides between more bulky amino acids and that a small
cavity is created at this position that can be filled by halothane.
However, the decrease in molecular volume of 20 Å3 created
by the G819A mutation is small compared with the 122 Å3 of
halothane, whereas the 145-Å3 decrease in volume caused by
the G819W mutation exceeds that of halothane. In that both the G819A
and G819W mutants are fully functional ion channels with
EC50 values for glutamate similar to that of wild-type
GluR6, it is clear that halothane must have some effect other than
filling a cavity. It is tempting to suggest that halothane enhances
receptor function by entering into a cavity and interacting with
residues one turn above or below Gly-819.
Ethanol and pentobarbital inhibit GluR6 function (25). Because
mutations of GABAA and glycine receptors that affect
volatile anesthetic actions also affect alcohol (but not barbiturate)
actions (5), we asked if mutation of Gly-819 would change ethanol and pentobarbital inhibition of GluR6. We found that a range of mutations did not alter the action of ethanol or pentobarbital. This suggests a
different mechanism of action, and perhaps different sites of action,
for these drugs as compared with those of the volatile anesthetics on
GluR6. Indeed, mutation of the TM2 region of AMPA receptors has been
shown to alter the action of pentobarbital (34). A distinct site and
mechanism of action could explain why alcohols and barbiturates inhibit
the function of GluR6 receptors, but volatile anesthetics have the
opposite effect.
In conclusion, we identified amino acid residues of the GluR6 receptor
that are important for volatile anesthetic potentiation of receptor
function. These results support the view that volatile anesthetics
interact with specific regions of membrane proteins rather than
nonspecifically with multiple regions of lipids or proteins. By analogy
to glycine and GABAA receptors (5), it is tempting to
speculate that Gly-819 is part of a hydrophobic pocket and provides an
anesthetic-binding site on GluR6 receptors. Unfortunately, it is not
currently possible to carry out the direct measurement of binding
required to test this hypothesis. Although there is little information
regarding the role of GluR6 in anesthesia, the creation of Gly-819
mutants in vivo using transgenic animal technology (35)
should be useful for answering this question.
We thank Drs. S. J. Mihic,
C. F. Valenzuela, M. P. Mascia, R. Cordoso, and V. Bleck for
helpful discussions and technical suggestions.