 |
 Previous Article | Next Article 
The Journal of Neuroscience, September 1, 2002, 22(17):7417-7424
Structural Evidence that Propofol Stabilizes Different
GABAA Receptor States at Potentiating and Activating
Concentrations
Daniel B.
Williams1, 3 and
Myles H.
Akabas1, 2
Departments of 1 Physiology and Biophysics and
2 Neuroscience, Albert Einstein College of Medicine, Bronx,
New York 10461, and 3 Integrated Program in Cellular,
Molecular, and Biophysical Studies, Columbia University, New York, New
York 10032
 |
ABSTRACT |
The GABAA receptor is a target of many general
anesthetics, such as propofol. General anesthetic binding sites are
distinct from the GABA binding sites. At low concentrations, the
anesthetics potentiate the currents induced by submaximal GABA
concentrations. At higher concentrations the anesthetics directly
activate GABAA receptors. In contrast, benzodiazepines,
such as diazepam, only potentiate currents induced by submaximal GABA
concentrations. Channel kinetic studies suggest that these drugs
stabilize different receptor states. We previously showed that the
accessibility of the anionic sulfhydryl reagent
p-chloromercuribenzenesulfonate (pCMBS ) applied extracellularly to cysteines
substituted for residues in the GABAA  1
subunit M3 membrane-spanning segment was state-dependent. The subset of
pCMBS-accessible, M3 segment cysteine mutants acts
as a reporter for receptor conformation. Here we show that
pCMBS, applied in the presence of a potentiating
concentration of propofol, reacts with a subset of 1
subunit, M3 segment, cysteine-substitution mutants (Y294C, V297C,
I302C, F304C). In the presence of a directly activating concentration
of propofol pCMBS reacts with a different subset
of the M3 cysteine-substitution mutants (Y294C, S299C, I302C, E303C,
A305C). These subsets are distinct from the subsets of M3
cysteine-substitution mutants that are reactive with
pCMBS in the absence and presence of GABA and in
the presence of diazepam. We hypothesize that distinct subsets of
reactive residues represent distinct conformations or ensembles of
conformations of the receptor. These results provide structural
evidence for at least five distinct receptor states, three
nonconducting states, resting, diazepam-bound and potentiating
propofol-bound, and two conducting-desensitized states, the activating
propofol-bound and GABA-bound states.
Key words:
acetylcholine; glycine; anesthesia; benzodiazepine; isoflurane; ethanol
| |
INTRODUCTION |
The GABA type A
(GABAA) receptor is an allosteric inhibitory
neurotransmitter-gated ion channel. The extracellular domain contains
two GABA binding sites that when occupied induce channel opening and
subsequent desensitization. The receptor also has binding sites for
allosteric modulators, including some general anesthetics,
benzodiazepines, and ethanol. The GABAA receptor is a target for the intravenous general anesthetic propofol, 2,6 di-isopropylphenol (Franks and Lieb, 1994 ; Krasowski and Harrison, 1999; Yamakura et al., 2001). At ~0.5 µM, propofol
potentiates currents induced by submaximal GABA concentrations but does
not directly activate GABAA receptors. At 20-fold
higher concentrations, propofol directly activates receptors, causing
channel opening in the absence of GABA (Sanna et al., 1995; Lam and
Reynolds, 1998). Propofol does not bind at the GABA binding sites. It
may bind in a crevice near the extracellular ends of the  subunit M2
and M3 membrane-spanning segments (Jones et al., 1995; Krasowski et
al., 1998). The effects of propofol on channel kinetics suggest that it stabilizes a doubly liganded, pre-open, nonconducting state
(Bai et al., 1999). Little is known, however, about the structure of
the propofol-bound receptor and how it differs from the resting state structure.
We previously showed that the reactivity of cysteines substituted for
1 M3 membrane-spanning segment residues with
pCMBS applied extracellularly is
state-dependent. The subset of
pCMBS-reactive residues provides a
reporter for the receptor state (Williams and Akabas, 1999). In the
resting state pCMBS only reacted with
cysteine (Cys) substituted for 1Ala291 and 1Tyr294, residues near the extracellular end
of M3. In the presence of GABA, pCMBS
reacted with Cys substituted for five more M3 residues
(1F296C, 1F298C,
1A300C, 1L301C, and
1E303C). Diazepam, a benzodiazepine, potentiates currents induced by submaximal GABA concentrations but does
not directly activate GABAA receptors. When
pCMBS was applied with diazepam, it
reacted with a subset of the M3 Cys-substitution mutants
(1A291C, 1Y294C,
1F296C, 1F298C, and
1L301C) that were accessible in the presence
of GABA (Williams and Akabas, 2000, 2001). We inferred that diazepam
binding induced or stabilized a distinct conformation or ensemble of
receptor conformations. Kinetic studies suggest this may be a singly
liganded state (Rogers et al., 1994; Lavoie and Twyman, 1996).
Although both diazepam and propofol potentiate GABA currents, they have
different effects on GABAA receptor kinetics
(Rogers et al., 1994; Bai et al., 1999; Ghansah and Weiss, 1999;
O'Shea et al., 2000). This implies that they stabilize different
states or ensembles of states. We hypothesized that the pattern of
pCMBS-reactive M3 Cys residues is a
marker for different states. Therefore, to provide structural evidence
that propofol stabilizes specific states, we investigated the effect of
coapplying pCMBS with either
potentiating or directly activating concentrations of propofol on the
1 M3 segment Cys-substitution mutants. Our results indicate that different subsets of M3 Cys mutants reacted with
pCMBS applied in the presence of
potentiating and directly activating concentrations of propofol. The
reactive Cys mutants were distinct from those that reacted in the
resting, GABA-bound and diazepam-bound states. We conclude that
propofol stabilizes a specific receptor conformation or conformations.
| |
MATERIALS AND METHODS |
Mutants and expression. The rat
1M3 segment cysteine-substitution mutants in
the pGEMHE plasmid were generated and characterized previously
(Williams and Akabas, 1999). In vitro mRNA transcription and
Xenopus oocyte preparation and injection were as described previously (Xu and Akabas, 1993; Williams and Akabas, 1999). Oocytes were injected with 50 nl of a 200 pg/nl solution of subunit mRNA in a
1:1:1 ratio of
1:1: 2S.
Electrophysiology. Two-electrode voltage-clamp recording
from Xenopus oocytes and data acquisition and analysis were
performed as described previously (Xu and Akabas, 1996; Williams and
Akabas, 1999). Oocytes were continuously perfused at 5 ml/min with
calcium-free frog Ringer's (CFFR) (in mM: 115
NaCl, 2.5 KCl, 1.8 MgCl2, and 10 HEPES, pH 7.5, with NaOH) at room temperature. Oocyte recording chamber volume was
~250 µl. Holding potential was  80 mV.
Experimental protocols. The sulfhydryl specific reagent used
in these experiments was p-chloromercuribenzenesulfonate
(pCMBS) (Sigma, St. Louis, MO). After
reaction with pCMBS,
-HgC6H4SO3
is covalently coupled to the reactive sulfhydryl.
To determine the irreversible effects of
pCMBS on the GABA-induced currents, the
following series of reagents were applied to two-electrode
voltage-clamped oocytes: 100 µM GABA, 20 sec; 100 µM GABA, 20 sec; EC50 GABA, 20 sec;
propofol alone, 20 sec; propofol + EC50 GABA, 20 sec; EC50 GABA, 20 sec; 0.5 mM
pCMBS ± propofol, (for propofol
concentration and duration of application see below); 100 µM GABA, 20 sec; 100 µM GABA, 20 sec;
EC50 GABA, 20 sec;
EC50 GABA, 20 sec (see Figs. 1 and
2 for examples). The applications of GABA
and reagents were separated by 3-5 min washes with CFFR to
allow complete recovery from desensitization. For wild-type and each
Cys mutant, propofol was applied at two concentrations; one that caused
potentiation but no detectable direct activation, and at 50 µM propofol, a concentration that caused direct
activation in all mutants. The potentiating concentration of propofol
used was 0.5 µM for all mutants except V297C where 0.1 µM propofol was used because direct activation was
observed with 0.5 µM propofol. To avoid accumulation of
propofol in the oocyte membranes and potentiation of subsequent
GABA-induced currents, we applied pCMBS
in the presence of the potentiating propofol concentration as three 20 sec applications interspersed by CFFR washes for a total of 1 min of
pCMBS application. For the directly
activating propofol concentration, as discussed more extensively in
Results, we could not avoid accumulation of propofol in the oocyte
membranes and potentiation of subsequent GABA-induced currents so we
simply applied pCMBS for 1 min in the
presence of activating propofol. Stock solutions of propofol in DMSO
were diluted into CFFR immediately before application. The percentage
of DMSO was never >0.02% and had no effect on GABA-induced current
(data not shown).
View larger version (15K):
[in this window]
[in a new window]
|
Figure 1.
Low concentrations of propofol potentiate
GABA-induced currents but do not directly activate the receptors.
Effects of propofol application on the currents recorded by
two-electrode voltage clamp from oocytes expressing the
1V297C (A) and
1L301C (B) mutants. Bars above the
current traces indicate the reagent applied and the duration of
application. Time between current traces is 3-5 min. Holding
potential, 80 mV. Note that propofol potentiates the GABA-induced
currents but does not elicit a current when applied by itself at these
concentrations. Also, the propofol effects wash out so that the final
GABA test current is similar to the initial current.
|
|
View larger version (24K):
[in this window]
[in a new window]
|
Figure 2.
The effect of coapplication of 0.5 mM
pCMBS with a potentiating concentration of
propofol on the subsequent GABA-induced currents. Currents recorded by
two-electrode voltage clamp from oocytes expressing the
1V297C (A), 1I302C
(B), 1E303C
(C), and 1F304C
(D) mutants. Bars above the current traces
indicate the reagent applied and the duration of application. Time
between current traces is 3-5 min. Holding potential, 80 mV. The
propofol concentration was 0.1 µM in
A and 0.5 µM in B-D.
Note that in A and D the subsequent
GABA-induced currents were potentiated; in B the
subsequent currents were inhibited, and in B-D the
subsequent currents were unchanged.
|
|
We infer that an irreversible change in the GABA-induced currents after
pCMBS application is attributable to the
covalent modification of a Cys by pCMBS.
Whether the change is potentiation or inhibition of subsequent GABA-induced currents does not affect this conclusion. At any given
position Cys modification by pCMBS may
alter ion conduction or gating kinetics. Potentiation after pCMBS modification is almost certainly
caused by a change in gating kinetics manifested as a reduction in the
GABA EC50. We have not examined the mechanistic
basis for the effects of pCMBS
modification in this paper because it is not relevant to the major
focus of this investigation. The percent effect of
pCMBS = {(IGABA,
after/IGABA, before) - 1} * 100. IGABA, after is the average peak current of the two GABA test pulses after the application of pCMBS, and
IGABA, before is the average of the
peak current of the initial two GABA applications. Test pulses of GABA
were applied at two concentrations, EC50
and >5 times EC50 (near-saturating GABA,
generally 100 µM except for L301C where 1 mM was used). Changes in the peak current induced
by the EC50 GABA test pulses are more sensitive
to effects of pCMBS modification on
gating kinetics, whereas changes in the peak current induced by the
near-saturating GABA test pulses are more sensitive to effects of
modification on conductance (Williams and Akabas, 1999).
For screening experiments, pCMBS was
applied for 1 min at 0.5 mM. This combination of time and
concentration were chosen because they were the maximal concentration
and duration that caused no significant increase in the leak
conductance of uninjected Xenopus oocytes. This limits our
ability to detect reactive residues. As discussed below, for a given
mutant, given the variability of responses, application of a reagent
must cause a net change in current greater than ~30% to be
statistically significantly different than wild type by a one-way
ANOVA (for n between 4 and 6). Thus, if complete
reaction caused 100% inhibition of the GABA-induced current, with a
detection threshold of 30% effect and the
pCMBS reaction conditions of 0.5 mM applied for 1 min, the slowest reaction rate
that we can detect must have a second order reaction rate constant >12
l · mol1 · sec1.
Measurement of reaction rates.
pCMBS reaction rates with the engineered
Cys were determined by the effect of sequential brief applications of
pCMBS as we have done previously
(Williams and Akabas, 1999, 2000). A test pulse of GABA was applied to
measure the GABA-induced current. Propofol + pCMBS (0.2-0.5
mM) was applied for 15-60 sec. After washout, a
test pulse of GABA was applied, and the GABA-induced current was
measured. The effect of five to eight brief, sequential applications of propofol + pCMBS were determined. The
magnitudes of the GABA test currents were normalized relative to the
initial test current. The normalized current was plotted as a function
of the cumulative duration of pCMBS
application and fitted with a single exponential function using Prism2
software (GraphPad, San Diego, CA). The second order rate constant was
calculated by dividing the pseudo-first order rate constant obtained
from the exponential fit by the pCMBS concentration.
Statistics and curve fitting. Data are expressed as the
percentage change of current after modification ± SEM. The
significance of differences between each mutant and wild type was
determined by one-way ANOVA using the Student-Newman-Keuls post
hoc test (SPSS for Windows; SPSS, Inc., Chicago, IL).
Dose-response curves were fit using Prism2 software.
It is important to recognize that
pCMBS-reactive residues were identified
based on the functional effect of modification. Functional effects were
determined by the statistical significance of the effect on a mutant
relative to the effect on wild type. For mutants where the average
effect after application of pCMBS was
small, whether the effect was judged to be significant depended, in
part, on the stringency of the one-way ANOVA post hoc test used. In our previous work on the 1 M3
segment, using the Student-Newman-Keuls post hoc test to
determine significance of effects, the effect of
pCMBS applied in the presence of GABA
was statistically significant at six residues,
1A291C, 1Y294C,
1F298C, 1A300C,
1L301C, and 1E303C.
With the less stringent Duncan post hoc test, an additional
residue 1F296C is judged to be reactive with
pCMBS applied in the presence of GABA
(Williams and Akabas, 1999). The choice of post hoc test is,
unfortunately, somewhat arbitrary. Thus, for Cys mutants where the
effects of complete reaction are small, it may be difficult to
determine functionally whether reaction has occurred.
| |
RESULTS |
Characterization of propofol effects on the M3 Cys mutants
The GABA EC50 for wild-type
112S receptors
was 2.1 ± 1.3 µM (n = 6) and for
the Cys-substitution mutants ranged between 0.5 ± 0.1 µM for 1A291C and
1Y294C and 48 ± 15 µM for 1L301C, as
reported previously (Williams and Akabas, 1999). Lacking a high-resolution structure of the membrane-spanning domain, it is
difficult to infer the structural basis for the changes in GABA
EC50 that resulted from Cys substitutions. Either
the mutations subtly alter the structure of the M3 segment or during
gating induced conformational changes the environment of these Cys
residue changes. Either way these changes somehow affect the kinetics of the transitions or the relative stability of different receptor states thereby altering the GABA EC50. For an
extensive discussion of the relationship between mutations and
EC50, see (Colquhoun, 1998).
For each Cys mutant, propofol dose-response relationships were
determined to identify the maximum propofol concentration that potentiated GABA-induced currents without directly activating the
receptors. For most Cys mutants the concentration used was 0.5 µM. A 0.5 µM concentration of propofol
potentiated EC20 to EC50
GABA currents in wild-type receptors by 46 ± 12%
(n = 7) and in the mutants potentiation ranged from
13% for 1V292C and 1S299C to 102% for
1Y294C (Table
1). For 1V297C,
direct activation occurred at 0.5 µM, and so
0.1 µM propofol was used. The effects of
application of potentiating concentrations of propofol washed out
completely within 5 min. It is difficult to establish an
EC50 for propofol potentiation because as the
propofol concentration increases, potentiation effects run into direct
activation, making it difficult to determine the maximum potentiation.
At propofol concentrations >50 µM the direct
activation current declines suggesting an inhibitory effect at high
propofol concentrations. The mechanism of this inhibition is
unknown.
All of the Cys mutants showed direct activation by 50 µM
propofol. For wild-type receptors, the current induced by 50 µM propofol was 41 ± 5% (n = 5) of
the current induced by saturating GABA. For the Cys mutants, the
current induced by 50 µM propofol relative to
the maximal GABA-induced current ranged from 24% for
1A295C to 146% for
1A300C (Table 1). Washout of 50 µM propofol terminated the propofol-induced
currents in all mutants, however, subsequent GABA-induced currents were
potentiated relative to the initial GABA test currents for over 30 min
after propofol washout. Addition of 10 mg/ml bovine serum albumin to
the wash buffer did not speed the washout of this propofol-induced
potentiation of subsequent GABA-induced currents. This limited our
ability to detect Cys mutants as reactive with
pCMBS plus 50 µM
propofol to those mutants where the effect on subsequent currents was
inhibition. Because of the very slow washout of propofol from the
oocyte membranes and the persistent potentiation of subsequent GABA
currents, we were unable to identify as reactive in the presence of
activating concentrations of propofol any position where the effect of
pCMBS modification was potentiation.
Reactions with pCMBS applied in the presence
of potentiating concentrations of propofol
For wild type and all of the M3 Cys mutants, the effects of a 1 min application of a potentiating concentration of propofol washed out
after a 5 min wash with CFFR buffer (Fig. 1). A 1 min application of
0.5 mM pCMBS with a
potentiating concentration of propofol had no effect on wild-type
112S receptors
(Williams and Akabas, 1999). When applied for 1 min in the presence of
a potentiating concentration of propofol,
pCMBS irreversibly altered the
subsequent GABA-induced currents of the mutants
1Y294C, 1V297C,
1I302C, and 1F304C
(Figs. 2, 3). The subsequent currents
were inhibited for the mutants 1Y294C and
1I302C. The subsequent currents were
potentiated for the mutants 1V297C and
1F304C (Figs. 2, 3).
1Y294C reacts with pCMBS in the resting state, thus, to
determine whether propofol altered the reactivity of
1Y294C, we measured the rate of reaction in the presence and in the absence of propofol. In the presence of propofol the second order reaction rate constant for
pCMBS with
1Y294C was 134 ± 20 l · mol1 · sec1
(n = 3). In the resting state the second order rate
constant was 47 ± 8 l · mol1 · sec1,
and in the presence of GABA the rate constant was 86 ± 14 l · mol1 · sec1
(Williams and Akabas, 1999). The increased reaction rate in the presence of a potentiating concentration of propofol implies that propofol altered the environment and/or the access pathway to this
residue compared with the resting and GABA-activated states.
View larger version (18K):
[in this window]
[in a new window]
|
Figure 3.
The irreversible effect of a 1 min application of
0.5 mM pCMBS applied in the presence
of a potentiating concentration of propofol on subsequent GABA-induced
currents of wild-type and mutant GABAA receptors. For each
mutant an EC50 GABA concentration was used for the test
pulses. Black bars indicate effects that are
statistically significantly different from the effect on wild type by a
one-way ANOVA. A negative effect indicates inhibition, and a positive
effect indicates potentiation of subsequent GABA currents. The mean and
SEM values are shown. For each mutant, the effects on three to six
oocytes are averaged.
|
|
The compound 2,6-di-tert-butylphenol is structurally similar to
propofol but is not an anesthetic (Krasowski et al., 2001). To
determine the specificity of propofol, we applied
pCMBS with 2,6-di-tert-butylphenol at
the same concentration as propofol on the reactive mutants. There was
no effect of a 1 min application of 2,6-di-tert-butylphenol plus 0.5 mM pCMBS on any of the four
Cys mutants that reacted with pCMBS in
the presence of propofol (Fig. 4). This
suggests that the propofol-induced changes in M3 Cys mutant reactivity
may be related to the anesthetic activity of propofol and not simply
caused by its hydrophobicity.
View larger version (13K):
[in this window]
[in a new window]
|
Figure 4.
The lack of effect of a 1 min application of 0.5 mM pCMBS applied in the presence of
2,6 di-tert-butyl-phenol (2,6 dtbp), a nonanesthetic
analog of propofol, on GABA-induced currents from oocytes expressing
1V297C (A) and
1A305C (B). The concentration of
2,6 di-tert-butyl-phenol was similar to a potentiating propofol
concentration, 0.5 µM in A, and similar to
an activating propofol concentration, 50 µM in
B. Time between current traces is 3-5 min. Holding
potential, 80 mV. Note that the initial and final GABA currents are
similar.
|
|
Reactions with pCMBS applied in the presence
of a directly activating concentration of propofol
A 1 min application of 50 µM propofol to wild-type
112S receptors
resulted in potentiation of subsequent submaximal GABA-induced test
currents but had no effect on currents elicited by saturating concentrations of GABA. Although the extent of this potentiation of
submaximal GABA-induced test currents diminished over time, it was
still significant after 30 min of washing. Similar effects were
observed with all of the mutants. We assume that during the 50 µM propofol application, it partitioned into the oocyte
membranes and then gradually leached out leaving a concentration
sufficient to potentiate subsequent submaximal GABA responses, but not
sufficient to directly activate. This limited our ability to detect Cys
mutants that were reactive with pCMBS
plus 50 µM propofol because we could only use saturating
GABA test pulses. Thus, only at Cys mutants where
pCMBS application inhibited subsequent
GABA-induced currents can we infer that reaction has occurred. If
pCMBS reaction potentiated subsequent
GABA-induced currents, an effect that we would only expect to see with
submaximal GABA test pulse, it would be difficult for us to distinguish
the effect from the persistent potentiation because of slow washout of
the propofol. Thus, all of the experiments with a directly activating
propofol concentration used only saturating concentrations of GABA test pulses.
For wild-type
112S receptors,
a 1 min application of 0.5 mM
pCMBS with 50 µM propofol
had no subsequent inhibitory effect. When applied for 1 min in the
presence of 50 µM propofol,
pCMBS irreversibly inhibited the
subsequent GABA-induced currents of the
mutants 1Y294C,
1S299C, 1I302C,
1E303C, and 1A305C
(Figs. 5, 6). As described above, because
1Y294C reacts with
pCMBS in the resting state, we measured
the rate of reaction in the presence of 50 µM propofol.
The second order reaction rate was 118 ± 23 l · mol1 · sec1
(n = 3), faster than the reaction rate in the absence
of GABA, 47 ± 8 l · mol1 · sec1
(Williams and Akabas, 1999). We also measured the
pCMBS reaction rate with
1E303C in the presence of 50 µM propofol to compare with the reaction rate
in the presence of GABA. The rate in the presence of propofol was
76 ± 9 l · mol1 · sec1
(n = 5); significantly slower than the rate of 220 ± 51 l · mol1 · sec1
in the presence of GABA (Williams and Akabas, 1999).
View larger version (15K):
[in this window]
[in a new window]
|
Figure 5.
The effect of coapplication of 0.5 mM
pCMBS with 50 µM propofol, a
directly activating concentration, on the subsequent GABA-induced
currents. Currents recorded by two-electrode voltage clamp from oocytes
expressing the 1I302C (A),
1E303C (B), and
1A305C (C) mutants. Bars above the
current traces indicate the reagent applied and the duration of
application. Time between current traces is 3-5 min. Holding
potential, 80 mV. Note that the subsequent GABA-induced currents
evoked by both saturating and EC50 GABA concentrations are
inhibited. The data in A and C suggest
that pCMBS modification reduced the GABA
EC50.
|
|
View larger version (18K):
[in this window]
[in a new window]
|
Figure 6.
The irreversible effect of a 1 min application of
0.5 mM pCMBS applied in the presence
of 50 µM propofol, a directly activating concentration,
on subsequent GABA-induced currents of wild-type and mutant
GABAA receptors. Saturating GABA concentrations were used
for the test pulses. Black bars indicate effects that
are statistically significantly different from the effect on wild type
by a one-way ANOVA. A negative effect indicates inhibition, and a
positive effect indicates potentiation of subsequent GABA currents. The
mean and SEM values are shown. For each mutant, the effects on three to
seven oocytes are averaged.
|
|
We tested whether coapplication of pCMBS
with 50 µM 2,6-di-tert-butylphenol, the anesthetically
inactive structurally related compound, had any effect on the reactive
mutants. A 50 µM concentration of 2,6-di-tert-butylphenol
did not induce a current in any of the mutants tested. A 1 min
coapplication of 0.5 mM
pCMBS plus 50 µM
2,6-di-tert-butylphenol did not inhibit the subsequent GABA-induced
currents of the mutants 1Y294C,
1S299C, 1I302C, 1E303C, and 1A305C
(Fig. 4). The hydrophobicity of the reagent coapplied with
pCMBS does not seem to affect the M3
Cys-substitution mutant reactivity. Thus, we infer that the increase in
the Cys mutant accessibility in the presence of an activating
concentration of propofol is related to its actions as a general anesthetic.
| |
DISCUSSION |
We have shown that pCMBS applied in
the presence of a potentiating concentration of propofol reacted with a
subset of M3 segment Cys-substitution mutants (Fig. 3, Table
2). pCMBS
reacted with a different subset of M3 segment Cys-substitution mutants
when applied with a directly activating concentration of propofol (Fig.
6, Table 2). These subsets of
pCMBS-reactive M3 segment
Cys-substitution mutants are different from the residue subsets that
react with pCMBS in the resting state,
i.e., in the absence of GABA, and in the GABA-bound and diazepam-bound
states (Table 2) (Williams and Akabas, 1999, 2000). The accessibility
of the M3 segment Cys-substitution mutants to reaction with
pCMBS depends on the state of the
GABAA receptor. Conformational changes during
gating and during drug binding appear to alter the structure surrounding the GABAA receptor M3
membrane-spanning segments. These structural changes create access
pathways from the extracellular solution into the interior of the
protein. This allows pCMBS to gain
access to M3 segment Cys-substitution mutants and thus, to react
(Williams and Akabas, 1999, 2000, 2001). We infer that the distinct
subsets of pCMBS-reactive
Cys-substitution mutants are markers or reporters for specific states
or ensembles of receptor states. A limitation of our approach is that
over the time course of our experiments (tens of seconds) the channels
undergo transitions between different states. Thus, we cannot know
whether the pCMBS accessibility occurs
in a single state or in multiple states. (Hereafter, when we refer to a
subset of residues being a reporter for a receptor state, we implicitly
acknowledge that it may be for an ensemble of states.) We hypothesize
that the accessibility of residues to react with
pCMBS differs in different states. Thus,
the fact that different patterns of accessible residues are obtained in
the presence of different drugs or agonists suggests that different
drugs stabilize specific states. Therefore, we infer that each of these
agonists or modulators, GABA, propofol, and diazepam, stabilize
distinct states.
View this table:
[in this window]
[in a new window]
|
Table 2.
Summary of 1 M3 segment Cys substitution
mutant accessibility to pCMBS applied in the presence of
the indicated reagentsa
|
|
Several kinetic models have been proposed to account for the results of
experimental studies of GABAA receptor kinetics
(Weiss and Magleby, 1989; MacDonald and Twyman, 1992; Maconochie et
al., 1994; Jones and Westbrook, 1995; Jones et al., 1998, 2001; Bai et
al., 1999; Jayaraman et al., 1999; Burkat et al., 2001). In general
these models have three nonconducting closed states (unliganded resting
state, monoliganded, and diliganded), leading to at least one open
conducting state. Single-channel data provides evidence for three open
states (Weiss and Magleby, 1989; MacDonald and Twyman, 1992). In
addition, there are other nonconducting, desensitized states.
Significant rates of entry into desensitized states can occur from the
diliganded closed state (Jones and Westbrook, 1995; Bai et al., 1999)
and in some models from the open state as well (Burkat et al., 2001).
Studies of the effects of diazepam on channel kinetics suggest that it
stabilizes a singly liganded closed state, increasing the association
rate for binding the first GABA molecule (Rogers et al., 1994; Lavoie
and Twyman, 1996). In contrast, studies of propofol at potentiating
concentrations suggest that it stabilizes a doubly liganded closed
state and slows entry into fast and slow desensitized states (Bai et
al., 1999). Our experiments provide structural evidence to support the
results of these kinetic studies. Thus, diazepam and propofol at
potentiating concentrations alter the
pCMBS accessibility of different subsets
of M3 Cys-substitution mutants. This implies that these two drugs
stabilize different states.
The subset of pCMBS-reactive residues
was different in the presence of GABA and directly activating propofol.
We previously suggested that reaction in the presence of GABA may be
occurring in a desensitized state (Williams and Akabas, 1999). Because
propofol reduces the extent of desensitization, the residues reactive
in the presence of activating propofol may be more representative of
the open state. Alternatively, we cannot exclude the possibility that
GABA and propofol may be stabilizing different open states.
The pCMBS-reactive residues are, at
least transiently, on the water-accessible surface of the protein
(Akabas et al., 1994; Karlin and Akabas, 1998). We infer this because
pCMBS reacts a thousand times faster
with the ionized thiolate form of cysteine
(S) compared with the unionized thiol
(SH) (Hasinoff et al., 1971), and only cysteines on the
water-accessible surface will ionize to any significant extent. Thus, a
question arises regarding how an access pathway was created for
pCMBS to reach residues in the interior
of the membrane-spanning domain of the protein? We previously showed
that in the presence of GABA the access pathway does not appear to pass
through the open channel because the
pCMBS reaction rates were not voltage
dependent (Williams and Akabas, 1999). Thus, we do not think that the
pCMBS-accessible M3 residues are part of
the channel lining. More likely, as the channel undergoes the
conformational changes from the closed to the open to the desensitized
states, spaces are formed between the membrane-spanning segments. These
spaces or crevices-cavities may transiently extend up to the
extracellular surface of the protein, allowing water and
pCMBS to enter the cavities.
High-resolution protein structures indicate that cavities in the
protein interior are water-filled, even if they are lined by
hydrophobic residues (Yu et al., 1999). Furthermore, the dynamic motion
of proteins allows the water molecules in these cavities to exchange
with bulk water (Englander and Kallenbach, 1983). Thus, in the crystal
structures of bacteriorhodopsin, the number of water molecules in the
protein interior changes in different photocycle intermediates,
suggesting that water can rapidly move into and out of the protein
interior as the protein undergoes conformational changes (Dencher et
al., 2000; Maeda et al., 2000; Gottschalk et al., 2001).
The formation of water-filled cavities in the membrane-spanning domain
during channel gating may play an important role in the mechanism of
general anesthetic potentiation of GABA-induced currents. Based on our
results, we infer that water-accessible cavities form around the M3
segment during channel gating and modulation by diazepam and propofol
(Williams and Akabas, 1999, 2000, 2001). These cavities may form as the
channel-lining M2 segments rotate as part of the conformational change
from the resting to the open state (Unwin, 1995; Horenstein et al.,
2001). If these cavities form or enlarge transiently during activated states of the receptor, and occupation of the cavity by an anesthetic is energetically more favorable than occupation by water, then an
anesthetic partitioning into the cavity would stabilize that state of
the receptor. Anesthetics have been shown to occupy pre-existing cavities in proteins of known crystal structure (Bhattacharya et al.,
2000; Eckenhoff et al., 2001; Whittington et al., 2001). Energetically
favorable occupation by an anesthetic of a protein cavity that is
present in the resting state or in a transient state would be expected
to stabilize the structure of that protein conformational state (Tanner
et al., 2001). For the GABAA receptor anesthetic
occupation of cavities formed during gating would stabilize activated
states of the receptor and thus may lead to the anesthetic-induced potentiation of GABA-induced currents that is observed. Consistent with
this, evidence has been presented that halothane, isoflurane, and
chloroform may occupy a common cavity (Jenkins et al., 2001). A binding
cavity for alcohols on GABAA and glycine
receptors may also exist (Wick et al., 1998). Thus, the formation of
water-filled cavities in the interior of the membrane-spanning domain
during channel gating may provide a molecular basis for allosteric
modulation of GABAA receptors by general anesthetics.
We infer that propofol stabilizes different receptor states at
potentiating and directly activating concentrations based on the
observation that different subsets of M3 Cys mutants are reactive with
pCMBS in the presence of the two
concentrations. This implies that there are at least two propofol
binding sites on the GABAA receptor consistent
with previous results (Krasowski and Harrison, 1999). The location of
these sites is unknown. Unlike diazepam, the subunit is not
necessary for either potentiation or activation by propofol (Jones et
al., 1995). Evidence has been presented that anesthetic binding sites
are formed by residues near the extracellular ends of the M2 and M3
segments. Mutation of the residues 1Ser270
(M2) and 1Ala291 (M3) alters potentiation by volatile anesthetics (Mihic et al., 1997; Koltchine et al., 1999; Jenkins et al., 2001), whereas mutation of the aligned residues in the
subunit alters potentiation by intravenous anesthetics (Sanna et
al., 1995; Belelli et al., 1997; Moody et al., 1997; Krasowski et al.,
1998). A role for the subunit in intravenous anesthetic binding has
also been suggested (Krasowski et al., 1997; Uchida et al., 1997; Lam
and Reynolds, 1998). We observed that Cys-substitution for
1 M3 segment residues altered the extent of
potentiation by 0.5 µM propofol (Table 1). Based on this
one cannot conclude that these subunit residues form part of the propofol binding site and that the mutations are directly altering affinity for propofol. More likely the mutations alter the transduction process or the relative stability of various receptor states, thereby
altering the efficacy of propofol (Colquhoun, 1998).
In summary, we have shown that propofol binding at both potentiating
and activating concentrations induced a conformational change in the
GABAA receptor membrane-spanning domain that
created a pathway for water and pCMBS to
gain access to engineered Cys residues in the
1 M3 membrane-spanning segment. The subsets of
M3 residues that were pCMBS-reactive in
the presence of potentiating and directly activating concentrations of
propofol were different, and we inferred that at the different
concentrations propofol stabilized two different states or ensembles of
states. These states were distinct from the resting state or the
GABA-bound or diazepam-bound states. The water-filled cavities that
form around the M3 segment during gating may have an important role in
forming the binding sites for general anesthetics and thus in the
mechanism of allosteric modulation of the GABAA
receptor by general anesthetics.
| |
FOOTNOTES |
Received March 20, 2002; revised April 23, 2002; accepted April 25, 2002.
This work was supported in part by National Institutes of Health Grants
GM61925, NS30808, and GM63266. We thank Drs. Neil Harrison, Andrew
Jenkins, and Jeffrey Horenstein for helpful discussions, Eric Goren for
preparation of oocytes, and Drs. Moez Bali, Amal Bera, and Jeffrey
Horenstein for comments on this manuscript.
Correspondence should be addressed to Dr. Myles Akabas, Department of
Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail: makabas{at}aecom.yu.edu.
D. B. Williams' present address: Laboratory for Molecular
Neuropharmacology, National Institute of Neurological Disorders and
Stroke, National Institutes of Health, Building 10, Room 5C103, 10 Center Drive, Bethesda, MD 20892.
| |
REFERENCES |
-
Akabas MH,
Kaufmann C,
Archdeacon P,
Karlin A
(1994)
Identification of acetylcholine receptor channel-lining residues in the entire M2 segment of the alpha subunit.
Neuron
13:919-927[Web of Science][Medline].
-
Bai D,
Pennefather PS,
MacDonald JF,
Orser BA
(1999)
The general anesthetic propofol slows deactivation and desensitization of GABA(A) receptors.
J Neurosci
19:10635-10646[Abstract/Free Full Text].
-
Belelli D,
Lambert JJ,
Peters JA,
Wafford K,
Whiting PJ
(1997)
The interaction of the general anesthetic etomidate with the gamma-aminobutyric acid type A receptor is influenced by a single amino acid.
Proc Natl Acad Sci USA
94:11031-11036[Abstract/Free Full Text].
-
Bhattacharya AA,
Curry S,
Franks NP
(2000)
Binding of the general anesthetics propofol and halothane to human serum albumin. High resolution crystal structures.
J Biol Chem
275:38731-38738[Abstract/Free Full Text].
-
Burkat PM,
Yang J,
Gingrich KJ
(2001)
Dominant gating governing transient GABA(A) receptor activity: a first latency and Po/o analysis.
J Neurosci
21:7026-7036[Abstract/Free Full Text].
-
Colquhoun D
(1998)
Binding, gating, affinity and efficacy: the interpretation of structure-activity relationships for agonists and of the effects of mutating receptors.
Br J Pharmacol
125:924-947[Web of Science][Medline].
-
Dencher NA,
Sass HJ,
Buldt G
(2000)
Water and bacteriorhodopsin: structure, dynamics, and function.
Biochim Biophys Acta
1460:192-203[Medline].
-
Eckenhoff RG,
Pidikiti R,
Reddy KS
(2001)
Anesthetic stabilization of protein intermediates: myoglobin and halothane.
Biochemistry
40:10819-10824[Medline].
-
Englander SW,
Kallenbach NR
(1983)
Hydrogen exchange and structural dynamics of proteins and nucleic acids.
Q Rev Biophys
16:521-655[Web of Science][Medline].
-
Franks NP,
Lieb WR
(1994)
Molecular and cellular mechanisms of general anaesthesia.
Nature
367:607-614[Medline].
-
Ghansah E,
Weiss DS
(1999)
Benzodiazepines do not modulate desensitization of recombinant alpha1beta2gamma2 GABA(A) receptors.
NeuroReport
10:817-821[Web of Science][Medline].
-
Gottschalk M,
Dencher NA,
Halle B
(2001)
Microsecond exchange of internal water molecules in bacteriorhodopsin.
J Mol Biol
311:605-621[Web of Science][Medline].
-
Hasinoff BB,
Masden NB,
Avramovic-Zikic O
(1971)
Kinetics of the reaction of p-chloromercuribenzoate with the sulfhydryl groups of glutathione, 2-mercaptoethanol, and phosphorylase b.
Can J Biochem
49:742-751[Web of Science][Medline].
-
Horenstein J,
Wagner DA,
Czajkowski C,
Akabas MH
(2001)
Protein mobility and GABA-induced conformational changes in GABA(A) receptor pore-lining M2 segment.
Nat Neurosci
4:477-485[Web of Science][Medline].
-
Jayaraman V,
Thiran S,
Hess GP
(1999)
How fast does the gamma-aminobutyric acid receptor channel open? Kinetic investigations in the microsecond time region using a laser- pulse photolysis technique.
Biochemistry
38:11372-11378[Medline].
-
Jenkins A,
Greenblatt EP,
Faulkner HJ,
Bertaccini E,
Light A,
Lin A,
Andreasen A,
Viner A,
Trudell JR,
Harrison NL
(2001)
Evidence for a common binding cavity for three general anesthetics within the GABAA receptor.
J Neurosci
21:RC136[Abstract/Free Full Text].
-
Jones MV,
Westbrook GL
(1995)
Desensitized states prolong GABAA channel responses to brief agonist pulses.
Neuron
15:181-191[Web of Science][Medline].
-
Jones MV,
Harrison NL,
Pritchett DB,
Hales TG
(1995)
Modulation of the GABAA receptor by propofol is independent of the gamma subunit.
J Pharmacol Exp Ther
274:962-968[Abstract/Free Full Text].
-
Jones MV,
Sahara Y,
Dzubay JA,
Westbrook GL
(1998)
Defining affinity with the GABAA receptor.
J Neurosci
18:8590-8604[Abstract/Free Full Text].
-
Jones MV,
Jonas P,
Sahara Y,
Westbrook GL
(2001)
Microscopic kinetics and energetics distinguish GABA(A) receptor agonists from antagonists.
Biophys J
81:2660-2670[Web of Science][Medline].
-
Karlin A,
Akabas MH
(1998)
Substituted-cysteine accessibility method.
Methods Enzymol
293:123-145[Web of Science][Medline].
-
Koltchine VV,
Finn SE,
Jenkins A,
Nikolaeva N,
Lin A,
Harrison NL
(1999)
Agonist gating and isoflurane potentiation in the human gamma- aminobutyric acid type A receptor determined by the volume of a second transmembrane domain residue.
Mol Pharmacol
56:1087-1093[Abstract/Free Full Text].
-
Krasowski MD,
Harrison NL
(1999)
General anaesthetic actions on ligand-gated ion channels.
Cell Mol Life Sci
55:1278-1303[Web of Science][Medline].
-
Krasowski MD,
O'Shea SM,
Rick CE,
Whiting PJ,
Hadingham KL,
Czajkowski C,
Harrison NL
(1997)
Alpha subunit isoform influences GABA(A) receptor modulation by propofol.
Neuropharmacology
36:941-949[Web of Science][Medline].
-
Krasowski MD,
Koltchine VV,
Rick CE,
Ye Q,
Finn SE,
Harrison NL
(1998)
Propofol and other intravenous anesthetics have sites of action on the gamma-aminobutyric acid type A receptor distinct from that for isoflurane.
Mol Pharmacol
53:530-538[Abstract/Free Full Text].
-
Krasowski MD,
Jenkins A,
Flood P,
Kung AY,
Hopfinger AJ,
Harrison NL
(2001)
General anesthetic potencies of a series of propofol analogs correlate with potency for potentiation of gamma-aminobutyric acid (GABA) current at the GABA(A) receptor but not with lipid solubility.
J Pharmacol Exp Ther
297:338-351[Abstract/Free Full Text].
-
Lam DW,
Reynolds JN
(1998)
Modulatory and direct effects of propofol on recombinant GABAA receptors expressed in Xenopus oocytes: influence of alpha and gamma2 subunits.
Brain Res
784:178-186.
-
Lavoie AM,
Twyman RE
(1996)
Direct evidence for diazepam modulation of GABAA receptor microscopic affinity.
Neuropharmacology
35:1383-1392[Web of Science][Medline].
-
MacDonald RL,
Twyman RE
(1992)
Kinetic properties and regulation of GABAA receptor channels.
Ion Channels
3:315-343[Medline].
-
Maconochie DJ,
Zempel JM,
Steinbach JH
(1994)
How quickly can GABAA receptors open?
Neuron
12:61-71[Web of Science][Medline].
-
Maeda A,
Tomson FL,
Gennis RB,
Kandori H,
Ebrey TG,
Balashov SP
(2000)
Relocation of internal bound water in bacteriorhodopsin during the photoreaction of M at low temperatures: an FTIR study.
Biochemistry
39:10154-10162[Medline].
-
Mihic SJ,
Ye Q,
Wick MJ,
Koltchine VV,
Krasowski MD,
Finn SE,
Mascia MP,
Valenzuela CF,
Hanson KK,
Greenblatt EP,
Harris RA,
Harrison NL
(1997)
Sites of alcohol and volatile anaesthetic action on GABA(A) and glycine receptors.
Nature
389:385-389[Medline].
-
Moody EJ,
Knauer C,
Granja R,
Strakhova M,
Skolnick P
(1997)
Distinct loci mediate the direct and indirect actions of the anesthetic etomidate at GABA(A) receptors.
J Neurochem
69:1310-1313[Web of Science][Medline].
-
O'Shea SM,
Wong LC,
Harrison NL
(2000)
Propofol increases agonist efficacy at the GABA(A) receptor.
Brain Res
852:344-348[Web of Science][Medline].
-
Rogers CJ,
Twyman RE,
Macdonald RL
(1994)
Benzodiazepine and beta-carboline regulation of single GABAA receptor channels of mouse spinal neurones in culture.
J Physiol (Lond)
475:69-82[Abstract/Free Full Text].
-
Sanna E,
Mascia MP,
Klein RL,
Whiting PJ,
Biggio G,
Harris RA
(1995)
Actions of the general anesthetic propofol on recombinant human GABAA receptors: influence of receptor subunits.
J Pharmacol Exp Ther
274:353-360[Abstract/Free Full Text].
-
Tanner JW,
Johansson JS,
Liebman PA,
Eckenhoff RG
(2001)
Predictability of weak binding from X-ray crystallography: inhaled anesthetics and myoglobin.
Biochemistry
40:5075-5080[Medline].
-
Uchida I,
Li L,
Yang J
(1997)
The role of the GABA(A) receptor alpha1 subunit N-terminal extracellular domain in propofol potentiation of chloride current.
Neuropharmacology
36:1611-1621[Web of Science][Medline].
-
Unwin N
(1995)
Acetylcholine receptor channel imaged in the open state.
Nature
373:37-43[Medline].
-
Weiss DS,
Magleby KL
(1989)
Gating scheme for single GABA-activated Cl- channels determined from stability plots, dwell-time distributions, and adjacent-interval durations.
J Neurosci
9:1314-1324[Abstract].
-
Whittington DA,
Rosenzweig AC,
Frederick CA,
Lippard SJ
(2001)
Xenon and halogenated alkanes track putative substrate binding cavities in the soluble methane monooxygenase hydroxylase.
Biochemistry
40:3476-3482[Medline].
-
Wick MJ,
Mihic SJ,
Ueno S,
Mascia MP,
Trudell JR,
Brozowski SJ,
Ye Q,
Harrison NL,
Harris RA
(1998)
Mutations of gamma-aminobutyric acid and glycine receptors change alcohol cutoff: evidence for an alcohol receptor?
Proc Natl Acad Sci USA
95:6504-6509[Abstract/Free Full Text].
-
Williams DB,
Akabas MH
(1999)
Gamma-aminobutyric acid increases the water accessibility of M3 membrane-spanning segment residues in gamma-aminobutyric acid type A receptors.
Biophys J
77:2563-2574[Web of Science][Medline].
-
Williams DB,
Akabas MH
(2000)
Benzodiazepines induce a conformational change in the region of the gamma-aminobutyric acid type A receptor alpha(1)-subunit M3 membrane-spanning segment.
Mol Pharmacol
58:1129-1136[Abstract/Free Full Text].
-
Williams DB,
Akabas MH
(2001)
Evidence for distinct conformations of the two alpha(1) subunits in diazepam-bound GABA(A) receptors.
Neuropharmacology
41:539-545[Web of Science][Medline].
-
Xu M,
Akabas MH
(1993)
Amino acids lining the channel of the gamma-aminobutyric acid type A receptor identified by cysteine substitution.
J Biol Chem
268:21505-21508[Abstract/Free Full Text].
-
Xu M,
Akabas MH
(1996)
Identification of channel-lining residues in the M2 membrane-spanning segment of the GABAA receptor alpha1 subunit.
J Gen Physiol
107:195-205[Abstract/Free Full Text].
-
Yamakura T,
Bertaccini E,
Trudell JR,
Harris RA
(2001)
Anesthetics and ion channels: molecular models and sites of action.
Annu Rev Pharmacol Toxicol
41:23-51[Web of Science][Medline].
-
Yu B,
Blaber M,
Gronenborn AM,
Clore GM,
Caspar DL
(1999)
Disordered water within a hydrophobic protein cavity visualized by x- ray crystallography.
Proc Natl Acad Sci USA
96:103-108[Abstract/Free Full Text].
Copyright © 2002 Society for Neuroscience 0270-6474/02/22177417-08$05.00/0
This article has been cited by other articles:
|
|
|
|
 
M. Ernst, S. Bruckner, S. Boresch, and W. Sieghart
Comparative Models of GABAA Receptor Extracellular and Transmembrane Domains: Important Insights in Pharmacology and Function
Mol. Pharmacol.,
November 1, 2005;
68(5):
1291 - 1300.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Jung, M. H. Akabas, and R. A. Harris
Functional and Structural Analysis of the GABAA Receptor {alpha}1 Subunit during Channel Gating and Alcohol Modulation
J. Biol. Chem.,
January 7, 2005;
280(1):
308 - 316.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. A. Gredell, P. A. Turnquist, M. B. MacIver, and R. A. Pearce
Determination of diffusion and partition coefficients of propofol in rat brain tissue: implications for studies of drug action in vitro
Br. J. Anaesth.,
December 1, 2004;
93(6):
810 - 817.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H.-J. Feng and R. L. Macdonald
Multiple Actions of Propofol on {alpha}{beta}{gamma} and {alpha}{beta}{delta} GABAA Receptors
Mol. Pharmacol.,
December 1, 2004;
66(6):
1517 - 1524.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. W. Lynch
Molecular Structure and Function of the Glycine Receptor Chloride Channel
Physiol Rev,
October 1, 2004;
84(4):
1051 - 1095.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. C. Bieda and M. B. MacIver
Major Role For Tonic GABAA Conductances in Anesthetic Suppression of Intrinsic Neuronal Excitability
J Neurophysiol,
September 1, 2004;
92(3):
1658 - 1667.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. A. Lobo, M. P. Mascia, J. R. Trudell, and R. A. Harris
Channel Gating of the Glycine Receptor Changes Accessibility to Residues Implicated in Receptor Potentiation by Alcohols and Anesthetics
J. Biol. Chem.,
August 6, 2004;
279(32):
33919 - 33927.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. D. W. Morris and J. Amin
Insight into the Mechanism of Action of Neuroactive Steroids
Mol. Pharmacol.,
July 1, 2004;
66(1):
56 - 69.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. J. Gallagher, L. Song, F. Arain, and R. L. Macdonald
The Juvenile Myoclonic Epilepsy GABAA Receptor {alpha}1 Subunit Mutation A322D Produces Asymmetrical, Subunit Position-Dependent Reduction of Heterozygous Receptor Currents and {alpha}1 Subunit Protein Expression
J. Neurosci.,
June 16, 2004;
24(24):
5570 - 5578.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Rusch, H. Zhong, and S. A. Forman
Gating Allosterism at a Single Class of Etomidate Sites on {alpha}1{beta}2{gamma}2L GABAA Receptors Accounts for Both Direct Activation and Agonist Modulation
J. Biol. Chem.,
May 14, 2004;
279(20):
20982 - 20992.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Bali and M. H. Akabas
Defining the Propofol Binding Site Location on the GABAA Receptor
Mol. Pharmacol.,
January 1, 2004;
65(1):
68 - 76.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. G. Newell and C. Czajkowski
The GABAA Receptor alpha 1 Subunit Pro174-Asp191 Segment Is Involved in GABA Binding and Channel Gating
J. Biol. Chem.,
April 4, 2003;
278(15):
13166 - 13172.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION
Compound via MeSH
