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The Journal of Neuroscience, December 1, 2001, 21(23):9083-9091
Agonist Trapping by GABAA Receptor Channels
Matt T.
Bianchi1 and
Robert L.
Macdonald2, 3, 4
1 Neuroscience Graduate Program, University of
Michigan, Ann Arbor, Michigan 48104-1687, and Departments of
2 Neurology, 3 Molecular Physiology and
Biophysics, and 4 Pharmacology, Vanderbilt University,
Nashville, Tennessee 37212
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ABSTRACT |
GABAergic IPSCs have a relatively slow decay (deactivation)
that appears to result from GABAA receptor channel openings
that occur well beyond the predicted duration of free GABA at central synapses. Open and desensitized states have been suggested to prevent
dissociation of agonist from the receptor, thus prolonging deactivation. However, simultaneous assessment of GABA binding and
channel gating has not been possible. We developed a functional assay
for occupancy of the GABA binding site or sites to test the GABA
"trapping" hypothesis. Deactivation currents were compared in the
absence and presence of bicuculline, a competitive antagonist that also
allosterically inhibits GABAA receptors. This provided a
model-independent, functional test of the hypothesis that GABA is
trapped on the receptor during gating: bicuculline could only inhibit
the channel if it was open but unbound by GABA. Although bicuculline
inhibited spontaneous and neurosteroid-activated GABAA receptor currents, it failed to alter the deactivation time course of
GABA-activated GABAA receptor currents. Protection of
deactivation current from bicuculline block indicated that GABA
remained bound to the receptors while the channel was open, thus
suggesting that all open states, as well as all closed and desensitized
states from which channel opening can occur, must be GABA liganded
states. Trapping may be specific to agonists, because the positive
allosteric modulator diazepam unbound from GABAA receptors
independent of GABA binding and channel activity.
Key words:
GABAA receptor; deactivation; inverse
agonist; GABA binding; concentration jump; diazepam
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INTRODUCTION |
At rest, ligand-gated ion channels
generally exist in stable closed conformations. Agonist binding
triggers conformational changes that culminate in transitions of the
channel among open, closed, and desensitized states. Much theoretical
and experimental work has focused on the relationship between agonist
binding and channel gating, yet their coupling remains poorly
understood. Early models of ligand-gated ion channels (see Scheme 1)
indicated that, in the simplest case, an agonist-binding step precedes
isomerization to the open state (Del Castillo and Katz, 1957 ). Implicit
in such a scheme is that while bound channels are visiting the open
state, agonist dissociation cannot occur, and thus, the agonist is
"trapped" on the receptor when the channel is open. In other words,
the equilibrium concept of "affinity" has no meaning (because it is infinite) for open channels. For more complex kinetic models, multiple
open, pre-open, and desensitized states may represent infinite affinity
states (Haas and Macdonald, 1999). Cyclic schemes that allow agonist
association and dissociation from all states imply a similar
phenomenon, represented as higher (although not infinite) affinity for
"active" states. In any case, agonist binding induces a
conformational change in the receptor complex that favors gating
transitions. These transitions are thought to reciprocally influence
the binding site.
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Although it has been difficult to explore experimentally the
interaction between binding and gating (Colquhoun, 1998), two studies
have addressed the concept of agonist trapping by
GABAA receptors. At central inhibitory synapses,
the postsynaptic response to vesicular release of GABA lasts longer
than predicted by the low affinity of the channels for GABA and the
relatively short burst duration of single-channel currents. Jones and
Westbrook (1995, 1996) proposed that desensitized state or states
transiently maintain synaptic GABAA receptors in
a bound state, thus prolonging the postsynaptic response to GABA
release. More recently, Chang and Weiss (1999a) combined binding and
electrophysiological analysis to provide evidence that channel opening
prevents dissociation of GABA from GABAC
receptors. However, any suggestion that agonist remains bound during
occupancy of specific states (e.g., open or desensitized) requires a
kinetic model, and therefore, interpretation is limited by the accuracy
of the model (Colquhoun, 1999).
This series of experiments was designed to investigate the question of
agonist trapping in a model-independent manner. A "double-jump" protocol allowed drug application specifically during channel deactivation. To probe the relationship between open and bound channels, we reasoned that application of an antagonist that bound to
the GABA binding site (a competitive antagonist) and also
allosterically inhibited open channels in the absence of GABA (via the
same site) would provide a functional assay for occupancy of the
GABAA receptor binding site or sites during
deactivation. Bicuculline is a competitive antagonist of
GABAA receptors (Macdonald and Olsen, 1994) that also allosterically inhibits channel activity in the absence of GABA (Barker et al., 1989; Ueno et al., 1997; Neelands et al., 1999).
In the presence of bicuculline during deactivation, any unliganded
receptors contributing to the deactivation current would be inhibited
by bicuculline, disproving the GABA-trapping hypothesis. If, however,
no acceleration of deactivation is produced by bicuculline, the
GABA-trapping hypothesis would be confirmed.
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MATERIALS AND METHODS |
Expression of recombinant GABAA
receptors. The cDNAs encoding rat  1,  3, and  2L,
GABAAR subunit subtypes were individually subcloned into the plasmid expression vector pCMVNeo. The L245S point
mutation in the 2L subunit was made using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and confirmed by sequencing. Oligonucleotide primers were synthesized by the University of Michigan DNA synthesis core facility (Ann Arbor, MI).
Human embryonic kidney cells (HEK293T; a gift from P. Connely, COR
Therapeutics, San Francisco, CA) were maintained in DMEM, supplemented with 10% fetal bovine serum, at 37°C in 5%
CO2 and 95% air. This cell line is a derivative
of HEK293 cells that constitutively express the SV-40 T antigen to
increase plasmid replication (DuBridge et al., 1987). Cells were
transfected with 4 µg of each subunit plasmid along with 1-2 µg of
pHOOK (Invitrogen, Carlsbad, CA) for immunomagnetic bead separation
(Greenfield et al., 1997), using a modified calcium phosphate
coprecipitation technique, as previously described (Angelotti et al.,
1993). The next day, cells were replated, and recordings were made
18-30 hr later.
Electrophysiology. Patch-clamp recordings were performed on
transfected fibroblasts bathed in an external solution consisting of
(in mM): NaCl 142; KCl 8;
MgCl2 6; CaCl2 1; HEPES 10;
and glucose 10, pH 7.4, 325 mOsm. Electrodes were formed from
thin-walled (whole-cell) or thick-walled (excised patch) borosilicate
glass (World Precision Instruments, Pittsburgh, PA) with a Flaming
Brown electrode puller (Sutter Instruments, San Rafael, CA),
fire-polished to resistances of 0.8-1.5 M (whole cell), or 4-12
M (excised patch) when filled with an internal solution consisting
of (in mM): KCl 153; MgCl2
1; MgATP 2; HEPES 10; and EGTA 5, pH 7.3, 300 mOsm. This combination of
internal and external solutions produced a chloride equilibrium
potential near 0 mV. Although large-conductance changes were observed
in some cells during this study, no evidence for chloride shifts was
detected in control experiments. I-V relations derived from
peak current and current after 10 sec of GABA (1 mM) application revealed similar reversal potentials (data not shown). Unless otherwise stated, cells and patches
were voltage-clamped at  10 to 60 mV using an Axon 200A amplifier
(Axon Instruments, Foster City, CA). No voltage-dependent effects were
observed between 10 and 60 mV. Unless otherwise stated, cells were
gently lifted from the recording dish soon after establishing the
whole-cell patch clamp configuration. Drugs were applied (via gravity)
to whole cells using a rapid perfusion system consisting of
three-barrel square glass connected to a Warner Perfusion Fast-Step
(Warner Instruments, Hamden, CT). The glass was pulled to a final
barrel size of ~250 µm. The solution exchange time was estimated
routinely by stepping a dilute external solution across the open
electrode tip to measure a liquid junction current. The 10-90% rise
times for solution exchange were consistently  1-2 msec. The exchange
around lifted cells is likely to be slower than the open tip
measurements. Although we did not quantify this, we inferred from the
current rise times that solution exchange occurred within 10 msec,
which is sufficiently fast for these experiments. For concentration
jumps with excised patches, a theta tube attachment was used, with
10-90% exchange times of 400 µsec.
Analysis of currents. Currents were low-pass filtered at
2-5 kHz, digitized at 10 kHz, and analyzed using the pClamp8 software suite (Axon Instruments). The deactivation time courses of
GABAA receptor currents were fit using the
Levenberg-Marquardt least squares method with one or two component
exponential functions of the form  an n, where
n is the best number of exponential components, a
is the relative amplitude of the component, and is the time
constant. A second component was accepted only if it significantly
improved the fit compared with a single exponential function, as
determined by an F test on the sum of squared residuals. Three component fits were not considered. The correlation coefficients of fitted curves were usually >90%. For comparison of deactivation time courses, a weighted summation of the fast and slow decay components (af *
f + as *
s) was used. For comparisons of the rate of
block onset, the fast exponential component was used (see Fig. 5 for
example). The bicuculline block of spontaneous currents could be
described with two exponential functions; however, because the relative
contribution was dominated by the fast component (>90%), we only used
its time constant for comparison. Note that because of limitations
associated with solution exchange around cells, the rate of block onset
represents an upper estimate of the macroscopic blocking rate (the
actual rate might be faster) (see Fig. 5C, for example).
Numerical data were expressed as mean ± SEM. Statistical
significance, using Student's t test (paired or unpaired as
appropriate) was taken as p < 0.05.
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RESULTS |
In a typical lifted cell transiently expressing 132L
GABAA receptors, current persists for hundreds of
milliseconds (slow deactivation) after removal of free GABA (Fig.
1A). In an outside-out patch containing a few GABAA receptor channels, a
brief pulse (5 msec) of 1 mM GABA elicited a
rapid inward current as channels synchronously opened. Repetitive
openings could be seen hundreds of milliseconds after the GABA pulse
was terminated (Fig. 1B).
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Figure 1.
GABAA receptor channel openings
outlast the duration of agonist exposure. A, Macroscopic
current response of a lifted HEK293T cell expressing 132L
GABAA receptors exposed to 1 mM GABA for 400 msec (solid bar). Despite precise control of the
solution bathing the cell by the concentration jump technique, the
current relaxation after agonist removal requires many hundreds of
milliseconds. The cell was voltage clamped at 15 mV.
B, Individual openings are observed for hundreds of
milliseconds after a 5 msec (arrow) pulse of 1 mM GABA delivered to an excised patch containing a few
132L GABAA receptors. The patch was voltage
clamped at 70 mV.
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The duration of deactivation currents is longer than predicted by the
low whole-cell GABA EC50 of these channels (~10
µM), and the short burst durations (~15 msec) and is
the basis for suggestions that conformations associated with channel
gating and desensitization may represent high-affinity states. Previous work has suggested that desensitized states (Jones and Westbrook, 1995;
Dominguez-Perrot et al., 1997; Haas and Macdonald, 1999) as well as
open states (Chang and Weiss, 1999a) may serve as transient high-affinity conformations, effectively slowing GABA unbinding and
thus prolonging deactivation. Thus, one hypothesis for this persistent
current is that GABA remains bound to or trapped by the open and
desensitized channels.
To test the hypothesis that GABA is trapped on the receptors during
deactivation, without making any assumptions about the number or
connectivity of kinetic states, we used bicuculline as a functional
assay for the occupancy of the GABA binding site or sites. We began by
characterizing bicuculline inhibition of 132L
GABAA receptor channels that are open in the
absence of GABA (Fig. 2). 132L
GABAA receptors opened spontaneously with low
probability. Bicuculline (100 µM) rapidly and reversibly
inhibited the spontaneous GABAA receptor current
in voltage-clamped lifted cells transfected with that isoform
(n = 4) (Fig. 2A).
132L(L254S) GABAA receptor channels had
frequent spontaneous openings and, consistent with a previous report
(Chang and Weiss, 1999b), these spontaneous openings were also rapidly
and reversibly inhibited by bicuculline (100 µM) in an excised patch (n = 4)
(Fig. 2B). (Although we consistently observed an
overshoot in the holding current after bicuculline washout for both
constructs, the basis for this phenomenon remains unclear.) The rate of
block onset reflects solution exchange time, the binding rate of
bicuculline, and the rate at which bound bicuculline inhibits the
channel. It has also been demonstrated that GABAA
receptor channels can be opened in the absence of GABA by neurosteroids
and that the neurosteroid-activated currents are bicuculline-sensitive
(Barker et al., 1989; Ueno et al., 1997). A lifted cell expressing
132L GABAA receptors was first jumped
from control solution into 10 µM alphaxalone,
then back into control (Fig. 2C). The rebound current after
termination of alphaxalone application likely indicated an additional
low-affinity site for open channel block. The same cell was jumped a
second time from control solution into alphaxalone, and then into 100 µM bicuculline alone, which strongly inhibited the deactivation current as indicated by the accelerated decay rate
(Table 1). Similar inhibition was
observed if 1 µM alphaxalone was used as the
agonist, a concentration that did not result in a rebound current (Fig.
2D). Thus, bicuculline clearly inhibited GABAA receptor channels in the absence of GABA.
We currently have no explanation for the slower rate of block onset
after activation by neurosteroid compared with block of spontaneous
currents, although this could indicate a state-specific interaction of
bicuculline with the channel.
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Figure 2.
Bicuculline inhibited GABAA
receptor currents in the absence of GABA. A,
132L channels open spontaneously with low probability. A 100 mM concentration of bicuculline (hatched
bar) rapidly and reversibly blocked the spontaneous activity in
a lifted cell expressing 132L GABAA receptors
voltage clamped at 30 mV. A transient "overshoot" current was
observed after bicuculline washout (dotted line in
A and B). B, 132L
GABAA receptors containing the L245S mutation in the 2L
subunit have a higher spontaneous opening probability. A 100 µM concentration of bicuculline (hatched
bar) rapidly and reversibly blocked the spontaneous activity in
an excised patch containing the mutant GABAA receptors
voltage clamped at 30 mV. C, GABAA
receptor currents activated by direct application of alphaxalone
(solid bar) were blocked by 100 µM
bicuculline (hatched bar). The cell was jumped first
from control solution into 10 µM alphaxalone and then
back to control solution (control wash), then the same
cell was jumped from control into 10 µM alphaxalone and
then into 100 µM bicuculline (bicuc
wash). The dotted line (in C and
D) shows the smaller holding current in the presence of
bicuculline. D, Inhibition of the deactivation current
was also observed when 1 µM alphaxalone was used, a
concentration that did not produce a rebound current.
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Next we used the double-jump protocol to activate the
GABAA receptors with GABA and then to apply
bicuculline only during current deactivation (in the absence of applied
GABA). We reasoned that if GABA remained bound to the channels that
were open during deactivation and if, once GABA dissociated from the
receptor, no rebinding of GABA occurred, then the current during
deactivation should not be inhibited by bicuculline. However, if GABA
could unbind from open channels or if unliganded receptors could reopen during the deactivation current, then bicuculline could bind and allosterically block opening. This would result in accelerated deactivation, because the bicuculline inhibition occurs faster (as
shown in Fig. 2, Table 1) than the deactivation rate after GABA
application. A 400 msec pulse of 1 mM GABA applied to a
lifted cell elicited a rapidly activating (<10 msec) current that
desensitized biphasically and deactivated slowly (deactivation ,
~300 msec) (Fig. 3A, Table
1). The deactivation time course after a 1 mM pulse of GABA was not significantly altered when the cell was washed
into 100 µM bicuculline (n = 7)
(Fig. 3A,E). Even when the activating concentration of GABA
was decreased to only 3 µM (near the GABA
EC50) (Fig. 3B), only a minor (9%;
p < 0.05) acceleration of the deactivation time
constant was observed when cells were washed into 100-200
µM bicuculline (n = 6) (Fig.
3B,E, Table 1). This minimal effect might be attributed to
block of spontaneous openings, which would be relatively more prevalent
at lower current amplitudes for a given amount of spontaneous activity.
In fact, a smaller holding current was observed during bicuculline wash (as well as picrotoxin wash), indicating block of spontaneous openings.
Also, when we used a very low concentration of GABA (100 nM), greater acceleration of deactivation was
observed during bicuculline wash (21.5%; n = 9; data
not shown). Alternatively, partially liganded channels might allow a
small degree of bicuculline inhibition. Monoliganded openings might
occur with higher probability at the lower GABA concentration, and if
bicuculline could act dominantly on the channel via the remaining site,
a small degree of inhibition would be observed. Although Ueno et al.
(1997) also suggested (based on the Hill coefficient of bicuculline
block) that binding of a single molecule of bicuculline was sufficient to inhibit GABAA receptor currents, further work
is required to test this possibility. Binding of bicuculline to the
receptor channel should have resulted in channel closure and
acceleration of deactivation after GABA application, because the onset
of bicuculline block in the absence of GABA was extremely fast (Fig.
2A,B, Table 1). The protection from bicuculline block
indicated that bicuculline could not bind to channels contributing to
the deactivation current, and thus we infer that such channels were
still GABA-bound.
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Figure 3.
Bicuculline failed to inhibit deactivation
currents after GABA application. A, Deactivation
currents after a concentration jump into 1 mM GABA
(solid bar) were identical whether the cell was washed
into control solution (open bar) or into 100 µM bicuculline (hatched bar).
B, A 100 µM concentration of bicuculline
(hatched bar) produced a small degree of inhibition in
the deactivation current after activation by a lower concentration of
GABA (3 µM; EC50 ~5 µM). The
dotted line shows the smaller holding current during
bicuculline wash. A portion of the trace (in the circle)
is expanded to show the small effect of bicuculline wash on the
deactivation current. C, The noncompetitive antagonist
picrotoxin (100 µM; hatched bar)
significantly inhibited deactivation currents after activation by 1 mM GABA. Note the smaller holding current during picrotoxin
wash. D, The benzodiazepine diazepam (1 µM; shaded bar) potentiated deactivation
currents after activation with 1 mM GABA. The dotted
line shows the larger holding current in the presence of
diazepam. E, Deactivation current pharmacology is
summarized as the percentage of change in the weighted time constant of
deactivation. The number of data points is indicated next to each
bar. Asterisks indicate significant
differences compared with control deactivation rate for each
condition.
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Trapped GABA should not, however, afford any protection from inhibition
by a noncompetitive antagonist applied during deactivation. Accordingly, when cells were jumped into 100 µM
picrotoxin, the deactivation current was substantially accelerated
(n = 5) (Fig. 3C,E, Table 1). Note the lower
holding current during the picrotoxin wash, indicating block of
baseline spontaneous openings.
If GABA was trapped on the channels during deactivation, drugs acting
at a separate modulator site such as the benzodiazepine site should
alter the deactivation time course. Benzodiazepines enhance subunit-containing GABAA receptor currents by
decreasing the EC50 for GABA, without affecting
efficacy (Rogers et al., 1994). Consistent with this proposed
mechanism, deactivation was slower when cells were washed into 1 µM diazepam after activation by GABA (Fig.
3D,E, Table 1). Similar enhancement was obtained whether 1 mM or 3 µM GABA was used
to activate the channels, and the effect was completely abolished by
coapplication of the benzodiazepine receptor antagonist flumazenil (10 µM) (n = 4; data not shown). This slowed deactivation was in fact caused by delayed unbinding of
GABA (and the resulting increase in "late" openings) because 100 µM bicuculline failed to block significantly
diazepam-enhanced deactivation currents (n = 3; data
not shown). Additionally, deactivation currents were accelerated during
wash into DMCM (600 nM) (n = 4) (Fig. 3E, Table 1), an inverse agonist at the
benzodiazepine site that reduces the affinity for GABA.
Under conditions where binding and rebinding of GABA could occur,
bicuculline was able to block GABAA receptor
currents (Fig. 4). During a prolonged (12 sec) application of GABA at a low concentration (3 µM,
near the EC50), bicuculline and GABA
coapplication blocked >90% of the current (n = 5)
(Fig. 4A). Inhibition onset was slow and similar to
the rate of deactivation during control wash, consistent with GABA
unbinding preceding bicuculline binding. A slow phase of
desensitization was observed during the 3 µM
GABA applications. The GABA current after removal of bicuculline was
larger than predicted by a control pulse in the same cell (Fig.
4A, gray trace), consistent with bicuculline
occupying the GABA binding site and preventing GABA-induced
desensitization during that time. Similar current profiles were
obtained when two GABA pulses were separated by control wash instead of
bicuculline coapplication (data not shown).
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Figure 4.
Bicuculline inhibited GABAA receptor
currents under conditions in which unbinding and rebinding of GABA
occurs. A, A 100 µM concentration of
bicuculline (hatched bar) blocked the current during a
long application of 3 µM GABA (solid bar)
to a lifted cell expressing 132L GABAA receptors.
The response to 3 µM GABA alone from the same cell is
superimposed in gray. Note the larger current after
removal of bicuculline. B, Current responses to 400 msec
jumps into 1 mM GABA in the same cell before and after
lifting the cell from the recording dish. Deactivation currents were
inhibited during bicuculline wash (hatched bar) before
the cell was lifted, and this deactivation current overlapped with that
observed in the same cell after it was lifted. Currents were normalized
to the amplitude at the offset of the GABA pulse. The larger vertical
scale value applies to the intact cell current response.
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A more striking example of GABA binding and rebinding came from a
comparison of double jump experiments in the same cells before and
after lifting them from the recording dish (Fig. 4B). Under conditions of imprecise solution exchange, such as might occur
around a cell adhering to the recording dish, some rebinding of GABA
might occur during the washout period. When concentration jumps using 1 mM GABA were applied to cells adherent to the
culture dish, current rise times were slower (>10 msec) than those
observed in lifted cells and patches, and the fast component of
desensitization ( < 15 msec) was difficult to resolve,
consistent with relatively poor rates of solution exchange. Bicuculline
partially inhibited the deactivation current (Fig.
4B) in double-jump experiments performed on the
intact cells. The half-time of decay was decreased by 24.6 ± 6.6% (n = 5; p < 0.01). However, when
the same cell was lifted off of the culture dish, thus ensuring more
precise solution exchanges (accompanied by faster current rise times
and increased resolution of fast desensitization), the bicuculline wash
no longer affected the current (n = 4). Moreover, the
control (no bicuculline) deactivation current in the lifted cells
overlapped the time course of deactivation current when the cell was
jumped into bicuculline before lifting.
Because 132L(L245S) mutant GABAA
receptors had increased spontaneous openings, they allowed an
additional test of GABA trapping (Fig.
5). A 400 msec pulse of 1 mM
GABA presumably saturated all of the GABA binding sites. At the instant
cells were subsequently jumped into bicuculline, there were no
unliganded channels available for bicuculline binding, and thus there
was no reduction in current. Because only the spontaneous openings
would be blocked, GABA unbinding would slow the onset of inhibition by
bicuculline (which was very rapid in the absence of GABA) (Fig. 2,
Table 1). Indeed, the extent of block increased with time (Fig.
5A), suggesting that fully liganded receptors were
protected, but after eventual unbinding of GABA, they resumed their
bicuculline-sensitive spontaneous openings. To illustrate this slow
onset of bicuculline block, the control traces were subtracted from the
bicuculline wash traces. Direct application of bicuculline blocked
spontaneous channel activity very rapidly (Fig. 5B, left,
C), although the initial rate of block onset ( fast) may have
been limited by solution exchange time around lifted cells (therefore
it represents an upper limit estimate for the rate of block onset). In
contrast, the block proceeded at a much slower rate during the washout
period (Fig. 5B, middle, subtracted trace, C). The traces
are overlaid to demonstrate the striking difference in onset of block
(Fig. 5B, right, C). The subtracted rate was similar to the
deactivation rate during control wash; further evidence that
deactivation of GABA-bound receptors was unaltered by the bicuculline
wash.
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Figure 5.
Onset of bicuculline block is limited by
GABA unbinding. A, Deactivation currents of
132L (L245S) GABAA receptors after a concentration
jump into 1 mM GABA (solid bar) were
partially blocked by bicuculline (hatched bar). Cells
were voltage clamped at 0 to 5 mV. The onset of block was
time-dependent (the separation of deactivation currents during control
wash and bicuculline wash increased with time). B, To
illustrate the time course of bicuculline inhibition, the traces were
subtracted (middle) for comparison with the direct
effect of bicuculline (left) in the same cell. The
traces are normalized and overlaid to demonstrate the slow onset rate
when bicuculline was applied during deactivation
(right). C, Summary chart showing
the onset rate of bicuculline inhibition for various conditions.
Although the time course of block was often best fit by a two
exponential function, only the faster time constant is shown, which
accounted for >90% of the decay. The number of cells is shown for
each condition. Note the logarithmic ordinate.
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These results demonstrated that GABA was trapped by open states and
thus raised a related question. Are allosteric modulators of the
GABAA receptor such as diazepam also trapped? To
investigate potential "activity dependence" or "trapping" of
diazepam binding, we first compared deactivation currents obtained with
the following double-jump protocols: (1) 3 µM GABA,
control wash; (2) 3 µM GABA, 1 µM diazepam
wash; (3) 3 µM GABA + 1 µM diazepam
coapplication, control wash; (4) 3 µM GABA + 1 µM diazepam coapplication, 1 µM diazepam
wash (Fig. 6). When GABA and diazepam
were coapplied, deactivation currents were slower than control
deactivation currents (Fig. 6) (3 µM GABA, control wash).
The relatively slow onset of enhancement in the diazepam wash (also see
Fig. 3D) was probably because diazepam had a slow
macroscopic on-rate (see below). When coapplication of 3 µM GABA and 1 µM
diazepam was followed by a 1 µM diazepam wash,
no additional effect on deactivation was observed. This suggested that
diazepam remained bound at least as long as GABA remained bound to the
receptor, that is, at least the duration of the deactivation current.
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Figure 6.
Diazepam unbinding is slower than GABA unbinding.
Diazepam unbinds at least as slowly as GABA from 132L
GABAA receptors. Deactivation currents are shown for
applications of 3 µM GABA (solid bar) with
control wash (open bar), 3 µM GABA with 1 µM diazepam wash (shaded bar), 3 µM GABA + 1 µM diazepam coapplication with
control wash, and 3 µM GABA + 1 µM diazepam
coapplication with 1 µM diazepam wash. Currents were
normalized to the amplitude at the offset of the GABA pulse.
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These results prompted us to determine specifically whether either GABA
binding or channel gating could influence the macroscopic unbinding of
diazepam. Therefore, we compared deactivation of diazepam-modulated
currents in the presence and absence of GABA, and in
GABAA receptors with short- and long-duration
mean open times. As stated earlier, 132L channels exhibited
low-probability spontaneous openings that were blocked by selective
GABAA receptor antagonists bicuculline and
picrotoxin (Figs. 2A, 3D). Direct application of 1 µM diazepam increased the
bicuculline-sensitive spontaneous channel activity, reaching peak
enhancement within 1 sec (10-90% rise time; ~700 msec)
(n = 7) (Fig.
7A1) (bicuculline sensitivity
not shown). The amplitude of this enhancement was generally <2% of
the maximal GABA-evoked response. After diazepam washout, this increase
in holding current relaxed with a time constant of ~4 sec (Fig.
7C). This deactivation reflected unbinding of diazepam,
because test pulses of GABA delivered at intervals after the removal of
free diazepam were enhanced within a similar time window (time constant
of 2.1 sec) (Fig. 7D). When a 1 sec pulse of 1 µM diazepam was coapplied during the current
evoked by 300 nM GABA (also with a 10-90% rise
time, ~700 msec), the subsequent current relaxation had a time
constant of ~4 sec (n = 4) (Fig. 7A2,C).
Similar results were obtained when a higher GABA concentration (2 µM) was used (data not shown). Next we tested the interaction of diazepam with 132L (L245S)
GABAA receptors, because they exhibited longer
spontaneous (and GABA-activated) mean open times (our unpublished
data). These channels deactivated significantly slower than wild-type
132L channels when brief (<5 msec) pulses of 1 mM GABA were applied to excised patches (Fig.
7B1), consistent with the significantly increased open times of the mutated channels compared with wild type. Direct application of
diazepam increased the spontaneous activity of 132L (L245S) GABAA receptor channels. This effect was
consistent with a previous report involving spontaneous gating caused
by an L to S mutation in the 3 subunit (Thompson et al., 1999),
although the basis for benzodiazepine modulation of spontaneous
GABAA receptor currents remains unclear. However,
the deactivation after a 1 sec pulse of 1 µM
diazepam was still ~4 sec (n = 4) (Fig.
7B2,C), similar to that observed in wild-type 132L
GABAA receptors in the absence or presence of
GABA. Thus, diazepam unbinding, as indicated by deactivation rate, was
not sensitive to differences in channel gating that strongly affect
GABA unbinding.
View larger version (24K):
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|
Figure 7.
Diazepam unbinding is independent of channel
gating. A1, In the absence of GABA, diazepam (1 µM) enhanced spontaneous activity in lifted cells
expressing 132L GABAA receptors.
A2, Diazepam enhanced the response to a steady-state
application of 300 nM GABA (different cell than in
B1). B1, Deactivation of 132L
and 132L(L245S) GABAA receptors after a 5 msec
pulse of 1 mM GABA (arrow). Longer mean open
times caused by this mutation slowed deactivation. B2,
In the absence of GABA, 1 µM diazepam enhanced
spontaneous activity in 132L(L245S) GABAA
receptors. C, Summary chart showing the rate of
deactivation after a 1 sec application of 1 µM diazepam
was indistinguishable under different GABA-binding and intrinsic gating
conditions. The number of cells is indicated in
parentheses. D, A 1 sec application of 1 µM diazepam applied in the absence of GABA enhances
subsequent responses to 1 µM GABA in lifted cells
expressing 132L GABAA receptors. Cells were washed
with control solution for the interpulse interval.
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|
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DISCUSSION |
This study strongly suggests that GABA is trapped on
GABAA receptor channels by open states and closed
or desensitized (preopen) states from which reopening can occur. Gating
transitions are thought to delay unbinding of GABA independent of the
microscopic affinity for GABA, resulting in a prolonged IPSC current
caused by reopening before dissociation eventually occurs. Several
studies have inferred a role for gating and desensitization in synaptic responses (Jones and Westbrook, 1995; Dominguez-Perrot et al., 1997;
Haas and Macdonald, 1999; Chang and Weiss, 1999a), and we have
identified a critical portion of TM1 involved in the functional coupling between desensitization and slow deactivation (Bianchi et al.,
2001). However, it has been difficult to simultaneously assess the GABA
binding site and channel gating. This study provides evidence for a
constraint on the relationship between agonist binding and channel
gating: GABA unbinding cannot occur from open and preopen states. Two
common ways to study ion channel function are through binding assays
and electrophysiological recording. Specific information about binding
has been inferred through interpretation of currents in the context of
a kinetic model. In many cases such models are empirical descriptions
of macroscopic data and are therefore of unknown relevance to the
behavior of individual channels. When binding is measured, nothing is
known about the function of detected receptors. This can be problematic
if conformational changes induced by binding affect dissociation
[resulting in errors of affinity estimates (Colquhoun, 1998)], or if
an unknown subset of receptors with intact binding sites are not
functional (Chang and Weiss, 1999a; Colquhoun, 1999). Furthermore, the
temporal resolution of conventional binding assays is orders of
magnitude slower than the time scale of channel transitions, limiting
the relevance of such measurements to steady-state behavior.
We developed a functional assay for occupancy of GABA binding sites.
Selective application of bicuculline during GABAA
receptor deactivation was used to probe GABA binding sites during
channel gating without making any assumptions about the nature of the gating process. Bicuculline blocked spontaneous channel activity in the
absence of GABA as well as openings triggered by neurosteroid binding
to a site distinct from that of GABA. However, after channel activation
by GABA, bicuculline had virtually no effect on
GABAA receptor deactivation currents. Bound GABA
effectively "protected" the channels from bicuculline inhibition
because bicuculline could not access its binding site. Although a
relatively high concentration of bicuculline was used [more than two
orders of magnitude higher than the IC50 found by
Ueno et al. (1997) in the presence of 3 µM GABA], we
cannot rule out the possibility that higher concentrations of
bicuculline would affect deactivation. We believe this is unlikely, because only minimal effects on deactivation were observed despite varying the GABA concentration by >300-fold. Interestingly, previous studies could not distinguish between a shared (or overlapping) binding
site for GABA and bicuculline, versus a separate binding site for
bicuculline that rendered the GABA binding site lower affinity. Our
data argues against the idea of a second site, because such a site
would be available for bicuculline to bind during deactivation and
would accelerate deactivation by favoring unbinding. However, we cannot
completely rule out the possibility of a very strong negative
cooperativity between structurally distinct GABA and bicuculline
binding sites.
The increased spontaneous activity of 132L (L245S) mutated
receptors offered an additional tool to test the idea that bound GABA
protected channels from bicuculline inhibition. In the absence of GABA,
the onset of bicuculline block was extremely fast (<10 msec). However,
when 132L (L245S) receptors were washed into bicuculline after
a pulse of saturating GABA, the onset of block was substantially
delayed (>500 msec; n = 5) (Fig. 5). This was expected
if the spontaneous openings of unliganded channels were the only ones
available for bicuculline block during the washout period. Thus, the
onset of bicuculline inhibition was limited by GABA unbinding, further
evidence that GABA remains trapped on the receptors.
In situations where GABA binding and unbinding were occurring,
bicuculline inhibition of GABAA receptor currents
was observed. In one case, channels activated by 3 µM
GABA (below the GABA EC50) were clearly sensitive
to bicuculline coapplication. During the coapplication of bicuculline,
the blocked channels no longer entered desensitized states, consistent
with the mutual exclusivity of GABA and bicuculline binding. This was
apparent in the strong rebound current after removal of bicuculline
(Fig. 4A). In the second case, we compared
deactivation and its sensitivity to bicuculline between single cells in
the intact and lifted configurations. Bicuculline washes partially
blocked the deactivation currents of cells adherent to the culture
dish. Lifting the same cells substantially improved the solution
exchange, as evidenced by faster rise times and increased resolution of
fast desensitization. Moreover, deactivation currents were no longer
sensitive to bicuculline. Therefore, when the probability of rebinding
of GABA was reduced, either by jumping an intact cell into excess
bicuculline, or by lifting the cell to achieve better solution
exchange, the deactivation time courses were the same.
It has been proposed that GABA binding induces some structural
rearrangement around the binding site (Wagner and Czajkowski, 2001). Whether such a rearrangement is a local consequence of GABA
binding or a result of channel gating is unknown. If channel gating
prevents dissociation of bound GABA via a "Venus fly trap or clam
shell" mechanism, then is it also true that channel gating in the
absence of GABA limits access to the GABA binding site? If so, the rate
of bicuculline block of open channels should be limited by channel
closure. However, spontaneous openings in lifted cells expressing
132L and 132L(L245S) receptors were blocked with a
fast time constant. Because the spontaneous open duration of wild type
and the mutated channels differed by at least fivefold (~300 µsec
and ~2 msec, respectively; our unpublished data), it was possible
that the bicuculline block was not limited by channel closure. However,
the solution exchange time around lifted cells might be too slow to
distinguish subtle differences in block rate. Also, the block of
neurosteroid-activated currents was slower than the block of
spontaneous whole-cell currents. We do not understand this difference,
but it may suggest that only certain states are available for block by
bicuculline in the absence of GABA. Block of spontaneous
132L(L245S) receptor currents in excised patches occurred with
a very fast time constant (~2 msec), but no comparison could be made
with block of spontaneous wild-type receptors in patches because the
spontaneous currents were undetectable or too small to measure
accurately. This time constant was in the range we observed for
spontaneous open durations, raising the possibility that channel
closure is required for block. Similar arguments could be made for the
interaction of GABA with spontaneously gating channels. We found that
current rise time was fast (~600 µsec; our unpublished data) when
patches containing 132L(L245S) GABAA
receptors were jumped into 1 mM GABA, similar to the rise times observed in wild-type receptors (Haas and Macdonald, 1999). Furthermore, all of the receptors became liganded during the GABA application (Fig. 5). These two observations suggested that channels could bind GABA even when they were open. However, these arguments assume that spontaneous and GABA-gated channel openings are associated with similar receptor conformations, which is not known except to the
extent that the single-channel conductance is the same (data not
shown). If the GABA binding site was accessible whether or not
spontaneous openings were occurring, then bicuculline might be able to
actively close open channels as well as prevent the reopening of closed
channels. Single-channel recordings of spontaneous activity can
distinguish among these possibilities, because the active closure of
open channels by bicuculline predicts shorter mean open durations.
The selective delivery of modulators during the deactivation current
presents a pharmacological tool that would be useful in several
contexts, such as for screening potential competitive antagonists,
where there is controversy between competitive and noncompetitive
actions, or where there is a proposed "mixed" mechanism. Moreover,
deactivation current modulation eliminates the potentially confounding
factor of agonist rebinding inherent in steady-state experiments. For
positive modulators such as benzodiazepines that have proposed
mechanisms associated only with GABA affinity, effects on
koff can be specifically investigated.
Although it is thought that diazepam does not affect
GABAA receptor gating (as indicated by
single-channel analysis), there has been some debate on whether diazepam increased GABA sensitivity through an effect on
kon or koff (Rogers et al., 1994; Lavoie and
Twyman, 1996). Our results indicated an effect on
koff, although we cannot exclude an
additional increase in kon. Note that
diazepam increased the spontaneous activity of 132L
GABAA receptors. Although this may indicate an
additional mechanism of action, the relatively small currents elicited
by direct application of diazepam cannot quantitatively account for the
enhancement of deactivation observed in the diazepam wash experiment
(Fig. 3D). Similar reasoning suggested that the benzodiazepine inverse agonist DMCM decreases GABA sensitivity at least
through an effect on koff. Although
the structural correlate of changing
koff is not known, it is possible that
the changes were not limited to the GABA binding site. For example,
diazepam altered the accessibility of engineered cysteine residues in
the third transmembrane domain that were distant from the GABA binding sites (Williams and Akabas, 2000). Despite the possibility that diazepam binding induces a distinct conformation of the receptor, its
unbinding (as indicated by deactivation rate) was unaffected by either
GABA binding or by channel opening. Interestingly, many of the residues
important for benzodiazepine binding (at the / interface) are
homologous to those important for GABA binding (at the /
interface) (Sigel and Buhr, 1997), and subunit interface structures
have been suggested to represent a general ligand-binding motif.
However, our data suggests a fundamental difference in the coupling of
gating-related conformations (such as the open state) and the
recognition sites for GABA and diazepam. Although the mechanism by
which channel gating detains GABA at its binding site remains poorly
understood, it appears that the process does not effectively generalize
to the benzodiazepine binding site to detain diazepam.
It remains to be seen how the binding of other modulators such as
barbiturates or neurosteroids may be affected by gating. It is also
unknown whether agonist trapping generalizes to other types of
ligand-gated channels. Certain competitive antagonists block
spontaneous channel activity in mutant neuronal acetylcholine (ACh)-gated receptors (Bertrand et al., 1997), and thus the
double jump paradigm could be used on ACh receptor channels. More
generally, it would be relevant to test classical competitive
antagonists for different ligand-gated channels to determine if
bicuculline-like allosteric inhibition (inverse agonism) is observed.
Not only will this allow phenomena such as agonist trapping to be
investigated for other channel families, but it will also compel a
re-evaluation of competitive antagonists in general as simply
"sitting" in the agonist binding site without any additional effect
on the receptor. This information is of practical interest because
competitive antagonists are often used in ligand-binding studies to
assay binding site availability and affinity without the bias of
efficacy intrinsic to the agonists themselves.
| |
FOOTNOTES |
Received June 11, 2001; revised Aug. 14, 2001; accepted Sept. 11, 2001.
This work was supported National Institutes of Health Grant R01-NS33300
(R.L.M.) and National Institute on Drug Abuse Training Fellowship
T32-DA07281-03 (M.T.B.).
Correspondence should be addressed to Dr. Robert L. Macdonald,
Department of Neurology, Vanderbilt University, 2100 Pierce Avenue,
Nashville, TN 37212. E-mail: Robert.Macdonald{at}mcmail.vanderbilt.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
