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J. Biol. Chem., Vol. 277, Issue 24, 21423-21430, June 14, 2002
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From the
Received for publication, October 26, 2001, and in revised form, March 21, 2002
We reported previously that tyrosine
62 of the Desensitization, an intrinsic biophysical characteristic of
ligand-gated ion channels, facilitates neurobiological adaptation to
prolonged or repeated exposure to agonist. The molecular mechanisms by
which this occurs have been subject to intense investigation but remain
poorly understood (1). However, the general consensus is that
agonist-induced conformational changes of the receptor protein induce
the initial, if not all, phases of the process (2, 3).
The Radioligand binding studies have revealed the existence of (at least)
two classes of agonist recognition sites on a single GABAAR
(10), which differ in affinity by more than 1 order of magnitude (see
also Refs. 11-13). By using the recombinant rat One approach to delineate the role of individual sites in receptor
function is to use site-directed mutagenesis to disrupt selectively
binding domains and to examine the consequences on receptor function.
By using this approach, we have recently identified an amino acid
residue (Tyr-62) of the In this study, we have expressed recombinant wild-type and mutant
GABAA receptors in Xenopus oocytes and have
developed methods to study aspects of the desensitization process.
Receptors carrying the Tyr-62 mutations retained the ability to be
desensitized upon prolonged exposure to agonists but with slightly
altered concentration dependence compared with the wild type. At low
GABA concentrations (1-10 µM), the Y62S mutant receptor
displayed the unusual property of first becoming desensitized but then
recovering from desensitization despite the continued presence of
agonist. Thus, under these conditions, this receptor (which lacks
measurable high affinity agonist-binding sites) seems to be unable to
maintain the desensitized state. Therefore, we propose a new model in
which the role of the high affinity sites is not to induce
desensitization but rather to stabilize the desensitized state once it
has been formed.
Site-directed Mutagenesis--
GABAAR cRNA Transcript and Oocyte Preparation--
Xenopus
oocytes were prepared as described (16). Capped cRNA transcripts for
GABAAR subunits were prepared from cDNA constructs that
were generous gifts from Dr. David Weiss. Oocytes were injected with
Two-electrode Voltage Clamp--
Electrophysiological
experiments were performed 2-7 days following oocyte injection.
Standard two-electrode voltage clamp techniques were carried out using
a GeneClamp 500B Amplifier (Axon Instruments Inc.) at a holding
potential of Concentration Dependence of Desensitization--
The current
amplitude elicited by a 30-s application of a saturating concentration
of GABA (1 mM) was first stabilized prior to experiments
such that a maximum variation of 5% was observed over three successive
applications. In desensitization experiments, changes in the current
amplitudes elicited by challenge applications of GABA (1 mM) or muscimol (2 mM), i.e.
concentrations that elicited a maximal response in all recombinant
receptors, were measured. Bath pre-perfusion with lower concentrations
of agonist was started after recovery of receptors from the
desensitization induced by the challenge application itself. The
relative current amplitude used in determination of IC50
values for desensitization was defined by the steady state current (I)
that was achieved in the presence of the indicated concentration of
agonist in the pre-perfusate (see example in Fig. 1). Data were
analyzed by iterative curve fitting using Equation 1 (GraphPad Prism
Software) as follows:
Characterization of Desensitization--
The assay protocols
were optimized to ensure that any observed desensitization was due only
to the presence of agonist in the bathing medium and not to other
variables. In experiments to measure the effects of frequency of
challenge and duration of pre-perfusion on current amplitude,
desensitizing GABA concentrations equivalent to their IC50
values (determined as above) were used in the pre-perfusion. Where
noted in the text, pre-perfusion was started either after washout of
the challenge concentration or immediately during the recovery phase
from the agonist challenge. Both pre-perfusion protocols gave the same
maximum depression of current amplitude under steady state conditions.
Rate of Recovery from GABA-induced Desensitization--
The rate
of recovery from desensitization was measured by first applying a
desensitizing concentration of GABA (its Emax or EC50 concentration as noted) and then monitoring the
magnitude of the peak current elicited by the same concentration of
GABA administered at different time intervals (1-30 min) after the initial challenge. The recovery of the current amplitude as a function
of time was fit by a single exponential model (GraphPad Prism, San
Diego, CA; www.graphpad.com) to give estimates of the rate and extent
of recovery. Similar experiments were carried out in the presence of
bath-perfused GABA (3 µM) to assess the effects of
pre-perfusion on the recovery from desensitization. Data were fitted to
Equation 2,
Statistical Analyses--
Data were analyzed by a one-way
analysis of variance followed by either a post hoc Dunnett's test or
Newman-Keuls to determine levels of significance. The data for
GABA-mediated current shown in Figs. 2 and 3 were analyzed by a one-way
repeated measures analysis of variance to compare the current before
and after bath pre-perfusion of the indicated concentration of
GABA.
Agonist-induced Desensitization
Fig. 1A shows
representative two-electrode voltage clamp recordings from recombinant
wild-type Fig. 1B shows the effects of bath perfusion of a lower
concentration of GABA (1 µM) which by itself induces only
a minimal chloride conductance (<2% of maximum). In contrast to the
results in Fig. 1A, there was no diminution of the response
to the first GABA challenge; rather a small, but reproducible, increase
in the current amplitude was observed. However, upon repeated
challenges, the current again declined to a steady state level, and
this was fully reversible upon removal of GABA from the perfusate.
These data suggest that channel activation may be required before the receptor can desensitize and, furthermore, that the extent of desensitization is dependent on the number of activations (see also
Ref. 17). Comparison of the data in Fig. 1, A and
B, demonstrates that the magnitude of steady state
desensitization is also dependent upon the concentration of GABA in the
perfusion medium (see below and Fig. 4).
Optimization of the Experimental Conditions to Investigate
Desensitization
The experimental protocol used here to study GABAA
receptor desensitization was adapted from that of Bartrup and
Newberry (17) who used whole cell patch clamp techniques to
study agonist-induced desensitization of the serotonin type 3 receptor
in NG108-15 cells. Because GABAA receptor desensitization
has not been quantified previously using the Xenopus oocyte
system, we have optimized the experimental procedures to ensure that
any desensitization observed was due to the presence of agonist in the
perfusate and was not affected by other experimental variables.
Frequency of Challenge Application--
The data in Fig.
2A illustrate the time
interval between challenges required to avoid
frequency-dependent effects in the wild-type receptor. As
in Fig. 1, currents to a maximally effective GABA concentration (1 mM) were recorded at regular time intervals, but in these
experiments, this interval was varied between 3 and 12 min as
indicated. After the third control challenge, the oocyte was perfused
with 3 µM GABA, a concentration that approximates its
IC50 for desensitization (see below). Responses to
subsequent regular challenges continued to be recorded prior to washout
of GABA from the perfusate. In these experiments, perfusion with the
lower concentration of GABA was started during the recovery phase from
the challenge application, i.e. immediately after receptor activation. Under these conditions, there was an immediate reduction in
the current amplitude to the next challenge (cf. Fig.
1B), again demonstrating the activation dependence of this
phenomenon (see below and also Ref. 17). As noted under
"Experimental Procedures," both preperfusion protocols
gave the same steady state levels of desensitization.
By using a 9- or 12-min interval between challenges, the control
responses were reproducible in amplitude, and there was no difference
in the magnitude of the inhibition observed during agonist perfusion or
in the recovery upon washout (Fig. 2A). A 3-min interval is
clearly not suitable for measuring the concentration dependence of
desensitization, because the receptor is unable to recover from the
desensitization induced by the challenge itself. This is shown by the
decrease in the amplitudes of successive control currents prior to
inclusion of the lower concentration of GABA in the bath. By using a
6-min interval, there were apparent frequency-dependent
effects on washout, i.e. the current did not immediately
return to control levels. Similar control experiments have been carried
out using muscimol (data not shown). In all cases, a 12-min interval
between challenges gave rise to reproducible effects in which the
apparent desensitization depends only on the presence of agonist in the
perfusion medium and on the number (see e.g. Fig.
1B) but not on the frequency of channel activations.
Pre-perfusion Time--
The effects of the time of pre-perfusion
were also investigated. Fig. 2B shows representative
experiments using wild-type receptors. In these experiments, responses
to 1 mM GABA were first stabilized; the oocytes were washed
and then perfused with 3 µM GABA for different times
prior to challenge. Increasing the pre-perfusion time from 1 to 12 min
showed that the extent of current depression was dependent upon
perfusion time, but there were no significant differences between 9 and
12 min, suggesting that desensitization reaches a steady state within 9 min. This also illustrates that unlike rapid application techniques
(18), gravity perfusion requires longer periods of equilibration to
achieve a stabilized desensitized state, which is in good agreement
with theoretical considerations of Edelstein et al. (19).
The data in Fig. 2 illustrate that a 12-min interval between challenges
gives rise to reproducible measurements of desensitization induced by
agonist in the pre-perfusate.
Desensitization of GABAA Receptors Carrying Mutations
of Tyr-62 of the Both mutant receptors desensitized upon bath perfusion with GABA
as shown in Fig. 3. The data shown in
this figure were obtained using perfusate GABA concentrations that
approximated the IC50 value for desensitization for each
receptor (see below). In receptors carrying the
Functional Consequences of the Loss of High Affinity Agonist
Binding to
-Aminobutyric Acid Type A Receptors
IMPLICATIONS FOR RECEPTOR DESENSITIZATION*
§ and
¶
Department of Pharmacology and ¶ Centre
for Neuroscience, University of Alberta, Edmonton,
Alberta T6G 2H7, Canada
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ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 subunit of the
-aminobutyric acid, type A
(GABAA) receptor is an important determinant of high
affinity agonist binding and that recombinant
1
2
2L
receptors carrying the Y62S mutation lack measurable high affinity
sites for [3H]muscimol. We have now examined the effects
of disrupting these sites on the macroscopic desensitization properties
of receptors expressed in Xenopus oocytes. Desensitization
was measured by the ability of low concentrations of bath-perfused
agonist to reduce the current responses elicited by subsequent
challenges with saturating concentrations of GABA. Wild-type receptors
were desensitized by pre-perfused muscimol with an IC50
~0.7 µM, which correlates well with the lower affinity
sites for this agonist that are measured in direct binding studies.
Receptors carrying the
2 Y62S and Y62F mutations desensitized at
slightly higher (2-7-fold) agonist concentrations. However, at low
perfusate concentrations, the Y62S-containing receptor recovered from
the desensitized state even in the continued presence of agonist. The
characteristics of desensitization in the wild-type and mutant
receptors lead us to suggest that the major role of the high affinity
agonist-binding site(s) of the GABAA receptor is not to
induce desensitization but rather to stabilize the desensitized state
once it has been formed.
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INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-aminobutyric acid type A receptors
(GABAARs)1 are
members of a receptor gene family (see Ref. 4) that includes the nicotinic acetylcholine receptors (nAChRs), glycine receptors, and the
serotonin type 3 receptor. These receptors are structurally and
functionally homologous and desensitize in the continued presence of
agonist (5, 6). GABAARs are large pentameric protein complexes composed of subunit isoforms from a number of classes (
1-6,
1-3,
1-3,
1-3,
,
, and
). Although the
precise subunit compositions of native receptors remain to be
established, a major subtype in the brain appears to be a combination
of
1,
2, and
2 subunits in a likely stoichiometry of 2:2:1
(7-9).
1
2
2 receptor
expressed in tsA201 cells, we found two classes of binding sites for
[3H]muscimol with Kd values of 8.1 and
430 nM (10). Because higher concentrations of muscimol are
required to activate this receptor subtype (EC50 of 7.1 µM when expressed in Xenopus oocytes), sites
of still lower affinity may be present (see below). Although the roles
of these multiple sites in receptor function require clarification, it
is generally accepted that the high affinity binding that is measured
under equilibrium conditions reflects binding to a desensitized
conformation(s) of the receptor (see Ref. 14). Furthermore, it is
assumed that agonist occupancy of these sites in the resting state of
the protein induces the conformational changes that lead to this
equilibrium desensitized state.
2 subunit of the GABAAR that is important for high affinity agonist binding (10). Mutation of
this residue to a phenylalanine (Y62F) caused a significant decrease in
agonist affinity, and mutation to a serine (Y62S) led to a loss of
measurable high affinity binding sites.
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EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 subunit
mutants were engineered using the Altered Sites II® in
vitro Mutagenesis System (Promega, Madison, WI) as described
previously (10, 15).
1,
2 (or
2 mutants), and
2L subunit cRNA (1 µg/µl total RNA) in a 1:1:1 ratio in a total volume of 50 nl.
Individual oocytes were maintained in 96-well plates at 14 °C in
modified Barth's medium (88 mM NaCl, 1 mM KCl,
0.5 mM CaCl2, 0.5 mM
Ca(NO3)2, 2.5 mM sodium pyruvate, 1 mM MgSO4, 2.4 mM
NaHCO3, 15 mM HEPES, pH 7.4) that was
supplemented with 100 µg/ml gentamicin.
60 mV. The microelectrodes were filled with 3 M KCl and had resistances of 0.5-2.0 megohms in frog
Ringer's medium (120 mM NaCl, 1.8 mM
CaCl2, 2 mM KCl, and 5 mM HEPES, pH
7.4). In all experiments, oocytes were continuously bathed in frog
Ringer's via a gravity perfusion system at a flow rate of ~5 ml/min.
GABA (Sigma) or muscimol (Sigma) was dissolved in the same medium.
where I is the peak amplitude of the current elicited by a given
concentration of agonist ([A]); Imax is the
maximum amplitude of the current; IC50 is the concentration
required for half-maximal inhibition; and n is the Hill
coefficient. The normalized data are presented as percent control to
construct concentration-response curves.
(Eq. 1)
where YO represents the base-line
response to GABA; YR is recovery; k
is the rate of recovery; and t is the time.
(Eq. 2)
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RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
2
2L receptors expressed in
Xenopus oocytes. In this experiment, maximally effective
concentrations of GABA (1 mM) elicited rapidly
desensitizing currents that were reproducible in amplitude when applied
at 12-min intervals (Fig. 1; see below). We have previously reported
that the EC50 for GABA-activated currents in this receptor
subtype is ~33 µM (10). After the challenge
applications, the oocyte was washed with Ringer's solution for 12 min
(to allow recovery from the desensitization induced by the challenge)
and then continuously bath-perfused with 10 µM GABA, a
concentration which itself evokes a significant current response
(<15% of maximum). Subsequent challenges at 12-min intervals showed
that the amplitudes of the evoked currents were reduced, declined to
steady state levels, and were fully reversible upon washout of GABA
from the perfusate. We interpret the observed reduction in current as
receptor desensitization induced by GABA in the perfusion medium.

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Fig. 1.
Representative recordings from application
(30 s) of GABA (
) to recombinant
1
2
2
GABAA receptors expressed in Xenopus
oocytes. The oocyte was challenged at regular time intervals
(12 min) using a concentration of agonist that elicited a maximum
response (1 mM). Perfusion of low concentrations of GABA in
the bath results in a decrease in the maximum amplitude
(IGABA) of the induced currents. This inhibition reaches a
steady state, and the currents return to control levels after washout
of agonist from the perfusate. A, bath pre-perfusion with 10 µM GABA causes an immediate reduction in the current
induced by the first challenge; B, perfusion of 1 µM GABA does not immediately desensitize the receptor,
but the apparent desensitization becomes pronounced with successive
challenge applications.

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Fig. 2.
A, inhibitory effects of GABA perfusion
on currents induced by 1 mM GABA in wild-type
1
2
2
recombinant GABAARs. Data represent the
mean ± S.E. of 4 independent experiments. Following the third
application (*) of GABA, 3 µM GABA was bath-perfused, and
the responses to 1 mM GABA challenges were recorded at 3- (
), 6- (
), 9- (
), and 12-min (
) intervals. By using a 9- or
12-min interval the data were not significantly different, but
frequency-dependent effects were observed at shorter
intervals (3 and 6 min). See text for further details. B,
effect of time of agonist perfusion on GABA-gated currents in wild-type
1
2
2 receptors. In each case, the response to GABA challenge
was stabilized prior to pre-perfusion of GABA in the bath for the
indicated duration. Following this application, the response to the
challenge concentration was measured again. Increased pre-perfusion
time significantly depressed GABA-gated current from control values,
but there was no significant difference between the 9-min pre-perfusion
time and the 12-min pre-perfusion time. Thus, in the oocyte recording
system, long pre-perfusion times (9-12 min) were required to achieve a
steady state desensitized response to the agonist. Data represent the
mean ± S.E. of (n) independent experiments.
2 Subunit
2 subunit Y62F
mutation, the desensitization characteristics measured in control
experiments (Fig. 3A) were very similar to those of
wild-type receptors. However, notable differences were observed using
the Y62S mutant (Fig. 3B). For this receptor, few frequency-dependent effects were apparent using challenge
intervals varying from 3 to 12 min. This indicates that this receptor
recovers more quickly (within ~3-min) from the desensitization
induced by the challenge than does the wild-type receptor.

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Fig. 3.
Experiments similar to those shown in
Fig. 2 were carried out using recombinant
GABAARs containing the Y62F (A) and Y62S
(B)
2 subunits. Data
represent the mean ± S.E. of 3 (Y62F) or 4 (Y62S) independent
experiments. Following the third application (*) of GABA, the agonist
was pre-perfused at concentrations of 5 and 25 µM,
respectively (approximating their IC50 values for
desensitization), to examine the effects of time (3 (
), 6 (
), 9 (
), and 12 min (
)) on functional recovery of the response from
the challenge concentration. Inhibition of GABA-evoked current is
significantly depressed during perfusion of agonist, and the Y62S
mutant receptor shows accelerated recovery from desensitization induced
by the challenge dose itself (see text for details).
Effect of Agonist Concentration on Receptor Desensitization
Experiments similar to those illustrated in Fig. 1 were carried
out to investigate the effects of agonist concentration on the steady
state level of desensitization. Fig.
4A shows the concentration dependence of both GABA- and muscimol-induced desensitization of the
wild-type receptor, and curve fitting gave apparent IC50 values of 3.3 and 0.7 µM, respectively (Table
I). In direct binding studies using
[3H]muscimol, we have reported that the wild-type
receptor expressed in tsA201 cells has (at least) two classes of
binding sites with affinities of about 8 nM and 0.43 µM (10). Although the latter value is subject to error
due to rapid ligand dissociation from lower affinity sites during
binding assays (see Ref. 13), the apparent KD value for this
population correlates well with the IC50 value for
muscimol-induced desensitization. Our preliminary interpretation is
that desensitization may be induced by occupancy of the lower affinity
binding sites measured in radioligand binding studies.
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The Y62F (Fig. 4B) and Y62S (Fig. 4C) mutations altered the concentration dependence for both GABA and muscimol-induced desensitization, the effects being greater in the case of the Y62S substitution (Table I). In both cases, muscimol was more potent than GABA in inducing desensitization. We have reported previously (10) that muscimol is also more potent in activation of these mutant receptors.
Receptors Carrying the
2 Subunit Y62S Mutation Do Not Maintain
the Desensitized State
During the course of the above experiments, we observed several
curious properties relating to the time dependence of desensitization of the Y62S mutant receptor. When the receptor was perfused with low
concentrations of GABA (
10 µM), i.e. below
its IC50 for steady state desensitization, the receptor
appeared to desensitize and then recover from this desensitization even
in the continued presence of the perfused agonist. The representative
traces in Fig. 5 illustrate this
phenomenon. In these experiments, the wild-type (Fig. 5A) or
Y62S (Fig. 5B) receptors were challenged successively with concentrations of GABA equivalent to their EC50 values for
activation (see Table I). In control experiments, we have shown that
the steady state level of desensitization is independent of whether the
EC50 (used in Fig. 5) or Emax (as in
Figs. 1-4) concentration of agonist is used as the challenge
application (data not shown). When the wild-type receptor was perfused
with 3 µM GABA, the currents, as expected from the
previous results, declined to a steady state level and returned to
control values upon washout. In contrast, the Y62S mutant receptor
showed desensitization during the pre-perfusion, but rather than
reaching a steady state residual current, the currents began to grow
again even in the continued presence of 3 µM GABA in the
perfusate. Thus this receptor, which we have shown previously to lack
measurable high affinity binding sites (10), appears unable to maintain
the desensitized state. Fig. 6 quantifies
this recovery phenomenon when the GABA concentration in the perfusate
was increased to 10 µM. In the wild type (Fig. 6A), the receptor desensitized, and this state was
maintained until washout of the GABA from the perfusate. In contrast,
the Y62S mutant desensitized but then recovered to control values in
the continued presence of perfused GABA (Fig. 6B). The
recovery phenomenon in the mutant is
concentration-dependent. Although it is clear at low
perfusate concentrations, i.e. 3-10 µM (Figs. 5 and 6), it is less obvious at higher concentrations. This is discussed further below.
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Rate of Recovery of Peak Amplitude as Function of Time
To define further the recovery of GABAARs from
desensitization, we have investigated the rate and extent of recovery
using wild-type and mutant Y62S receptors. In these experiments, a
desensitizing concentration of GABA was applied, and the amplitude of
the peak current to a subsequent challenge administered at times
varying from 1 to 30 min after the initial application was measured.
Fig. 7 shows the complete recovery from
desensitization of wild-type (Fig 7A) and mutant (Fig.
7B) receptors from successive challenges at their respective
EC50 concentrations. The Y62S mutant receptor recovers
~2-fold faster than the wild type, and curve fitting by a single
exponential model (Table II) gave
half-times for recovery of 0.8 and 1.5 min, respectively. These results
are in agreement with the data presented in Figs. 2 and 3, which show
that the mutant receptor recovers more quickly from desensitization
induced by the challenge application.
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Similar experiments were carried out in the presence of 3 µM bath-perfused GABA, i.e. similar to the
conditions used in Fig. 5. In the case of wild-type receptors, 3 µM GABA did not significantly alter the rate of recovery,
but at longer time intervals, the current induced by the challenge
application was reduced, reflecting the ability of the bath perfused
GABA to "lock" a proportion of the receptors in a desensitized
state. As expected from the previous results, the Y62S mutant receptor
displayed different recovery properties (Fig. 7B). The
receptor initially desensitized, but despite the continued presence of
bath-perfused GABA (3 µM), the magnitude of the currents
returned to control levels (Fig 7B) within about 20 min.
This again demonstrates that low concentrations of GABA in the
perfusate are able to induce desensitization but are unable to maintain
the receptor in a desensitized state. First impressions of the data in
Fig. 7B suggest that the rate of recovery may be slowed in
the presence of GABA in the perfusate. However, this is unlikely to be
due to a direct effect on the kinetics of recovery from the
desensitization induced by the challenge application itself because no
such behavior was seen in other experiments, e.g. Fig.
3B. It is more likely that these data reflect the same
phenomenon as depicted in Fig. 5B, i.e. that the
slow recovery to control levels is dictated by the time scale of the reversal of the transient desensitization process.
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DISCUSSION |
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The identification of molecular domains that contribute to receptor activation and desensitization is a primary goal in structural studies of GABAARs. The number of agonist-binding sites on a single GABAAR has not been unequivocally defined, owing in large part to the inherent complexity of this receptor family. Early binding studies using native brain membranes suggested that there were at least two classes of binding sites of high (KD = 10-30 nM) and low (KD = 0.1-1.0 µM) affinity (11, 13). Although these observations may have arisen from the heterogeneity of GABAARs that are now known to exist in mammalian brain, more recent studies using recombinant, presumably homogeneous, receptor subtypes suggest that individual receptors carry both high and low affinity sites for agonists (10, 20). In addition to these sites, which are amenable to measurement in direct binding studies, many investigators have suggested (11) that there may also be "ultralow" affinity sites (KD >10 µM) to explain the higher concentrations of GABA that are required to activate the GABAAR ion channel. Thus agonist binding is complex, and irrespective of the true number of binding sites carried by each receptor, it is clear that the functional roles of the multiple classes of sites have not been adequately addressed. In this report, we use site-directed mutagenesis to selectively disrupt the high affinity sites to probe their roles in receptor function.
The major goal of the present study was to relate the occupancy of binding sites to the effects on GABAAR desensitization. Although the Xenopus oocyte perfusion system is not a rapid application system and, as such, does not allow biophysical analysis of the microscopic rates of desensitization, it appears well suited for this type of analysis in which the concentration dependence of desensitization is measured under essentially equilibrium conditions. The experimental procedures have been optimized (see Figs. 2 and 3) to avoid potential artifacts, and as discussed below, the system has been validated by the close agreement of a number of the present results with those reported for other receptor systems using more rapid perfusion and other techniques such as patch clamp analysis to study wild-type and mutant receptors with respect to desensitization (17, 21). Further analysis of these mutant receptors using rapid application techniques will be important in elucidating the detailed mechanisms underlying desensitization and recovery.
Several models of receptor activation and desensitization have been proposed to account for conformational transitions of allosteric proteins. Adaptations of the Monod-Wyman-Changeux model (22) suggest that within a population of receptors, there exist two receptor conformations in which there is an equilibrium between a low affinity resting state and a high affinity desensitized state. This two-state model and its variants, which have been developed mainly to describe the transitions of the peripheral nAChR, propose that agonist binding to the resting state promotes both channel activation and desensitization and predict that the heterogeneity observed in binding studies arises from a dynamic equilibrium between different conformational states of the same receptor-ligand complex. Similarly, some investigators have suggested that the two populations of agonist sites detected in binding studies of GABAARs also reflect the presence of interconvertible states. However, we and others (11) have suggested that the sites are independent, in accordance with our early model of distinct sites being involved in activation and desensitization of the Torpedo nAChR (see Ref. 23).
One of the basic predictions of two-state models, developed from the
original Monod-Wyman-Changeux model, is that desensitization may occur
without channel activation (for review see Ref. 29). In the present
study, bath perfusion of agonist reproducibly inhibited GABA-gated
chloride conductances at recombinant wild-type GABAARs in a
concentration-dependent manner. However, the present data suggest that desensitization may require channel activation. Agonist perfusion did not reduce the amplitude of the response to the first
challenge application unless (a) the concentration of
agonist in the perfusate was sufficiently high to cause significant
receptor activation (Fig. 1A) and/or (b) agonist
perfusion took place during the recovery phase from the desensitization
induced by the agonist challenge, i.e. immediately after
channel activation (Fig. 2A). Low concentrations of bath
perfused agonist (e.g. 1 µM as in Fig. 1B) did not diminish the response to the first challenge
even when the perfusion time was prolonged (data not shown). These observations are similar to those previously reported by Bartrup and
Newberry (17) who used whole cell patch clamp techniques to study the
concentration dependence of desensitization of the serotonin type 3 receptor in NG108-15 cells. Such activation dependence is also evident
in the current traces reported by Corringer et al. (24)
investigating the desensitization properties of an
7 nAChR-serotonin
type 3 chimera expressed in Xenopus oocytes. In one chimera,
10 nM nicotine did not reduce the amplitude of the response
to the first 1 µM nicotine challenge (approximately the
EC50 for this mutant receptor), but subsequent challenge
responses were progressively reduced to a steady state level. A higher
perfusate concentration (0.1 µM) caused an immediate
inhibition, possibly because some receptor activation had occurred
during the perfusion.
The concentration dependence for desensitization of wild-type receptors by muscimol and GABA gave IC50 values of 0.7 and 3.3 µM, respectively (Fig. 4A). The latter value agrees well with the estimate of 1-2 µM reported by Overstreet et al. (25) for GABA-induced desensitization of native receptors in hippocampal slices. Measured IC50 values are also in agreement with estimates of the affinities of the lower affinity population of sites measured in binding studies. We reported previously that the lower affinity KD for [3H]muscimol binding to this receptor subtype expressed in tsA201 cells is 0.43 µM (10). The affinity for GABA binding to this population of sites was ~1 µM, as measured indirectly from its ability to potentiate [3H]flunitrazepam binding. Whereas this correlation may be fortuitous, it is clear that the IC50 values do not correlate with the affinities of the high affinity sites measured under equilibrium conditions (KD of 8.1 and 119 nM for muscimol and GABA, respectively). The association of desensitization phenomena with the lower affinity sites contradicts the speculation of many investigators that it is the high affinity agonist-binding sites that mediate desensitization (see Ref. 27).
Intriguingly, we recently obtained a very similar result when studying the nAChR from Torpedo electric organ but using a quite different technique, i.e. rapid agonist-induced flux measurements using native Torpedo membrane vesicles (28). Under equilibrium conditions, it is well established that this receptor carries two high affinity sites for agonists (KD of ~100 nM for carbamylcholine), and it is generally assumed that occupancy of these sites in the resting state of the receptor leads to the equilibrium desensitized state (29). However, saturation of these sites with carbamylcholine under equilibrium conditions did not diminish the flux response induced by subsequent exposure to a higher (activating) concentration of this ligand. Thus, as for the GABAA receptor above, occupancy of the high affinity sites per se does not desensitize the receptor. In contrast, the concentration dependence for desensitization, measured by the inhibition of the flux response upon subsequent challenge, gave an IC50 for carbamylcholine of 15.5 µM. This correlates well with occupancy of intermediate affinity sites that we have so far detected only under non-equilibrium conditions (30, 31). The parallels between the two receptor systems suggest that the underlying mechanisms for agonist-induced desensitization may be common to all members of this receptor family.
Desensitization of the two mutant receptors, Y62F and Y62S, occurred at 2.5-7.5-fold higher concentrations of both GABA and muscimol, with the Y62S mutant being more affected. These decreases in apparent affinity parallel those of these mutations on agonist-induced receptor activation (10). In control experiments, the desensitization properties of the Y62F-containing receptor were otherwise very similar to those of the wild-type. We reported previously (10) that this mutation has a mixed effect on [3H]muscimol binding, reducing the two populations of sites observed in the wild-type receptor to a single resolvable population of intermediate affinity. Thus, it would appear that the changes in agonist binding affinity may be due to perturbation of components of desensitization, which could account for the decrease in agonist sensitivity.
The data in Fig. 3B were the first indications that other
desensitization characteristics of the Y62S mutant receptor were different from their wild-type counterparts because this receptor appeared to recover from desensitization more quickly, i.e.
within about 3 min under the experimental conditions employed. However, the most interesting property of this mutant is illustrated in Figs.
5B and 6B, i.e. that low
concentrations of agonist desensitize the receptor, but this reverses
despite the continued presence of agonist in the perfusate. The initial
desensitization observed is clearly due to the presence of GABA in the
perfusate. In Fig. 5B, it is shown that under control
conditions, the currents induced by the challenge concentration fully
recover within the first 9-min assay point used. Because the main
difference between the wild-type receptor and the
2 Y62S mutant is
the presence of high affinity sites in the former and their absence in
the latter, we conclude that it is the agonist occupancy of the high
affinity sites that stabilizes the equilibrium desensitized state.
The above results have provided new insights into possibly conserved mechanisms of this receptor family. However, many quantitative issues remain to be resolved. The number of agonist sites on a single receptor molecule has not been established, and the likely allosteric interactions between sites of different affinities inevitably complicate analysis and interpretation. In binding studies, the Y62S mutant receptor appeared to carry only one population of sites for GABA with an estimated KD of about 1 µM (10), i.e. not significantly different from the lower affinity sites measured in the wild-type receptor. Similar GABA concentrations were required to induce transient desensitization of the Y62S mutant (see Figs. 5 and 6) suggesting that, as in the wild type, it is the occupancy of these sites that initiates the desensitization process. However, in the case of the mutant receptor, in which the high affinity sites are disrupted, the desensitized state is not "locked," and the receptor recovers. As noted under "Results," this recovery phenomenon is observed only at relatively low perfusate concentrations (1-10 µM). At higher concentrations, the observed desensitization displays similar properties to that of the wild type, albeit with a higher IC50 value (24.7 µM for GABA; Table I). This value does not correspond to either the apparent affinity measured in equilibrium binding studies or the EC50 for activation (178 µM) (10). Indeed for all three receptors studied here, desensitization occurred at concentrations that were ~10-fold lower than those required for receptor activation. It remains to be established whether this reflects the presence of distinct low affinity sites that mediate channel opening as we have proposed for the Torpedo nAChR (see Ref. 28).
In conclusion, we have investigated the functional consequences of
mutations to the high affinity agonist-binding sites of recombinant
GABAARs expressed in Xenopus oocytes.
Desensitization of the wild-type
1
2
2 receptor has been
characterized with respect to agonist concentration dependence, rate of
recovery, and apparent dependence on receptor activation. The reduction
in affinity for [3H]muscimol that occurs upon
substituting Tyr-62 of the
2 subunit by phenylalanine and serine
(10) is paralleled by an increase in the concentration dependence of
agonist-induced desensitization. The most intriguing observation is
that the Y62S mutant receptor, which lacks measurable high affinity
binding sites, recovers from desensitization even in the continued
presence of agonist. This suggests that the high affinity binding site
plays a major role in conferring stability to the desensitized state,
perhaps by virtue of a "locking" mechanism. Confirmation of this
novel result will undoubtedly require crystallization of native
GABAAR and the use of high resolution biophysical
techniques to monitor conformational transitions. Nevertheless, in the
present study, we have begun to unravel the importance of high affinity
agonist binding domains in GABAAR function.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Prof. Ian L. Martin, Drs. David A. Wagner, and Martin Davies for invaluable discussions and Prof. Jeremy J. Lambert and colleagues for introducing us to the Xenopus oocyte expression system. We also thank Dr. David S. Weiss for providing the GABAA receptor rat cDNA clones and Eugene Chomey for preparation of Xenopus laevis oocytes.
| |
FOOTNOTES |
|---|
* This work was supported in part by the Canadian Institutes of Health Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by the Natural Sciences and Engineering Research Council of Canada, the NeuroScience Canada Foundation, and the Alberta Heritage Foundation for Medical Research. Present address: Dept. of Physiology, University of Wisconsin, 1300 University Ave., Madison, WI 53706.
To whom correspondence should be addressed: Dept. of
Pharmacology, School of Medical Sciences, University of Bristol,
Bristol BS8 1TD, UK. Tel.: 44-117-928-8092; Fax: 44-117-9250168;
E-mail: susan.dunn@bristol.ac.uk.
Published, JBC Papers in Press, April 3, 2002, DOI 10.1074/jbc.M110312200
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
GABAARs,
-aminobutyric acid type A receptors;
GABA,
-aminobutyric acid;
GABAA,
-aminobutyric acid, type A;
nAChRs, nicotinic
acetylcholine receptors.
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