Previous Article | Next Article 
The Journal of Neuroscience, October 1, 2002, 22(19):8411-8421
Coupled and Uncoupled Gating and Desensitization Effects by Pore
Domain Mutations in GABAA Receptors
Michaela
Scheller1 and
Stuart A.
Forman2
1 Klinik fuer Anaesthesiologie der Technischen
Universitaet Muenchen, Klinikum rechts der Isar, D-81675 Munich,
Germany, and 2 Department of Anesthesia and Critical
Care, Massachusetts General Hospital, Boston, Massachusetts 02114
 |
ABSTRACT |
GABAA receptors are allosteric ligand-gated ion
channels. Agonist-induced gating and desensitization have been proposed
to be coupled via pore domain structures. Mutations at two
1 subunit pore-domain (transmembrane domain 2)
residues enhance GABA sensitivity, leucine-to-threonine at position 264 (9'), and serine-to-isoleucine at position 270 (15'). We investigated
the role of these residues in gating, desensitization, and deactivation
of
1
2
2L GABAA receptors using rapid GABA concentration jumps and patch-clamp electrophysiology. GABA EC50 values for
1(L264T)
2
2L and
1(S270I)
2
2L currents were,
respectively, ~80-fold and 13-fold lower than the wild-type
EC50. Unlike wild type, both mutant receptors displayed significant picrotoxin-sensitive currents in the absence of GABA, indicating that they enhance gating efficacy. Both mutants displayed current activation rates that matched wild type at 1 µM
GABA and above. Desensitization of wild-type and
1(S270I)
2
2L currents displayed indistinguishable rates and amplitudes, whereas
1(L264T)
2
2L currents
desensitized extremely slowly. Deactivation of wild-type currents
displayed two rates and slowed after partial desensitization, whereas
currents from both mutants deactivated slowly with single rate
constants that were unaffected by desensitization. These results
indicate that both
1(L264T) and
1(S270I)
mutations increase the gating efficacy of receptors by slowing channel
closing, which accounts for nearly all of the similar changes that they
produce in macrocurrent dynamics. Because the
1(S270I)
mutation uncouples its gating effects from those on rapid
desensitization, these two processes are necessarily associated with
movements of distinct receptor structures (gates). The effects of the
1(L264T) mutation suggest that the conserved leucines
may play a role in gating-desensitization coupling.
Key words:
GABA receptor; acetylcholine receptor; ion channel; gating; desensitization; pore; electrophysiology
 |
INTRODUCTION |
GABAA
receptors (GABAARs) are the major
inhibitory neurotransmitter receptors in the mammalian CNS and targets
for sedatives, anxiolytics, anti-epileptics, and general anesthetics.
GABAA receptors are pentameric ligand-gated ion
channels, part of the superfamily, including nicotinic acetylcholine
(nACh), 5-hydroxytryptamine type 3 (5-HT3), and
glycine receptors (Ortells and Lunt, 1995
). Each homologous
GABAA receptor subunit contains a large
extracellular N-terminal domain and four hydrophobic domains predicted
to form transmembrane elements (TM1 to TM4) (Barnard et al., 1998
). The chloride-conducting pore of the receptor is thought to be surrounded by
predominantly helical TM2 domains (Karlin and Akabas, 1995
). Mutations
in TM2 domains can affect chloride conductance, sensitivity to
blockers, and channel gating (Gurley et al., 1995
).
In voltage-clamp recordings of multiple GABAA
channels, rapid GABA application activates a chloride current and
initiates multiphasic current desensitization to nonconducting states.
Both gating and desensitization of ligand-gated receptors display
allosterism; they occur with low probability in the absence of agonists
and with high probability after agonist binding, driven by increased affinity (Neubig et al., 1982
; Jackson et al., 1990
; Galzi and Changeux, 1994
; Chang and Weiss, 1999
). Fast receptor desensitization may help determine the magnitude and shape of GABAergic IPSCs, both by
truncating activation and prolonging deactivation (Jones and Westbrook,
1995
; Overstreet et al., 2000
; Burkat et al., 2001
).
Auerbach and Akk (1998)
proposed that nACh receptor gating and
desensitization are movements of independent gates that are energetically coupled. A number of studies has suggested that GABAA receptor desensitization is negatively
coupled to gating, i.e., desensitization proceeds only from closed
states (Jones and Westbrook, 1995
; Haas and Macdonald, 1999
; Li and
Pearce, 2000
). However, nonequilibrium single-channel studies indicate that GABAA receptors desensitize from both closed
and open states with comparable rates (Burkat et al., 2001
). Structural
studies implicate TM2 domains in gating dynamics and suggest that they form gating structures in nACh and GABAA
receptors (Xu and Akabas, 1996
; Wilson and Karlin, 2001
). Less is known
about structures involved in fast desensitization. Bianchi et al.
(2001)
reported that desensitization of GABAA
receptors containing
2 versus
subunits was
unaffected by swapping their highly homologous TM2 domains. Bianchi and
Macdonald (2001)
found that mutation of a conserved leucine in the
GABAA receptor
subunit TM2 domain both enhanced gating and slowed the apparent rate of desensitization, attributing this to negative gating-desensitization coupling. It is
unknown whether other mutations within TM2 domains that affect gating
also alter desensitization, as implied by coupled mechanisms.
We focused on two single amino acid mutations that are known to enhance
GABA sensitivity in recombinant
1
2
2L
GABAA receptors,
1(L264T) and
1(S270I). L264 is one of the highly conserved
TM2-9' leucines found in all superfamily subunits. Replacing these 9' leucines with serines or threonines in GABAA,
nACh, or 5-HT3 receptors apparently stabilizes
open states whether agonists are bound or not, decreasing agonist
EC50 values, prolonging channel openings, and
increasing activation in the absence of agonist (Revah et al., 1991
;
Yakel et al., 1993
; Filatov and White, 1995
; Labarca et al., 1995
;
Tierney et al., 1996
; Chang and Weiss, 1998
, 1999
; Dalziel et al.,
2000
). The
1(S270I) mutation alters receptor modulation by alcohol and anesthetics (Mihic et al., 1997
; Koltchine et
al., 1999
; Ueno et al., 1999
). Like
1(L264T),
1(S270I) reduces GABA
EC50, but whether it stabilizes open states is
unknown. Activation, rapid desensitization, and deactivation kinetics
of
1
2
2L
GABAA receptors containing these mutations have
not been reported previously. Given the proximity of these two TM2
residues and the similar impacts of the mutations on GABA
EC50, we hypothesized that both mutations would
cause similar changes in molecular dynamic behavior.
We used submillisecond GABA concentration jumps to elicit macrocurrents
from voltage-clamped patches and cells expressing GABAA receptors, deriving both equilibrium and
kinetic activation, desensitization, resensitization, and deactivation
data. Receptor activity in the absence of GABA was assessed
electrophysiologically using high concentrations of picrotoxin. Our
results indicate that both mutations enhance conduction gating. The
1(S270I) mutation alters gating but not
desensitization, indicating that the status of the
GABAA receptor conduction gate can be uncoupled
from desensitization. In contrast, the
1(L264T) mutation affects both conduction
gating and desensitization. Thus, some, but not all, parts of the pore domain that affect the gating mechanism also couple to desensitization.
 |
MATERIALS AND METHODS |
Site-directed mutagenesis. cDNAs encoding
1,
2, and
2L subunits of the human
GABAA receptor in pCDM8 vectors (Invitrogen, Carlsbad, CA) were supplied by Dr. Paul J. Whiting (Merck Sharp and
Dohme Research Labs, Essex, UK). cDNAs for
1(S270I) and
1(L264T) were constructed using high-fidelity PCR oligonucleotide-directed mutagenesis. The presence of the mutations and absence of stray mutations in the cDNAs were confirmed by dideoxynucleotide sequencing.
Transient expression of recombinant GABAA
receptors. Human embryonic kidney cells (HEK293) [American
Type Culture Collection (ATCC), Rockville, MD] were cultured in
minimum essential Eagle medium (ATCC), supplemented with 10% horse
serum and 1% penicillin-streptomycin (all from Life Technologies,
Grand Island, NY), and maintained at 37°C in a 5%
CO2 incubator. Cells were plated on
protamine-coated glass coverslips. Transient expression of receptor
channels was achieved by transfecting (Chen and Okayama, 1987
) cells
with cDNA mixtures for GABAA receptors encoding
wild-type
1
2
2L,
1(S270I)
2
2L, or
1(L264T)
2
2L
at a w/w ratio of 1
:2
:5
(or 1
:2
for
-less controls).
Cells were cotransfected with an expression plasmid (
H3-CD8; Jurman
et al., 1994
) for the lymphocyte surface antigen CD8-
, which was a
gift from Dr. Gary Yellen (Harvard Medical School, Boston, MA). Cells
were cultured for 36-72 hr after transfection and incubated briefly
with polystyrene microspheres and precoated with anti-CD8 antibody
(Dynabeads M-450 CD8; Dynal, Great Neck, NY). Transfected cells
expressing CD8, which correlated with the presence of GABA-activated
currents, were identified by adhering microspheres using phase-contrast microscopy.
GABAA receptor
electrophysiology. For electrophysiology, culture medium was
replaced by an extracellular solution containing (in
mM): 162 NaCl, 5.3 KCl, 0.67 Na2HPO4, 0.22 KH2PO4, 15 HEPES, 5.6 glucose, and 2 CaCl2, adjusted to a pH of 7.30 with NaOH. Patch pipettes were fabricated from borosilicate glass
(Fisher Scientific, Pittsburgh, PA), fire polished to open tip
resistances of 2-5 M
, and filled with intracellular solution
containing (in mM): 140 KCl, 2 MgCl2, 11 EGTA, 10 HEPES, and 10 glucose,
adjusted to a pH of 7.30 with KOH. Extracellular solutions containing
GABA, diazepam, or picrotoxin (all from Sigma, St. Louis, MO) were
prepared shortly before experiments. Experiments were performed at room temperature (20-22°C) using standard outside-out or whole-cell patch-clamp techniques. For whole-cell recordings, the smallest cells
(diameter of
6 µm; to increase mechanical stability and to decrease
solution exchange time) were lifted from the coverslips. Patches or
cells were voltage clamped at
50 mV during recordings. Pipette
capacitance and series resistance were compensated in the whole-cell
mode. Currents through the patch-clamp amplifier (Axopatch 200A; Axon
Instruments, Foster City, CA) were filtered (eight-pole bessel, 2-5
kHz) and digitized at 2-20 kHz using commercial software (pClamp 8.0;
Axon Instruments).
Rapid GABA application. Solutions were applied to patches or
whole cells using a piezo-driven quad (2 × 2) capillary tube capable of switching between four flowing solutions (Forman, 1999
). Solution exchange times for switching between adjacent solutions were
0.1-0.5 msec, measured as the 10-90% rise time for open pipette junction currents. This is also the effective time for solution changes
for excised patches. Whole-cell activation times at saturating GABA
concentrations were measured to be <1.5 msec with cells <6 µm in
diameter Larger cells slowed down solution exchange times and were not
studied. Gravity-driven solutions in each of the four capillary lumens
could be changed in ~60 sec using upstream selector valves coupled to
reservoirs. A continuous flow of external solution through the
recording chamber prevented accumulation of transmitter in the bath.
Standard protocols repetitively applied different concentrations of
GABA (1 nM to 10 mM) as pulses of variable
duration, as appropriate for each experiment (for details, see
Results). A 20-40 sec interval between agonist pulses was used for
recovery of channel activity from desensitization. Ensemble averages of between 4 and 10 GABA-activated responses were stored for each experiment (e.g., different low GABA concentrations) in a single patch
or cell. For each type of receptor, at least three different patches or
cells from at least two different batches of transfected cells were
studied in each experimental condition (GABA concentration response,
activation, desensitization, or deactivation).
Data analysis. GABA-evoked ensemble average currents were
baseline corrected by subtracting leak currents (and currents
attributable to spontaneously active channels) recorded in each
sweep before GABA application. For the two concentration internally
controlled GABA-response experiments, current amplitudes in low GABA
(ILow) just preceding the switch to
control saturating GABA (1-10 mM) were
normalized to the peak current just after the solution switch (ISat). Peak current data from
single-concentration sweeps were similarly normalized to a paired
control sweep. Normalized currents were fitted with logistic (Hill)
equations of the following form:
|
(1)
|
where
Imax/ISat
is the maximum normalized current, EC50 is the
concentration eliciting a half-maximal response, and nH is the Hill coefficient of activation.
Activation rates were derived from multiexponential fits to the rising
phase of currents (inverted) after jumping into GABA. Exponential fits
for activation were improved by restricting the data set to points
between ~10% activation (beyond the "foot") and into the
desensitization phase when it was present. The positive amplitude
component of the fit was taken as the activation rate, as
desensitization represented negative exponential components. For fast
activation at GABA
10 µM, only patch currents were
analyzed. For lower GABA concentrations, large currents were most
readily recorded from whole cells. Although solution exchange was
slower at cells than at patches, the apparent solution exchange rate was readily detected at high GABA (1-10 mM) in cells.
Thus, by comparing activation rates at low and high GABA, we could
discern when activation at low GABA was limited by GABA binding rather than by solution exchange.
Desensitization rates were derived from exponential fits to the
decaying current phase between the peak of activation and the
termination of GABA application. A constant term, representing steady-state desensitization, was unconstrained in the fits.
Deactivation rates were derived from exponential fits to the decaying
current after removal of GABA. Depending on the number of exponential
components, single, double, or triple exponential functions were fitted
to these current decay phases using a Levenberg-Marquardt search
protocol and least-squares minimization. The number of exponential
components was increased until the addition of another component did
not significantly improve the fit using an F test (p < 0.01). Two-tailed Student's t
tests were used for statistical comparisons of the various properties
of mutant and wild-type receptors. Nonlinear and linear least-squares
fits and statistical analyses were performed using commercial software
[Origin v5.0 (Microcal, Northampton, MA) or Clampfit v8.0 (Axon
Instruments)]. All results are reported as means ± SD.
Kinetic modeling. Model-generated current data were
calculated using MATLAB software (The Mathworks, Natick, MA) based on state matrix (Q matrix) differential equations and methods that have
been described by Colquhoun and Hawkes (1995)
. Iteration time intervals
for the calculations were 0.1-1 msec. Model-generated current data
were imported into Origin worksheets and analyzed by the same methods
used for electrophysiologic recordings.
Evaluation of GABAA receptor subunit
expression. GABAAR expression studies in
HEK293 cells may be subject to artifacts attributable to endogenous
expression of subunits (Ueno et al., 1996
). We found that untransfected
HEK293 cells had no endogenous GABA-activatable currents. Whole-cell
currents recorded from cells transfected with wild-type and mutant
cDNAs were generally above 500 pA, suggesting that combinations with
endogenous subunits do not contribute significantly to our results. In
addition, some investigators (Boileau and Czajkowski, 1999
) have
suggested that the
subunit may not be efficiently incorporated into
GABAARs under some conditions, whereas others have reported that
is efficiently incorporated into
GABAARs when it is cotransfected with
and
subunits (Verdoorn et al., 1990
; Hadingham et al., 1992
; Krampfl et
al., 1998
). Several results lead us to conclude that
2L was efficiently incorporated into the
GABAARs we studied. First, the GABA
EC50 of GABAARs from cells transfected with only
1 and
2 cDNAs (
-less) was consistently and
significantly lower than the EC50 determined from
cells transfected with all three subunits [3.9 ± 0.23 µM for
1
2 (n = 5) vs 25 ± 0.7 µM for
1
2
2L].
We did not detect any evidence for a mixture of two populations of
receptors in GABA concentration responses. Second, we found no
difference in the GABA sensitivity and kinetic behavior of
GABAAR currents from cells that were transfected
with our usual cDNA mix versus those from cells in which the relative stoichiometry of
2L cDNA was increased
fourfold. Finally, we found that submaximal GABA-activated
currents (EC5-10) from cells expressing
1
2
2L
receptors were enhanced threefold (n = 4) by 3 µM diazepam, which is known to require a
subunit (Pritchett et al., 1989
), whereas similar experiments in
1
2 receptors showed no enhancement by 3 µM diazepam
(n = 3).
 |
RESULTS |
Both
1(L264T) and
1(S270I) mutations
enhance receptor gating
GABA-dependent peak currents
The equilibrium between resting receptors and GABA-activated open
receptors depends on both GABA binding-unbinding and gating efficacy
and is reflected in the GABA-dependent peak currents elicited by rapid
GABA concentration jumps. Previous studies in oocytes and HEK293 cells
have reported enhanced GABA sensitivity for receptors containing
1(L264T) or
1(S270I)
mutations (Chang et al., 1996
; Koltchine et al., 1999
; Scheller and
Forman, 2001
), but none have studied both mutants in the
1
2
2L
subunit background or used submillisecond GABA concentration jumps. For
each type of GABAA receptor, we assessed relative
peak-activated currents over a range of GABA concentrations using a
two-concentration protocol, in which currents were initially activated
with a low concentration of agonist until the peak current amplitude
was reached, followed by a rapid jump to saturating GABA (1-10
mM). The peak current ratio for low versus saturating GABA
(ILow/ISat) was thereby assessed within a few milliseconds in a single internally controlled sweep, minimizing the effects of both current rundown and
desensitization on this measurement. Initial GABA pulse durations of 10 msec up to 1.5 sec were used to reach peak currents, because of the
large variability in activation rates over the GABA concentration range
tested (1 nM to 10 mM).
Representative current traces for the wild-type and mutant
GABAA receptor are shown in Figure
1. Peak control currents varied between
patches or cells because of varying numbers of active receptors. Some
variability in the
ILow/ISat ratios was
attributable to the use of slightly different initial pulse lengths
(resulting in variable desensitization), as well as differences in
currents from patches and small cells, which displayed different
apparent activation and desensitization rates. However, these
variations were not large, because EC50 values
derived from independent experiments in patches and cells differed by
<30%. Combined data from all wild-type patches and cells studied
(Fig. 1, circles) were fit using Equation 1 (Materials and
Methods) with an EC50 of 25 µM.

View larger version (17K):
[in this window]
[in a new window]
|
Figure 1.
GABA-dependent peak
currents of wild-type
1 2 2L,
1(L264T) 2 2L, and
1(S270I) 2 2L
GABAA receptors. Left panels,
Traces are examples of currents recorded from
voltage-clamped membrane patches expressing wild-type or mutant
GABAA receptors, elicited with a two-concentration GABA
activation protocol. Initial activation at a low GABA concentration was
followed by a rapid jump to 1 mM GABA in the same sweep and
then reversal of these steps. Solid lines above
traces indicate timing of the different GABA
applications and the low GABA concentration used (micromolar). Longer
exposures to low GABA were used with
1(L264T) 2 2L receptors
because of their very high sensitivity to GABA and slow
desensitization. Right, Relative peak currents at low
versus saturating (1-10 mM) GABA were derived from traces,
by calculating the ratio of currents immediately before
(ILow) and after
(ISat) the jump from low to high
GABA. Combined normalized data (average ± SD) from patches and
cells are plotted. The lines through data
points represent Equation 1 (see Materials and Methods) fitted
to data. Wild type (circles), EC50 of 25 µM; nH = 1.3. 1(L264T) 2 2L
(triangles), EC50 of 0.30 µM;
nH = 0.83. 1(S270I) 2 2L
(squares), EC50 of 1.3 µM;
nH = 1.3.
|
|
Incorporation of the
1(L264T) mutation
in
1
2
2L
GABAA receptors resulted in a large increase in
sensitivity to GABA, with concentrations in the low micromolar range
eliciting peak currents that were near maximal (Fig. 1). Activation of
1(L264T)
2
2L GABAA receptors was very slow at the lowest range
of GABA tested, requiring GABA applications of up to 1.5 sec to reach
steady-state current. However, desensitization of these receptors was
also very slow, so relative peak currents were very consistent between patches and cells. The combined data (Fig. 1, triangles)
were fitted with an EC50 of 0.30 µM, ~80-fold lower than the wild-type value.
GABAA receptors containing the
1(S270I) mutation also showed increased
sensitivity to GABA activation (Fig. 1). GABA concentrations near 1 µM elicited approximately half the current seen with 1 mM GABA. Combined patch and cell data for
1(S270I)
2
2L
GABAA receptors (Fig. 1, squares) were
fitted with an EC50 of 1.3 µM, ~20-fold lower than the wild-type value.
Although the single-sweep internally controlled concentration-response
method is convenient and minimizes uncertainty caused by rundown,
it may result in EC50 values and Hill
coefficients that differ from those derived from traditional
concentration-response measurements, in which peak currents at low and
saturating agonist concentrations are measured in separate sweeps. For
comparison with the two-concentration method, normalized peak
GABA-elicited responses were recorded from patches expressing wild-type
or mutant GABAA receptors using
single-concentration pulses. In patches expressing both wild-type and
1(L264T)
2
2L
GABAA receptors, EC50
values derived using the single-concentration method (Table 1) were very similar to those derived
from internally controlled sweeps. For
1(S270I)
2
2L
GABAA receptors, the EC50
derived using single-concentration sweeps was 2.1 µM, 1.6 times the EC50 derived from internally controlled
sweeps. These significantly different results from the two
normalization methods are expected when current activation with agonist
concentrations near EC50 is slow compared with
desensitization, which is the case for the
1(S270I) mutant but not for wild-type or the
1(L264T) mutant (see below).
Spontaneous activation
Spontaneous receptor activation represents a measure of the
equilibrium between resting and open channels that is independent of
GABA binding. TM2 domain mutations that affect the open-closed gating
equilibrium appear to affect gating equally whether or not GABA is
bound (Chang and Weiss, 1999
). We estimated spontaneous receptor
activation electrophysiologically by measuring the portion of the
holding current at zero GABA that could be inhibited by high
concentrations of the channel blocker picrotoxin (1 mM). To
estimate spontaneous current as a fraction of total receptor activity,
the picrotoxin-sensitive current, IPTX
Ihold, was normalized to the
difference between IPTX and the
maximal GABA-activated current (1-10 mM GABA) in
the same patch: (IPTX
Ihold)/(IPTX
IGABA).
Holding currents for voltage-clamped (
50 mV) cells and patches
expressing wild-type
1
2
2L
or
1(S270I)
2
2L
channels were generally very small compared with peak GABA-activated
currents. In comparison, cells and patches expressing
1(L264T)
2
2L
channels consistently displayed large holding currents. The application of picrotoxin did not perceptibly affect wild-type holding currents (Fig. 2, left). Because the
baseline noise level in our whole-cell recordings was 1-2 pA, we were
able to detect a change of ~1 pA in holding currents. The absence
of any picrotoxin-induced change in wild-type holding
current, associated with maximal GABA-activated currents up to 5 nA,
indicates that wild-type channels open spontaneously <0.01% of
the time. In contrast, picrotoxin dramatically reduced
1(L264T)
2
2L
holding currents (Fig. 2, middle), indicating that 36% of
1(L264T)
2
2L
receptors were conducting in the absence of GABA (Table 1). In
addition, picrotoxin significantly decreased the holding current in
patches and cells expressing
1(S270I)
2
2L receptors (Fig. 2, right; Table 1), indicating that 0.5% of
these receptors were conducting in the absence of GABA.

View larger version (19K):
[in this window]
[in a new window]
|
Figure 2.
GABA-independent activation estimated from
picrotoxin-sensitive "leak" currents. Membrane patches expressing
wild-type or mutant GABAA receptors were voltage clamped at
50 mV and exposed to rapidly applied pulses of 1 mM
picrotoxin (upward traces) or 1 mM GABA
(downward traces). Both mutant receptors exhibited
picrotoxin-sensitive holding currents, whereas wild type did not. The
fraction of receptors active in the absence of GABA was estimated from
the ratio (IPTX Ihold)/(IPTX IGABA). Results are summarized in
Table 1.
|
|
Gating enhancement by mutations is attributable to slow
channel closure
The rate of current onset recorded during initial GABA application
(activation) is a function of GABA binding rates and channel opening
rates for ligand-bound receptors. At very high GABA, the activation
rate approximates the channel opening rate. At lower GABA, activation
is limited by concentration-dependent binding and, at very low
concentrations, may reflect channel closing or unbinding steps
(Celentano and Wong, 1994
; Maconochie et al., 1994
). To discern whether
mutant effects on gating were associated with changes in GABA
association or channel opening rates, we studied current activation
over a wide range of GABA concentrations.
For wild-type and mutant channel currents, activation rates increased
with increasing concentrations of GABA (Fig.
3). At 1 µM
[GABA]
1 mM, wild-type current activation rates increased in
direct proportion to GABA. The slope of an error-weighted fitted line
through these data are 0.95 ± 0.065 × 107
M
1sec
1,
the apparent rate of GABA binding. Above 1 mM GABA,
activation rates show asymptotic behavior, with peak rates in patches
ranging from 2000 to 5000 sec
1 at 1-10
mM GABA. At the lowest GABA concentrations that elicited currents suitable for analysis (0.2-0.5 µM), we observed
an asymptotic low rate for wild-type current activation (Fig. 3,
left inset). The average low GABA activation rate for
wild-type currents was 8 sec
1, similar
to that reported for granule cell GABAA currents
(Maconochie et al., 1994
).

View larger version (17K):
[in this window]
[in a new window]
|
Figure 3.
GABA-dependent activation rates for wild-type and
mutant GABAA receptors. Panels are log-log
plots of average activation rates versus GABA concentration. Each
point represents at least three experiments. The
lines connecting data points between 0.5 µM and 1 mM GABA represent linear
least-squares fits. Horizontal lines represent average
rates at low and high GABA within the depicted ranges. Examples of
current activation traces at both very low GABA and high GABA are shown
as insets to each panel.
Left, Wild type
( 1 2 2L);
10 6 M GABA ( 1 = 28.6 sec 1); 10 2 M
GABA ( 1 = 4600 sec 1).
Middle,
1(L264T) 2 2L;
10 7 M GABA ( 1 = 3.4 sec 1); 10 2 M
GABA ( 1 = 3700 sec 1).
Right,
1(S270I) 2 2L;
10 7 M GABA ( 1 = 7.3 sec 1); 10 2 M
GABA ( 1 = 3000 sec 1).
Average activation parameters for all experiments are reported in Table
1.
|
|
Activation rates for
1(L264T)
2
2L
and
1(S270I)
2
2L
GABAA receptor currents were nearly identical to
those from wild type at equal GABA concentrations >0.5
µM (Fig. 3, middle and right), despite eliciting larger proportions of maximal peak currents. In the 1 µM to 1 mM range, the
apparent GABA binding rates for
1(L264T)
2
2L
and
1(S270I)
2
2L
GABAA receptors are 0.8 ± 0.15 × 107
M
1sec
1
and 0.9 ± 0.11 × 107
M
1sec
1,
respectively. In addition, maximal activation rates for both mutant
receptors, measured at 1-10 mM GABA in patches,
closely match those of wild-type receptors (Table 1). The low GABA
current activation rate asymptotes for
1(L264T)
2
2L
and
1(S270I)
2
2L GABAA receptor currents were significantly lower
than that for wild type (~4 and 5 sec
1, respectively; p
0.01).
The
1(L264T) mutation slows desensitization, whereas
the
1(S270I) mutation does not
GABA receptor current decay in the continuous presence of
saturating GABA (1-10 mM) was studied using long GABA
pulses (
1.5 sec). Fast desensitization rates in both wild-type and
1(S270I)
2
2L receptors were assessed in excised outside-out patches. Desensitization of
1(L264T)
2
2L
receptors was also studied in whole cells using GABA pulses of
20 sec
in length.
Desensitization of GABA currents elicited from wild-type
1
2
2L
GABAA receptors was variable, displaying two to
three exponential components (Fig. 4,
left) and a nonzero constant term, representing the
steady-state current after prolonged desensitization. After GABA pulses
of 5 sec, wild-type currents desensitized to a steady-state value
averaging 8 ± 5.3% of peak (n = 12). Three
exponential components significantly improved nonlinear least-squares
fits over two components in 5 of 12 patches (F test at
p
0.01). The rates and fractional amplitudes for the
three resolved components were as follows: 
= 70 ± 29 sec
1,
Afast = 0.2 ± 0.16;

= 6 ± 5.1 sec
1,
Aint = 0.2 ± 0.11;

= 0.7 ± 0.35 sec
1,
Aslow = 0.5 ± 0.16. When these
five traces were fitted with only two exponential components, the
results were similar to those for the remaining seven traces, averaging
~20 and 1 sec
1, respectively, for fast
and slow decays (Table 1). Relative amounts of fast and slow
desensitization varied from patch to patch, but on average, fast
desensitization accounted for less than half of the current decay.

View larger version (13K):
[in this window]
[in a new window]
|
Figure 4.
Desensitization kinetics of wild-type and mutant
GABAA receptors. Examples of currents recorded from
voltage-clamped membrane patches during long desensitizing pulses of
1-10 mM GABA. Lines above
traces indicate the application of GABA. Exponential
fits to the desensitizing current phases are shown overlaid on the
currents. Left, Wild type
( 1 2 2L). Current
decay during desensitization was best fitted with two exponentials
[ = 18 sec 1 (36%);
 = 0.97 sec 1 (64%)].
Middle,
1(L264T) 2 2. Because of the
small degree of desensitization during the 6 sec GABA pulse, we were
unable to fit an exponential function to these data.
Right,
1(S270I) 2 2L.
Desensitization was best fit by two exponentials
[ = 18 sec 1 (25%);
 = 0.67 sec 1 (75%)].
Average desensitization parameters for all experiments are reported in
Table 1.
|
|
The
1(L264T) mutation dramatically reduced and
slowed desensitization (Fig. 4, middle). After GABA (1 mM) pulses of 5 sec, desensitization reduced
current from peak values by only 19 ± 8.3% (n = 8), and GABA pulses of
20 sec produced <40% desensitization. Desensitization in
1(L264T)
2
2L
currents was consistently slow in both patches and whole cells,
although some currents showed several apparent phases of
desensitization. Based on these results,
1(L264T)
2
2L
GABAA receptors desensitize with a time constant of >10 sec and to a much lower degree than wild type (Table 1).
Desensitization of GABA currents elicited from
1(S270I)
2
2L
GABAA receptors was very similar to that observed
in wild-type currents, displaying two to three exponential
components (Fig. 4, right). After 5 sec in 1 mM GABA,
1(S270I)
2
2L
currents decayed to 11 ± 7.7% of peak (n = 7).
In four of seven patches, F test analysis indicated
significantly (p
0.01) improved fits with three exponentials versus two. The average rates and fractional amplitudes for the three resolved components were as follows: 
= 80 ± 43 sec
1,
Afast = 0.2 ± 0.08;

= 9 ± 5 sec
1,
Aint = 0.2 ± 0.12;

= 0.7 ± 0.24 sec
1,
Aslow = 0.5 ± 0.14. Two
component fits for these four patches were similar to the remaining
three patches (Table 1). Neither the fitted rates of current decay nor
the relative amount of fast and slow components for
1(S270I)
2
2L
currents significantly differ from wild-type values (Table 1).
Recovery from desensitization is slowed by the
1(S270I) mutation
Recovery of GABAA receptor response after
desensitization was studied in patches expressing wild-type and
1(S270I)
2
2L
GABAA receptors using a double-pulse protocol
(data not shown). An initial 2 sec pulse at 1 mM GABA was
applied, resulting in ~70% desensitization, followed by a variable
period of recovery (20 msec to 40 sec) and a second pulse of 1 mM GABA to elicit a maximal response from nondesensitized
receptors. To correct for possible rundown or slow recovery, the second
peak was normalized to the first peak for each sweep. Because
desensitization of
1(L264T)
2
2L
GABAA receptors was minimal, their recovery from
desensitization was not studied.
Two phases of resensitization were seen with wild-type receptors. Fast
resensitization accounted for 25-35% of recovery, with time constants
ranging from 60 to 150 msec (Table 1). Slow resensitization of
wild-type receptors was characterized by a time constant of 13 sec.
GABAA receptors containing the
1(S270I) mutation displayed only a single slow
resensitization phase with a time constant of 20 sec (Table 1). Thus,
the resensitization of
1(S270I)
2
2L was significantly slower than that of wild type.
Both
1(L264T) and
1(S270I) mutations
slow deactivation and remove desensitization-deactivation coupling
The current decay after removal of GABA represents transitions
from active ligand-bound receptor states to inactive unbound states,
including channel closure and GABA dissociation. In addition, desensitized GABAA receptors may reopen before
GABA dissociation, prolonging deactivation (Jones and Westbrook, 1995
).
We assessed deactivation rates for wild-type and
1(S270I)
2
2L
GABAA receptor currents in excised patches after
maximal GABA pulses of 10-3000 msec. Because of its slow
desensitization, longer GABA pulses were sometimes used to study
1(L264T)
2
2L deactivation.
Representative traces displaying deactivation of maximally activated
wild-type and mutant GABAA receptor currents are
shown in Figure 5. For
1
2
2L
currents, deactivation was best fitted with two exponentials (Fig. 5,
left; Table 1), in agreement with previous studies on
1
2
2L
receptors expressed in HEK293 cells (Tia et al., 1996
). The majority of
wild-type current deactivation followed a fast time course with a rate
of ~50 sec
1, followed by a slower
deactivation at ~7 sec
1 (Table 1). As
reported previously (Jones and Westbrook, 1995
), the relative amount of
fast deactivation in wild-type receptors decreased with longer GABA
pulses. After 3 sec GABA application, the relative amplitude of the
slow deactivation component increased to 60 ± 5.1%
(n = 7), whereas the rates of fast and slow
deactivation were unaffected by pulse duration.

View larger version (14K):
[in this window]
[in a new window]
|
Figure 5.
Deactivation kinetics of wild-type and mutant
GABAA receptors. Examples of current decays (deactivation)
recorded from voltage-clamped membrane patches after removing GABA
after pulses of saturating (1 mM) GABA. Exponential fits to
deactivating current phases are shown overlaid on the currents.
Left, Wild type
( 1 2 2L).
Deactivation after a 10 msec pulse of 1 mM GABA
(solid trace) was best fitted by two exponential
components with  = 35 sec 1 (87%) and  = 6.5 sec 1 (13%). Deactivation after a 1000 msec
desensitizing pulse of 1 mM GABA (dotted
trace) shows slower apparent deactivation. The data were best
fitted by two exponential components with  = 45 sec 1 (44%) and  = 5.3 sec 1 (56%). Middle,
1(L264T) 2 2. Deactivation
after a 100 msec pulse of 1 mM GABA was best fitted by a
single exponential with  1 = 1.4 sec 1. No significant change in deactivation rate
was observed using a 5 sec pulse of GABA. Right,
1(S270I) 2 2L. Deactivation
after a 10 msec pulse of 1 mM GABA was best fitted by a
single exponential with  1 = 2.3 sec 1. No significant change was observed using a 1 sec pulse of GABA. Average deactivation parameters for all experiments
are reported in Table 1.
|
|
Deactivation kinetics for both
1(L264T)
2
2L
and
1(S270I)
2
2L
GABAA receptor currents displayed only single
rates that were very slow compared with wild-type deactivation
(Fig. 5, middle and right; Table 1). Deactivation
of
1(L264T)
2
2L
currents proceeded at a rate of ~1
sec
1, over 40 times slower than the
dominant deactivation rate in wild type. Deactivation of maximally
activated
1(S270I)
2
2L currents was ~17-fold slower than wild-type fast deactivation (~3
sec
1). Furthermore, deactivation of
mutant receptor currents did not change after prolonged exposure to
GABA (desensitization).
 |
DISCUSSION |
We studied macrocurrent kinetics in
GABAA receptors containing TM2 mutations that
enhance apparent GABA sensitivity. Compared with wild type, both
1(L264T) and
1(S270I)
mutations slow deactivation and induce increased spontaneous activation
in the absence of GABA without changing apparent activation kinetics.
One major difference between currents from the two mutant receptors was observed:
1(S270I)
2
2L
receptors desensitized like wild type, whereas
1(L264T)
2
2L
receptors display very slow desensitization.
To help interpret and illustrate how these mutations alter
GABAA receptor molecular transitions, we
considered a simple, semiquantitative allosteric-kinetic model (Fig.
6A). The model consists
of three types of states: resting closed (R), open (conducting; O), and desensitized (D), each of which can bind GABA (G). It incorporates known GABAA receptor state transitions, including
gating from both ligand-bound and unliganded states (Chang and Weiss,
1999
), and desensitization from both closed and open states (Burkat et al., 2001
) but contains fewer states than other allosteric models (Hall, 2000
; Pearce, 2001
).

View larger version (30K):
[in this window]
[in a new window]
|
Figure 6.
A simple allosteric-kinetic model of
GABAA receptor states and transitions. A, A
schematic incorporating three types of interchanging states: resting
(R), open (O), and desensitized
(D). Each of these states can bind GABA
(G), forming states RG, OG, and DG. Only O and OG
states are conducting and assumed to have equal conductance. The model
incorporates allosteric gating of GABAA receptors (Chang
and Weiss, 1999 ) by linking the gating of liganded and unliganded
receptors (maintaining a fixed ratio between closing rates and
'). In addition, the model allows desensitization from both closed
and open states at equal rates (kdes)
as shown by Burkat et al. (2001) . The major states involved in binding,
gating, and desensitization are highlighted in bold
letters. Note that each state in the schematic is an element in
one three-state cycle and two four-state cycles, resulting in highly
constrained equilibrium behavior. For calculations of model-generated
currents, the model was further constrained by making all three GABA
binding rates (kon) equal. The
"wild-type" kinetic model (18 rate constants) was fully defined by
eight input rate values and the '/ ratio:
kon = 107
M 1sec 1;
k = 500 sec 1;
= 3000 sec 1; = 200 sec 1; kdes = 2 sec 1; k = 200 sec 1; k = 0.5 sec 1; ' = 5 sec 1;
'/ = 5. Other rate constants were calculated from
constraints on the model. Rate constants were not "fitted" to
our experimental results, and some rate constants were assigned to
enable display of multiple features of model-generated traces on a
single time scale. B-E, Model-generated macrocurrent
traces were calculated under conditions mimicking a 1 sec baseline (0 GABA) period, a 2 sec GABA pulse (concentrations labeled in
micromolar), and 2 sec of deactivation at 0 GABA. B,
Wild-type model-generated currents at various GABA concentrations
display features qualitatively similar to experimental currents,
including low activation at zero GABA
(P0 = 0.005), fast activation,
desensitization, and fast deactivation. C, Decreasing
20-fold does not alter the maximal rate of desensitization and
slows the deactivation rate approximately sevenfold. There is increased
activation at zero GABA (P0 = 0.091),
and EC50 is reduced approximately fivefold. These changes
qualitatively reflect those produced by the 1(S270I)
mutation. D, Decreasing koff
20-fold does not alter the maximal rate of desensitization and slows
the deactivation rate approximately sevenfold. Compared with wild
type, activation at zero GABA is unchanged
(P0 = 0.005), and EC50 is
reduced approximately sevenfold. E, Decreasing both and kdes 20-fold increases activation at
zero GABA (P0 = 0.091) and reduces
EC50 ~18-fold. The apparent rate and extent of
desensitization and the rate of deactivation are reduced. These changes
qualitatively reflect those produced by the 1(L264T)
mutation.
|
|
Both
1(L264T) and
1(S270I) mutations
stabilize open states
The many similar changes in equilibrium and kinetic behavior
caused by
1(L264T) and
1(S270I) mutations suggest that they affect a
common molecular transition. Indeed, all of the common effects of these
two mutations can be explained by slowing channel closing rates. The
reduced EC50 values of the mutant
receptors could be attributable to enhanced microscopic binding
(reduced KG
koff/kon),
gating efficacy (reduced
/
), or both (Colquhoun, 1998
). Our allosteric-kinetic model (Fig. 6A)
illustrates that reductions in either KG
(koff) or
(
) result in reduced
EC50 values, unaltered desensitization at high
GABA, and slow deactivation (Fig. 6C,D). The only
clearly distinguishing feature between currents generated by these two
altered models is the amount of spontaneous activity they display in
the absence of GABA, because increased activation in the absence of
GABA is produced by increased gating efficacy but not by increased GABA
binding affinity. Additional support for this interpretation comes from
other studies reporting increased spontaneous gating by
GABAA receptors containing either
(S270) or
(L264) mutations (Chang and Weiss, 1999
; Findlay et al., 2000
; Ueno
et al., 2000
).
Although both
1(L264T) and
1(S270I) mutations produce qualitatively
similar changes in GABAA receptor macrocurrent
gating, the impact of
1(L264T) on spontaneous
activation and EC50 is clearly larger than that
of
1(S270I). Importantly, the relative EC50 values and the amounts of spontaneous gating
activity (Table 1) for these mutants are in very good accord with
quantitative predictions for doubly liganded ion channels (Forman and
Zhou, 1999
) and the allosteric gating model proposed by Chang and Weiss (1999)
. The EC50 for doubly liganded ion channels
is predicted to depend on the square root of the closing rate/opening
rate ratio (
/
; Forman and Zhou, 1999
). Because gating probability in the absence of ligand is directly proportional to gating probability of ligand-bound receptors [
/(
+
)], EC50
should also be proportional to the inverse square root of spontaneous
activation probability (Chang and Weiss, 1999
). Thus, if altered gating
probability (efficacy) is the basis for our mutation-induced
EC50 changes, the sevenfold ratio of
EC50 values for
1(S270I)
and
1(L264T) mutants predicts that, compared
with
1(S270I)
2
2L,
