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INTRODUCTION |
The GABAA1
receptors are the major inhibitory neuronal ion channels in the
mammalian central nervous system. Two subunits termed
and
have
initially been purified from bovine brain (1) and the corresponding
DNAs have been cloned (2). Many mammalian subunits have been cloned
since (3-8). These subunits show a high degree of homology to subunits
of the nicotinic acetylcholine receptors, the glycine receptor and and
the serotonin (type 3) receptor. The GABAA receptor is the
site of action of many drugs, among them the benzodiazepines (for
review see Ref. 9). The binding site for the channel agonist GABA and
that for benzodiazepines are thought to be located at subunit
interfaces in homologous positions (for reviews see Refs. 10 and
11).
The second transmembrane segment of these subunits lines the ion
channel. In GABAA receptor
1 subunits, residues
Val256, Thr260, Thr261,
Leu263, Thr264, Thr267,
Ile270, Ser271, and Asn274 have
been reported to be exposed to the channel lumen (12). Picrotoxin is
likely to interact with residue Val256 (13, 14). Mutation
in a homologous residue in Drosophila receptors confers
cyclodiene resistance (15). A leucine residue is strictly conserved in
the middle of the M2 region of all subunit isoforms and is located at
position 263 of the
1 subunit. Substitution to a serine
in
1,
2, or
2 resulted in
an abnormally high apparent GABA affinity for channel opening (16).
Some point mutations of this leucine on
1,
1,
2, or
1 subunits
resulted in spontaneous open channels (17-21).
During work aimed at the understanding of the site in M2 involved in
the recognition of tert-butylbicyclophosphorothionate (22),
we investigated the properties of chimeric
1
3 receptor subunits coexpressed with
1 subunits. The results indicated an importance of
Ala252 and Leu253 for the
tert-butylbicyclophosphorothionate binding affinity. Here,
we show that substitutions of
3A252 and
3L253 result in reduced picrotoxin affinity. For
3L253F affinity is more than 100-fold reduced, and a
huge transient opening of the channel upon removal of picrotoxin was
evident. Such a transient channel opening has not been observed for any
other mutation before, and our mathematical model suggests that this is
a consequence of the strongly decreased affinity for picrotoxin.
Residue Val256, neighboring Phe257, the
homologue on the
1 subunit, has been shown to covalently interact with other noncompetitive blockers acting at the picrotoxin binding site (14). Leucine 253 of the
3 subunit
(
3L253) may therefore be part of the contact site for
picrotoxin together with
1V256. In addition, we report
that single point mutation
3L253F confers abnormal
gating properties to
1
3 receptors. These
include spontaneous opening of the channels and a very high GABA
sensitivity for channel gating, Thus, our work points to the
involvement in GABAA receptor channel gating of more
N-terminally located amino acid residues than previously suggested.
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EXPERIMENTAL PROCEDURES |
Amino Acid Residue Numbering--
Residues are numbered
according to the mature rat sequences.
Construction of Receptor Subunits--
The cDNAs coding for
the
1,
3, and chimeric subunits of the
rat GABAA receptor channel have been described elsewhere
(22, 23). Site-directed mutagenesis was done using the QuikChangeTM mutagenesis kit (Stratagene). In vitro synthesized sequences
have been verified by DNA sequencing.
Functional Expression and Characterization--
Xenopus
laevis oocytes were prepared, injected, and defolliculated, and
currents were recorded as described (24, 25). Briefly, oocytes were
injected with 50 nl of capped, polyadenylated cRNA dissolved in 5 mM K-HEPES, pH 6.8. This solution contained the transcripts
coding for the different subunits at concentrations of 75 nM. RNA transcripts were synthesized from linearized
plasmids encoding the desired protein using the mMessage mMachine kit
(Ambion) according to the recommendations of the manufacturer. A
poly(A) tail of ~300 residues was added to the transcripts by using
yeast poly(A) polymerase (Amersham Pharmacia Biotech). The cRNA
combinations were coprecipitated in ethanol and stored at
20 °C.
Transcripts were quantified on agarose gels after staining with Radiant
Red RNA Stain (Bio-Rad) by comparing staining intensities with various amounts of molecular weight markers (RNA Ladder; Life Technologies, Inc.). Electrophysiological experiments were performed by the two-electrode voltage clamp method at a holding potential of
80 mV.
GABA and picrotoxin (Fluka) were applied for 20 s, and a washout period of 3-15 min was allowed to ensure full recovery from
desensitization. The perfusion solution (6 ml/min) was applied through
a glass capillary with an inner diameter of 1.35 mm, the mouth of which was placed about 0.4 mm from the surface of the oocyte. The rate of
solution change under our conditions has been estimated 70% within
less than 0.5 s (25). Current responses have been fitted to the
Hill equation: I = Imax/(1+(EC50/[A])n) where
I is the peak current at a given concentration of GABA (A),
Imax is the maximum current, EC50 is
the concentration of agonist eliciting half-maximal current, and
n is the Hill coefficient. Currents were measured using a
modified OC-725 amplifier (Warner Instruments Corp.) in combination
with a xy recorder or digitized using a MacLab/200 (AD Instruments).
Kinetic Modeling--
We analyzed the gating behavior of the
mutated channel using a kinetic model that omits all states that only
become marginally populated (26). For arguments described later, the
model consists of four states: two different states for closed channels
(C, R), one state for open channels (O) and one state for channels
blocked by picrotoxin (O.PTX). The triangular three state model in the absence of picrotoxin (O, C, R) requires six microscopic rate constants. They are calculated using the experimentally determined current decay constants from the open to the closed states
(
1 and
2), the equilibrium constant for
the spontaneously open state, and the law of detailed balancing. The
remaining rate constants were used to adjust the simulation to the
data. The binding of picrotoxin to the receptors adds four new rate
constants to the system. They have to provide the reopening constant
measured in the presence of picrotoxin (
), and they have to satisfy
the law of detailed balancing. The additional two rates were used to
fit the data. Therefore, from the 10 rate constants in total there are
only four that can be used to reconcile the simulations with the data.
The kinetics of application and washout of picrotoxin is not taken into
account. They are assumed to happen instantaneously. This assumption is
justified because the processes studied here develop at a much slower
time scale. The integration of the differential equation system was
performed using the subroutines of the Matlab 5.2.0 library (MathWorks
Inc.).
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RESULTS |
Expression of Receptors Containing Chimeric
Subunits--
Fig. 1 shows the
structure of wild type and chimeric subunits. The chimera consist of
N-terminal sequences of the
3 subunit fused to
C-terminal sequences of the
1 subunit. They differ from each other in the residues forming the ion channel pore region. Each of
the chimeras was coexpressed together with the
1 subunit in Xenopus oocytes. Mature receptors incorporated in the
surface membrane were analyzed by using the two-electrode voltage clamp technique. Heteromeric
1CH7 receptors failed to respond to GABA (<10 nA), and an apparent outward current could be measured during application of 1 mM picrotoxin alone (Fig.
2A). Interestingly, a strong
transient inward current was detected during washout of picrotoxin in
the absence of GABA (Fig. 2A). Wild type
1
3 receptors were activated by GABA and
showed no response to the application of picrotoxin alone (Fig.
2B). Injection of cRNA coding for CH7 alone did not result
in ion currents induced by picrotoxin or GABA (data not shown),
indicating that the channel with these unusual properties was formed
from
1CH7. Three additional chimeras were coexpressed
together with the
1 subunit to localize residues important for the unusual gating behavior. Only
1CH74
also displayed the picrotoxin washout current found in
1CH7 receptors (Table I).
An amino acid comparison between the chimera revealed that a
threonine-valine-phenylalanine motif was common to CH7 and CH74 and
absent in the other two constructs. Three mutant subunits were
constructed by individually introducing these three residues of
1 into the homologous positions of
3,
with the aim to identify the amino acid residue responsible for the
abnormal properties.

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Fig. 1.
Schematic drawing of the
1, the
3, and chimeric subunits.
N, N terminus; C, C terminus; boxes
represent transmembrane regions M1-M4. 1 sequences are
represented by thin line and open boxes.
3 sequences are represented by a thick line
and gray boxes. CH7 is composed of the N-terminal amino
acids 1-250 of the 3 subunit and the amino acids
255-428 of the 1 subunit. The amino acid sequences of
the M2 regions of 1 and 3 are compared.
The position of leucine 253 in the 3 subunit is marked
by an asterisk. A schematic representation of the M2 regions
of wild type and chimeric subunits is drawn in the lower
part of this figure. The chimera consist of the N terminus of the
3 subunit fused to the C terminus of the
1 subunit and differ from each other by the sequences
present in the M2 regions. The upper part of the figure is
taken from Ref. 22.
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Fig. 2.
Electrophysiological properties of
1CH7 and of
1 3
receptors. Currents were recorded under voltage clamp
conditions at 80 mV from oocytes injected with 1 and
CH7 (A), with 1 and 3
(B), or with 1 and 3L253F (C)
subunits. 1 mM GABA and 1 mM picrotoxin
(Ptx) were applied for 20 s each (A and
B, bars) or 1 min (C). Two additional
identical experiments in each case gave similar results. The same
behavior of the channels was observed by using lower concentrations of
picrotoxin in three additional experiments and shorter application
times in 17 additional experiments.
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Expression of Point Mutated Receptors--
Wild type and mutant
3 subunits were coexpressed together with the
1 subunit in Xenopus oocytes. All three
mutant receptors expressed GABA-activated chloride currents. Receptors
with a leucine to phenylalanine substitution in the channel
pore-forming region at position 253 (
1
3L253F) could be activated by very low
concentrations of GABA (30 nM). Repeated applications
elicited increasingly smaller current amplitudes even when a washout
period of up to 15 min was used (data not shown). For this reason it
was technically not possible to measure the apparent affinity of GABA
for channel opening in
1
3L253F. Dose
response curves for wild type
1
3,
1
3V251T, and
1
3A252V revealed 3.5- and 6.0-fold
increases in the apparent affinities to GABA for these mutated
receptors (Table II), respectively.
Response to Picrotoxin--
Application of 1 mM
picrotoxin in the absence of GABA did not induce any apparent outward
or inward currents in
1
3V251T and
1
3A252V receptors. In contrast, in
oocytes expressing
1
3L253F receptors,
picrotoxin application resulted in apparent outward currents (Fig.
2C) similarly to oocytes expressing
1CH7. The mutated channel
1
3L253F also showed a
huge transient inward current upon washout of picrotoxin (Fig.
2C). At 1 mM picrotoxin, the amplitude of the
transient inward current was 11 times (average of three determinations)
the amplitude of the apparent outward current.
To exclude that the current properties upon injection of cRNAs coding
for
1 and
3L253F resulted from homomeric
3L253F, we also expressed this mutated subunit alone. No
detectable signal could be obtained with 300 µM
picrotoxin (two independent batches of oocytes).
A voltage ramp protocol was used to measure ion currents in mutant
1
3L253F channels before and during and
the application of picrotoxin and about 5 s after its removal
(Fig. 3A). The voltage ramp
had a duration of 0.13 s, such that the amplitude of the inward
current showed little change during the application of the ramp.
Picrotoxin application resulted in a reduction of the membrane
conductance, indicating that part of the receptors are spontaneously in
an open conformation. All three curves obtained had the same
intersection at
38 ± 1 mV (three experiments). Therefore, it
can be concluded that the spontaneous current and the transient inward
current have the same ion permeability. Replacing the 95 mM
Cl
with 9.5 mM Cl
and 84.5 mM acetate
in the outside medium resulted in
an about 60 mV shift to the right in the reversal potential of the
transient inward current (not shown), in line with a chloride selective
conductance. From the reversal potential of
38 mV determined at an
extracellular chloride concentration of 95 mM, an
intracellular chloride concentration of ~23 mM may be
estimated. The same voltage ramp protocol was used before and during
the application of a subsaturating concentration of GABA to oocytes
expressing the wild type
1
3 receptor
(Fig. 3B). The intersection of the two curves was found
at
25 ± 2 mV (three experiments), indicating an intracellular
chloride ion concentration of about 37 mM. The difference
in the intracellular chloride concentration in oocytes expressing wild
type and the point mutated receptor can be explained by a more negative
membrane potential of the oocyte than the chloride reversal potential. Under conditions where the permeability for this ion is increased, the
membrane potential drives chloride ions out of the cell.

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Fig. 3.
Voltage dependence of ion currents of
1 3L253F
(A) and
1 3
(B) receptors. Continuous voltage ramps of 130 ms
duration were generated from a holding potential of 80 to +50 mV, and
ion currents were recorded. The experiment was first performed in the
absence of picrotoxin (medium), then in the presence of 0.3 mM picrotoxin, and finally during the washout of the drug
in A and first during perfusion with medium and then during
perfusion with medium containing 0.3 µM GABA in
B. Two additional experiments in each case gave similar
results.
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As mentioned above, the channel opens to a certain degree spontaneously
producing an inward current. The initial response to the application of
picrotoxin is an apparent outward current reflecting channel closure.
The picrotoxin concentration dependence of the peak current amplitude
is illustrated in Fig. 4A. It
increases between 10 and 1000 µM picrotoxin and shows no
saturation up to 1 mM picrotoxin (Fig. 4C). The
maximum apparent outward current may, however, be estimated, assuming
an infinite membrane resistance for the case where all channels are in
the closed state. Assuming that most channels are in the open state
upon removal of picrotoxin, it can be estimated that less than 9% of
the channels are spontaneously open prior to the application of
picrotoxin. The shape of the late apparent outward current response
during perfusion with picrotoxin depends on the concentration of
picrotoxin used (Fig. 4A). In the presence of low
concentrations, only a transient inhibition of the current could be
detected, followed by reopening of the channels. The time course of
this reopening seemed picrotoxin concentration independent in the range
of 10-300 µM. Because of the small amplitudes of this
component, it could only be estimated. Assuming a mono-exponential time
course reopening was characterized by a
in the range of ~6-13 s
(not shown).

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Fig. 4.
The size of the apparent outward current and
the transient inward current in
1 3L253F
receptors both depend on the concentration of picrotoxin.
A, current traces obtained by applying increasing
concentrations of picrotoxin (bars) for 1 min. B,
computer simulations describing the concentration dependence of the
transient inward current evoked by picrotoxin withdrawal in the mutated
1 3L253F GABAA receptor. The
assumed concentrations of picrotoxin were 10, 30, 100, 300, and 1000 µM. The application time was 60 s. The rate
constants are described in the legend to Fig. 7 and assume the
following values: k1 = 5.0 × 10 2 µM 1 s 1,
k2 = 1.5 s 1,
k3 = 8.1 × 10 2
s 1, k4 = 8.1 × 10 3 s 1, k5 = 3.0 × 10 4 µM 1
s 1, k6 = 1.0 × 10 1 s 1, k7 = 5.0 × 10 2 s 1,
k8 = 5.5 × 10 2
s 1, k9 = 1.0 × 10 3 s 1, and k10 = 9.0 × 10 3 s 1. C, size of
the apparent outward current amplitudes and the inward current
amplitudes from three different oocytes. Data are given as the
means ± S.D.
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A large transient inward current was observed during washout of
picrotoxin. The concentration dependence on picrotoxin of this current
is illustrated in Fig. 4C. Its amplitude increases using
increasing concentrations of picrotoxin and does not saturate up to 1 mM picrotoxin. Half of the amplitude observed at 1 mM picrotoxin was observed at about 300 µM
(Fig. 4C). Fig. 5A
shows that the size of this current increases with the duration of
picrotoxin application. For this experiment 300 µM
picrotoxin was applied during different time intervals between ~1 and
60 s. The time dependence of the increase in inward current
amplitude is well fitted with a mono-exponential function with
= 9.1 ± 2.2 s (n = 3). Reclosure of the
channel after transient opening was independent of the picrotoxin
concentration and followed a bi-exponential time course with
f = 5.5 ± 2.8 s and
s = 23.3 ± 12.0 s (means ± S.D., three experiments at five
different concentrations each).

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Fig. 5.
The size of the washout current depends on
the duration of picrotoxin application in
1 3L253F
receptors. A, 300 µM picrotoxin was
applied for ~1, ~2, 4, 8, 16, 30, and 60 s (bars).
Two additional experiments gave similar results. B, computer
simulation of the dependence of the inward current on the time span of
picrotoxin application. Duration of 300 µM picrotoxin
perfusion increases from 1 s to 2, 4, 8, 16, 30, and 60 s.
The rate constants were as described in the legend to Fig.
4B.
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The picrotoxin sensitivity of GABA-activated currents was also measured
for wild type
1
3 receptors and mutant
1
3V251T and
1
3A252V receptors. A GABA concentration
eliciting 10-15% of the maximum current was used in these
experiments. Because
1
3V251T and
1
3A252V receptors displayed an increased
apparent affinity to GABA (Table II), a lower concentration of agonist had to be used for these two mutant receptors.
1
3A252V receptors displayed an about
10-fold reduced sensitivity to picrotoxin compared with wild type and
1
3V251T receptors (Fig.
6).

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Fig. 6.
Decreased picrotoxin sensitivity of
GABA-activated currents in
1 3A252T
receptors. A, current traces in the absence and in the
presence of increasing concentrations of picrotoxin (Ptx,
upper bars). 1 µM GABA was used for oocytes
expressing wild type receptors, and 0.3 µM GABA was used
for oocytes expressing 1 3V251T or
1 3A252V (lower bars).
B, relative current amplitudes at the end of 20 s of
picrotoxin application. Data are given as the means ± S.D. of
three experiments.
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Kinetic Modeling--
To explain the following two phenomena,
namely the reopening of the channels during continuous application of
picrotoxin and the transient inward current after its removal, we
performed computer simulations based on the model shown in Fig.
7. Rate constants used are given in the
figure legends of Figs. 4B. It is assumed that about 5% of
the channels are spontaneously open, which determines the equilibrium
state of the system and the initial conditions for the simulations. The
channels switch between this open state and the closed states giving
rise to the observed inward resting current. Washout of picrotoxin is
assumed to lead to fast dissociation from the receptor without
rebinding. This results in the immediate emptying of all bound states.
The number of picrotoxin less closed states is then given by the
relaxation process upon picrotoxin removal. The double exponential
decay determined in the experiments requires a minimal model which
consists of three states: an open state (O) and two closed states (C
and R). Applying picrotoxin to the system leads to a pronounced
reduction in the number of open channels. The resulting initial
decrease of the inward current (apparent outward current) is followed
by the relaxation into a new equilibrium state. We propose that only
the state (O.PTX) (binding of picrotoxin to the open channels) is
kinetically relevant because after withdrawal of picrotoxin these
channels are able to produce a transient inward current. If in contrast
picrotoxin binding to closed channels would be dominant, no transient
inward current could be observed, because the channels would already be
closed. Fig. 5B presents simulations of experiments where
the duration of 300 µM picrotoxin application is varied.
Because the equilibrium is on the side of the state (O.PTX) because of
the excess of picrotoxin, the longer the transient filling of the state
(O.PTX), the larger the transient inward current amplitude after
removal of picrotoxin. Fig. 4B shows simulations of the current during perfusion with different concentrations of picrotoxin. The amplitude of the initial apparent outward current is limited at
high picrotoxin concentration because of saturation of channel closure.
The following relaxation process into the new equilibrium state depends
only weakly on the concentration of picrotoxin (largest relaxation
constant
= 13-16 s), which is in agreement with the experiments. The simulations revealed that the closed state (R) serves
as a reservoir for states C and O so that after binding of the channels
to picrotoxin the two latter states become partly refilled by channels
from state R. Obviously the higher the concentration of picrotoxin, the
larger the population of the state (O.PTX), and as a consequence the
amplitude of the rebound current augments with increasing picrotoxin
concentration. The true k2 that describes the
transition rate from O.PTX to O is obscured by the rate of solution
change (see "Experimental Procedures"). The model is constructed in
such a way that the decay of the inward current after removal of
picrotoxin is double exponential with time constants
1 = 8 s and
2 = 12 s. Both, the picrotoxin
concentration dependence (Fig. 4B) and the time dependence
(Fig. 5B) of the responses of the mutated channel to
picrotoxin exposure and removal predicted by the model should be
compared with the experimentally observed behavior (Figs. 4A
and 5A). Except for the initial peak of the apparent outward
current predicted from the model and almost absent in the experimental
traces, the agreement between model and experiment is remarkable. The
difference is probably mostly due to the limited rate of solution
change (see "Experimental Procedures"), which has not been taken
into account in the simulations.

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Fig. 7.
Model used in the computer simulations.
The states O, C, R, and O.PTX denote the open state, the closed state,
the resting state, and the state where picrotoxin is bound to the open
conformation, respectively. Note that the state O.PTX is nonconducting
because of the channel blocker picrotoxin. The value of the rate
constants are given in the legend to Fig. 4B.
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DISCUSSION |
We studied alterations in the channel lining part of the
recombinant
1
3 GABAA
receptor. A mutant ion channel was found that displayed a transient
chloride current upon removal of the channel blocker picrotoxin. A
single point mutation is able to confer this unusual property to the
receptor, namely, of leucine 253 in the M2 region of the
3 subunit to a phenylalanine, which is present in the
homologous position of all
subunits. The point mutated
3 was coexpressed together with the
1
subunit. These receptors can open spontaneously in the absence of GABA
and are blocked by picrotoxin in a dose-dependent fashion.
Washout of picrotoxin results in a strong transient inward current. The
amplitude of this current is dependent on the concentration of
picrotoxin and the time of its application. The point mutated receptors
could be activated by very low concentrations of GABA. Obviously, this mutation strongly affects gating of the channel and its interaction with picrotoxin.
It is interesting to compare the newly identified position with other
positions within M2 that are important for receptor function and
modulation. Based on radioligand binding experiments to chimeric
receptors, it has been suggested that
3L253 is important for the binding of another channel blocker
tert-butylbicyclophosphorothionate (22). This position is 12 amino acid residues N-terminal to the one on the
3
subunit implicated in the action of loreclezole (27). It is located 6 residues C-terminal to the predicted cytoplasmic entry point into the
membrane and is 6 residues N-terminal to the conserved leucine in the
center of the M2 region. Point mutations of this leucine also resulted
in spontaneous currents (17-20). The spontaneous currents were in some
cases antagonized by GABA (18, 19) and were sensitive to picrotoxin
(18-21). Spontaneous channel activity has also been reported with
expression of the rat or mouse
1 subunit alone (28, 29),
the mouse
3 subunit alone (30), and a combination of rat
5 and
subunits (31). Using the mentioned amounts of
cRNA, no spontaneous currents were observed for homomeric wild type
3 and mutant
3L253F receptors. In all the
mentioned cases an off-current upon picrotoxin washout was never
reported. Our model presented below will predict why this is not the case.
The structure of the GABAA receptor in M2 is presumably
close to that of other members of the ligand-gated ion channel family. The above cited studies and studies of the homologous nicotinic acetylcholine receptor suggested that the centrally conserved leucine
is structurally critical for channel gating and might even be a part of
the channel gate (32). Other studies place the gate more N-terminal to
the cytoplasmic end of the channel pore (13). Because
1
3L253F receptors display an altered
channel gating our results support this proposal.
Valine 256 on the
1 subunit has been proposed to be
exposed to the channel lumen (13) and to be in direct contact with picrotoxin (13, 14). A homologous residue has also been implicated in
the action of the cyclodiene insecticide resistance in invertebrate GABA receptor subunit Rdl (15). Because of the 5-fold
symmetry of the channel pore, it is likely that the homologous residue on the
3 subunit, alanine 252, is also facing the
channel pore and might be close to the bound picrotoxin entity. Our
results indeed show that replacement of alanine by valine in this
position (
3A252V) results in an about 10-fold reduced
affinity for picrotoxin. Mutation of the adjacent leucine 253 to
phenylalanine even more drastically reduces the picrotoxin affinity. It
cannot completely be ruled out that the substitution might have some
indirect effects on the picrotoxin binding pocket. However, it is
tempting to speculate that picrotoxin is in direct contact with
3L253. In apparent contrast to this speculation, Xu and
Akabas (12) found that the homologous phenylalanine 257 on the
1 subunit after cysteine substitution is not accessible
to sulfhydryl reagents. However, this residue is on the same side of
the
-helix as reactive positions and is also adjacent to them. Side
chains other than cysteine might be at least partially accessible to
the channel lumen and could be in contact with the picrotoxin molecule.
We propose a kinetic model for
1
3L253F
describing its interaction with picrotoxin. It makes a number of
predictions that are discussed in the following. Mutant
1
3L253F receptors assume an open and two
closed states in the absence of GABA. The open state is inferred from
the presence of spontaneously open channels, and the closed states are
required because of the double exponential decay of the transient
inward current upon picrotoxin removal. Picrotoxin is mostly bound to
open channels that become nonconducting because of the bound channel
blocker resulting in an apparent outward current. Reopening of channels
in the presence of picrotoxin finds a simple explanation with the help
of our model. The binding of picrotoxin to the open (O) and closed
channel (C) is faster than the transition between the two closed states
(R and C). After emptying states O and C by adding picrotoxin, the
channels in the desensitized state R serve as a reservoir that slowly
releases channels to the open state via state C. The equilibrium of
this process is reached in about 1 min (Figs. 4 and 5). The regain of
open channels is responsible for the increase of the inward current in
the presence of picrotoxin.
The huge transient inward current after picrotoxin washout is the
result of the decreased affinity (fast transition of O.PTX to O), and
it depends on both the duration of picrotoxin exposure and on its
concentration (Figs. 4 and 5). Because of the almost instantaneous
transition of O.PTX to O upon picrotoxin removal, the amplitude of the
initial washout current is approximately proportional to the population
of state O.PTX. Therefore, we can use this amplitude to estimate the
overall equilibrium affinity constant to picrotoxin. It is higher than
300 µM in the present case for
1
3L253F receptors, although the local
affinity constant of the reaction between O and O.PTX is 20 µM in our model. The former value should be compared with
3 µM picrotoxin that reduces GABA induced currents in
1
3 to about 50% (Fig.
4C).
As mentioned above, substitution of the centrally conserved leucine in
GABA receptors in some cases also resulted in the formation of
spontaneously open channels. Our model shows that the large amplitude
of the fast transient inward current requires a faster transition from
O.PTX to O as compared with all other transition rates of the model.
Increasing the time constant for this process reduces the amplitude and
slows down the time course for the transient population of the open
state. Both effects are observed for other GABAA receptors
with substitution of the centrally conserved leucine (18-21).
Therefore, the model suggests that for these mutated GABAA receptors the dissociation of picrotoxin from the blocked channel (k2) is slowed down. The relative amplitude of
this fast transient inward current is also limited by the fraction of
channels that are in the open state in the absence of picrotoxin. The
larger this fraction of spontaneously open channels is, the smaller the predicted inward current. Both factors may contribute to a different degree to the lack of an transient inward current in the initially cited cases of spontaneous currents.
In summary, we report two important observations. The first is that we
describe two point mutations in the M2 region of the
3
subunit close to the putative cytoplasmic end that both result in an
increase of apparent GABA affinity. One of these,
3L253F results additionally in spontaneous channel opening and is thus structurally critical for channel gating. Our results therefore strongly suggest that the region important for channel gating has to be
extended at least four amino acid positions toward the N terminus as
compared with previous conclusions (18). The second observation is that
these point mutations result in a reduced affinity for picrotoxin. The
residue
1V256 has been shown to covalently interact with
other noncompetitive blockers acting at the picrotoxin binding site
(14). Mutation of the homologous residue
2A252 shows a
10-fold effect. However, mutation of the neighboring residue
3L253 has drastic effects on picrotoxin affinity and may
therefore together with
1V256 form part of the contact site for picrotoxin.