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The Journal of Neuroscience, April 1, 1999, 19(7):2474-2488
Chlorpromazine Inhibits Miniature GABAergic Currents by Reducing
the Binding and by Increasing the Unbinding Rate of GABAA
Receptors
Jerzy W.
Mozrzymas1, 2,
Andrea
Barberis1,
Krystyna
Michalak2, and
Enrico
Cherubini1
1 Neuroscience Program and Istituto Nazionale Fisica
della Materia Unit, International School for Advanced Studies, 34 014 Trieste, Italy, and 2 Department of Biophysics, Wroclaw
University of Medicine, 50 368 Wroclaw, Poland
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ABSTRACT |
Recent studies have emphasized that nonequilibrium conditions of
postsynaptic GABAA receptor (GABAAR) activation
is a key factor in shaping the time course of IPSCs (Puia et
al., 1994
; Jones and Westbrook, 1995). Such nonequilibrium, resulting
from extremely fast agonist time course, may affect the interaction between pharmacological agents and postsynaptic GABAARs. In
the present study we found that chlorpromazine (CPZ), a widely used antipsychotic drug known to interfere with several ligand and voltage-gated channels, reduces the amplitude and accelerates the decay
of miniature IPSCs (mIPSCs). A good qualitative reproduction of the
effects of CPZ on mIPSCs was obtained when mIPSCs were mimicked by
responses to ultrafast GABA applications to excised patches. Our
experimental data and model simulations indicate that CPZ affects
mIPSCs by decreasing the binding
(kon) and by increasing the unbinding
(koff) rates of GABAARs.
Because of reduction of kon by CPZ, the
binding reaction becomes rate-limiting, and agonist exposure of
GABAARs during mIPSC is too short to activate the receptors
to the same extent as in control conditions. The increase in unbinding
rate is implicated as the mechanism underlying the acceleration of
mIPSC decaying phase. The effect of CPZ on GABAAR binding
rate, resulting in slower onset of GABA-evoked currents, provides a
tool to estimate the speed of synaptic clearance of GABA. Moreover, the
onset kinetics of recorded responses allowed the estimate the peak
synaptic GABA concentration.
Key words:
chlorpromazine; GABAA receptors; miniature
IPSCs; patch-clamp; binding and unbinding rate constants; hippocampus
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INTRODUCTION |
Phenothiazines (PTZs) are a family
of compounds commonly used in the treatment of psychiatric disorders.
The mechanism whereby these drugs exert their therapeutic effects
appears to be through blockade of dopamine receptors (Snyder et al.,
1974; Seeman, 1980). However, these substances have been shown to
affect also a number of other physiologically important sites. It is
known, for instance, that PTZs compete for serotonin and 
-adrenergic
and histamine receptors (Peroutka and Snyder, 1980). More recently,
electrophysiological studies have shown that PTZs interfere with a
number of ligand- and voltage-activated channels (Gould et al., 1983;
Sand et al., 1983; Changeux et al., 1986; Dinan et al., 1987; Zorumski
and Yang, 1988; Ogata et al., 1989; Müller et al., 1991; Bolotina et al., 1992; Benoit and Changeux, 1993; Lidsky et al., 1997). In
particular, it has been reported that PTZs block in a noncompetitive manner the responses evoked by exogenous application of GABA (Zorumski and Yang, 1988) and reduce the amplitude of IPSCs (Agopyan and Krnjevic, 1993), but the mechanism of these effects has not been elucidated. It is likely that the noncompetitive block of
GABAA receptors by PTZs, reported by Zorumski and Yang
(1988), is one of the processes underlying the reduction in amplitude
of IPSCs observed by Agopyan and Krnjevic (1993). However, in the
experiments of Zorumski and Yang (1988), GABA application system was
too slow to mimic the time course of the agonist in the synapse, making it difficult to directly refer these results to the PTZs effects on
synaptic currents.
A downregulation of synaptic inhibition may play an important
role in the induction of epileptic activity. Thus, a further understanding of the effects of PTZ on inhibitory synaptic transmission appears to be particularly important because the use of PTZs may be
associated (especially at high doses) with adverse effects, including
seizures (Toone and Fenton, 1977; Itil and Soldatos, 1980).
The aim of the present work was to study the mechanisms underlying the
effects of chlorpromazine (CPZ), a widely used PTZ, on miniature IPSCs
(mIPSCs) in cultured hippocampal neurons. We report that CPZ reduces
the amplitude of mIPSCs in a dose-dependent manner and accelerates
their decay. The mechanism of CPZ action on GABAA receptors
was studied using the ultrafast GABA applications to excised patches
that fairly mimicked the synaptic currents. The results have been
expressed in terms of a kinetic model, and it is suggested that the
effects of CPZ are caused by a reduction in binding and increase in
unbinding rates of GABAA receptor channel.
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MATERIALS AND METHODS |
Cell culture. Primary cell culture was
prepared as described previously (Andjus et al., 1997). Briefly,
hippocampi were dissected from 2- to 4-d-old rats, sliced and
digested with trypsin, mechanically triturated, centrifuged twice at
40 × g, plated in the Petri dishes, and cultured for
up to 12 d. Experiments were performed on cells between 5 and
12 d in culture.
Electrophysiological recordings. Currents were recorded in
the whole-cell and outside-out configurations of the patch-clamp technique using the EPC-7 amplifier (List Medical, Darmstadt, Germany).
In the case of whole-cell recordings of the synaptic and GABA-evoked
currents, the series resistance (Rs) was
in the range of 4-8 M
, and 50-70% of Rs
compensation was accomplished. Both mIPSCs and GABA-elicited currents
in the excised patch configuration were recorded at a holding potential
(Vh) of 
70 mV. In the whole-cell configuration, GABA-evoked responses were often very large (>1 nA). To
diminish a possible distortion caused by
Rs, the electrical driving force for
chloride ions was reduced by setting the Vh at
30 mV. The intrapipette solution contained (in mM) CsCl
137, CaCl2 1, MgCl2 2, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid
(BAPTA) 11, ATP 2, and HEPES 10, pH 7.2 with CsOH. The composition of
the external solution was (in mM) NaCl 137, KCl 5, CaCl2 2, MgCl2 1, glucose 20, and HEPES 10, pH
7.4 with NaOH. mIPSCs were recorded in the presence of tetrodotoxin
(TTX; 1 µM) and kynurenic acid (1 mM) to
block fast sodium spikes and glutamatergic EPSCs. All the
experiments were performed at room temperature 22-24°C.
The current signals were stored on a video recorder after
pulse-code modulation. For the analysis requiring a high temporal resolution (e.g., rise time kinetics of synaptic or evoked currents), the signals were low-pass filtered at 10 kHz with a Butterworth filter
and sampled at 50-100 kHz using the analog-to-digital converter CED
1401 (Cambridge Electronic Design Limited, Cambridge, UK) and
stored on the computer hard disk. Otherwise, for analysis of slower
events, the cutoff frequency of the filter and the sampling rate were
lowered accordingly. The data and acquisition software were kindly
given by Dr. J. Dempster (Strathclyde University, Glasgow, UK).
Two different perfusion systems for GABA applications were used: the
multibarrel RSC-200 perfusion system (Bio-Logic, Grenoble, France) and
the ultrafast system based on the use of a piezoelectric-driven theta
glass application pipette (Jonas, 1995). The head of the multibarrel
system was modified to improve the speed of drug application on cells
adhering to the bottom of Petri dishes. This system was used either to
evoke the whole-cell GABA-induced currents or to exchange the solution
around the cell from which the synaptic activity was recorded. Judging
from the onset of the liquid junction potentials, a complete exchange
of the solution around the open-tip electrode occurred within 10-20
msec. A better indication of the exchange time around the cell was
given by the rise time of whole-cell responses evoked by high
concentrations of GABA (>1 mM). Because it is known that
with those concentrations the rise time of GABA responses is less than
or close to 1 msec (Maconochie et al., 1994; see also Results and
Discussion), the observed rate of rise of the whole-cell current
(15-30 msec) was mainly determined by the speed of the solution
exchange. The piezoelectric translator used for ultrafast perfusion
system was from Physik Instrumente (Waldbronn, Germany), and theta
glass tubing was from Hilgenberg (Malsfeld, Germany). For this
perfusion system, the open-tip recordings of the liquid junction
potentials revealed that complete exchange of solution occurred within
80-250 µsec.
When studying the effect of CPZ on GABA-evoked currents, GABA was
applied in the presence of CPZ, after pre-equilibration at the same CPZ
concentration for at least 4 min.
Analysis. The decaying phase of the currents was fitted with
a biexponential function in the form:
|
(1)
|
where Afast,
Aslow are the fraction of the fast and slow
component, respectively, As is the steady-state
current, and 
fast and slow are the fast
and the slow time constants. In the case of analysis of normalized
currents, the fractions of kinetic components fulfilled the
normalization condition: Afast + Aslow + As = 1. When
fitting the IPSCs or deactivation currents, the steady-state component
As was omitted.
To compare the time duration of the agonist presence in the case of
different patterns of concentration time course, the effective exposure
E was defined as following:
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(2)
|
where c(t) is the concentration and t = time. In the case of the square-like (with amplitude A and
time of application tappl) and
exponential time course (A · exp(t/)) the
effective exposure would be: A · tappl and A · , respectively.
The kinetic modelling was performed with the Bioq software kindly
provided by Dr. H. Parnas from the Hebrew University, Jerusalem. The
Bioq software converted the kinetic model (Fig. 8) into a set of
differential equations and solved them numerically assuming, as the
initial condition, that at t = 0, no bound or open
receptors were present. Various experimental protocols were
investigated by "clamping" the agonist concentration time course in
the form of square-like pulses (ultrafast perfusion experiments) or of an exponentially decaying function (to model the synaptic clearance). The solution of such equations yielded the time courses of
probabilities of all the states assumed in the model. The fit to the
experimental data were performed by optimizing the values of rate
constants for a given experimental protocol.
Data are expressed as mean ± SEM. Student's unpaired
t test was used for comparison of data.
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RESULTS |
CPZ reduces the amplitude of mIPSCs
mIPSCs were recorded in the whole-cell configuration of the
patch-clamp technique, at a holding potential of 70 mV, in the presence of TTX (1 µM) and kynurenic acid (1 mM) in 9 of 15 cells. mIPSCs were
GABAA-mediated because they were reversibly blocked by
bicuculline (10 µM, data not shown). mIPSCs had a mean
amplitude of 77.2 ± 15 pA (n = 9). CPZ applied
in the bath at concentrations of 10-100 µM induced a
dose-dependent decrease in mIPSC amplitude (Fig.
1A,B).
As shown in the graph of Figure 1E, in the presence of 10, 30, and 100 µM CPZ, the synaptic currents were
decreased to 0.74 ± 0.1, 0.69 ± 0.12, and 0.09 ± 0.07 (n = 5) of the control value, respectively.
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Figure 1.
CPZ decreases the amplitude and accelerates the
decay of mIPSCs. A, Average of 57 mIPSCs recorded from
the same cell (Vh = 70 mV). The decaying
phase of the trace was fitted with Equation 1 (Materials and Methods),
assuming As = 0. The values of time
constants are specified below the trace.
B, Average of 76 mIPSCs recorded in the presence of 30 µM CPZ (from the same cell as in A).
Traces were fitted with Equation 1. C, Superimposed,
normalized current records shown in A (thick
line) and B (thin line). A clear
acceleration of the decaying phase in the presence of 30 µM CPZ is evident. D, The normalized
traces shown in C are presented in an expanded time
scale. Note that CPZ had no effect on the onset of mIPSC.
E, Dose dependence of the effect of CPZ on mIPSC
amplitude (open columns) and area (hatched
columns). Amplitudes and areas were normalized (dotted
line) to the control values. The CPZ concentrations are
indicated above the bars. In this and in the following
figures, bars above the columns represent SEM.
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CPZ accelerates the decay of mIPSCs
The effectiveness of synaptic inhibition does not depend only on
the amplitude but also on the time course of synaptic currents. Especially in the case of IPSC, the decay kinetics is particularly important for temporal integration of synaptic signals as the decaying
phase of IPSCs are known to be long lasting, often exceeding hundreds
of milliseconds (Edwards et al., 1990; Pearce, 1993; Puia et al., 1994;
Jones and Westbrook, 1995).
Similar to previous reports (Edwards et al., 1990; Jones and Westbrook,
1995), the decay of mIPSCs recorded in our experiments could be well
fitted with the sum of two exponentials (Fig. 1A, Table 1). To compare the decay kinetics
in control conditions and in the presence of CPZ, the synaptic events
were averaged, and the traces were fitted with theoretical curves (Fig.
1A,B). When superimposing the
normalized traces (Fig. 1C), a clear acceleration of the
decaying phase in the presence of 30 µM CPZ is evident. This effect was dose-dependent, being negligible at 10 µM, but gradually increased at higher CPZ concentrations
(Table 1). The acceleration of mIPSC decay by CPZ was a consequence of
a decrease in the area and in the time constant of the slower component
(Table 1). Thus, a decreased amplitude and accelerated decay of mIPSCs are the two factors whereby CPZ reduces the charge transfer associated with a single mIPSC (charge transfer calculated as the integral of
mIPSC). As shown in Figure 1E, the CPZ-induced
increase in the decay rate of mIPSCs contributes substantially to the
decrease in the mIPSC area (charge transfer), especially at higher
(
30 µM) concentrations of CPZ.
We have also examined whether CPZ affects the rise time of mIPSCs. We
found that in control conditions and in the presence of CPZ (10-100
µM) the 10-90% rise time was not significantly (p > 0.5) different (Fig. 1D,
Table 1).
CPZ affects whole-cell GABA-induced currents
To further explore the mechanisms underlying CPZ-induced
decrease in the amplitude and acceleration of mIPSC decay kinetics, the
effect of this drug was studied on the whole-cell currents evoked by
applications of GABA from a multibarrel perfusion system (see Materials
and Methods). These experiments were performed on the same neurons from
which mIPSCs were recorded. The rise time of the whole-cell current
induced by high doses of GABA (1 mM) was in the range of
15-30 msec. Figure 2A
shows a typical example of the current evoked by GABA (1 mM, applied for 1.2 sec) at a holding potential of 30 mV.
Application of the same GABA concentration in the presence of 100 µM CPZ led to a significant decrease in the amplitude and
in the onset rate of GABA-evoked current (Fig.
2B,C). On average, the amplitude of
GABA-induced currents in the presence of CPZ (100 µM) was
0.73 ± 0.03 of the control value (Fig. 2D).
This effect was independent of GABA concentration in the range from 5 to 1000 µM, suggesting a noncompetitive type of block.
The most striking discrepancy between the effect of CPZ on mIPSCs and
on the currents evoked by exogenous GABA application was the amount of
their block by CPZ. As shown in Figure 2D, whereas 100 µM CPZ reduced the mIPSCs amplitude by ~90%, the
same CPZ concentration induced only ~30% depression of GABA-evoked
responses. CPZ (100 µM) increased also the time-to-peak
of GABA-evoked (1 mM) current from 21 ± 3.4 to
54 ± 7.4 msec (Fig. 3C;
n = 3). However, it is known that when GABA (1 mM) is applied with a fast perfusion system (Maconochie et
al., 1994; Puia et al., 1994), the time-to-peak is close or <1 msec.
This indicates that the rise time observed in our experiments reflects
mainly the speed of drug application. The estimated rise time of the
agonist concentration in the synaptic cleft (Clements, 1996) is at
least two orders of magnitude faster than that obtained by our
perfusion system. It is thus likely that the interaction of CPZ with
mIPSCs occurs at a much faster time scale and for this reason cannot be
reproduced by a conventional perfusion system. To test this
possibility, a series of experiments was performed using an ultrafast
perfusion system.
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Figure 2.
CPZ affects the whole-cell currents evoked by GABA
using the multibarrel perfusion system. A,
B, Example of whole-cell currents evoked by GABA (1 mM, bars) in control
(A) and in the presence of 100 µM
CPZ (B) at Vh = 30
mV. C, Same traces as in A (thick
line) and B (thin line) are
normalized and superimposed. Note the slower rate of current onset in
the presence of CPZ. D, CPZ affects the amplitude of the
whole-cell GABA-evoked currents to a much smaller extent than that of
mIPSCs (0.73 vs 0.09 of the control values, respectively).
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Figure 3.
Currents evoked by brief GABA applications, using
the fast perfusion system, mimic mIPSCs. A, Example of
current evoked by brief GABA application (1 mM for 2 msec,
see inset above the current trace). The decaying phase
of GABA-evoked current was fitted with Equation 1 (Materials and
Methods), assuming As = 0; the values of
time constants are specified below the trace.
B, Example of a mIPSC with a biexponential curve fitting
(Eq. 1, As = 0) to the decaying phase.
C, Example of the rising phases of currents evoked by
different GABA concentrations. The duration of GABA application was
long enough to reach the peak current value. The rising kinetics
saturates at ~3 mM GABA.
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GABA applied by a ultrafast perfusion system mimics
synaptic currents
The open-tip recordings of the liquid junction potentials showed
that using the ultrafast perfusion system (see Materials and Methods),
a complete exchange of solution occurred within 80-250 µsec. These
values are similar to those reported by Jonas (1995) and Maconochie et
al. (1994). Figure 3A shows a typical current response to 1 mM GABA applied for 2 msec to an excised patch at a holding
potential of 70 mV. Similar to what observed for mIPSCs, the currents
were characterized by a biphasic decay described by two time constants
(Table 2). The fast decay time constant
(fast) of mIPSCs and of GABA responses was not
significantly different (p > 0.3; Tables 1, 2).
However, the time constant of the slower component
(slow) was significantly longer
(p < 0.05) in the case of currents evoked by
fast GABA applications. The latter finding is similar to the
observation of Jones and Westbrook (1995) who also found that the decay
of GABA responses is slower than that of the synaptic currents. The
rate of the rising phase was dependent on GABA concentration and
similar to what was observed by Jones and Westbrook (1995), saturated
at ~3 mM GABA (Fig. 3C). Thus, the above
analysis shows that currents evoked by fast GABA applications reproduce
qualitatively the major kinetic properties of mIPSCs. Taking this into
account, such GABA responses were used as a tool to explore the
mechanism of the effect of CPZ on the kinetics of mIPSCs.
The currents elicited by fast GABA applications (1 mM GABA
for 2 msec) were also recorded in the presence of CPZ. Figure
4A shows typical
responses to fast GABA applications in control conditions in the
presence of 30 and 100 µM CPZ. On average, CPZ (30 and 100 µM) reduced the current amplitude to 0.71 ± 0.15 (n = 10) and 0.32 ± 0.09 (n = 17) of the control values (Fig. 4C), respectively. As
shown in Figure 4C, the percentage of the amplitude
reduction by 30 µM CPZ was very similar to that observed
for mIPSCs but with 100 µM CPZ, the reduction of the
current was smaller. However, with respect to the block of mIPSCs by
CPZ, a much better agreement was obtained when mIPSCs were modelled by
current responses evoked by fast GABA applications rather than by
slower multibarrel perfusion (compare Figs. 2D,
4D). This observation indicates that indeed the
effect of CPZ on GABAA receptors strongly depends both on speed and time duration of agonist application.
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Figure 4.
CPZ decreases the amplitude and accelerates the
decay kinetics of the currents evoked by brief applications of GABA.
A, Examples of currents evoked by brief GABA pulses (1 mM for 2 msec, inset above the current
traces) in control conditions (0 CPZ) and in the presence of 30 and 100 µM CPZ. B, Superimposed, normalized
current records shown in A (thick line,
control; thin line, in the presence of 100 µM CPZ). A clear acceleration of the decaying phase in
the presence of CPZ is evident. C, CPZ decreases the
amplitude of currents evoked by brief GABA applications (hatched
bars) in a dose-dependent manner. The comparison is made with
the effect of CPZ on mIPSC amplitude (open bars). While
at 30 µM, the effect of CPZ on amplitude of mIPSCs and of
GABA-evoked currents is similar; at 100 µM CPZ, mIPSC
amplitude is affected to a larger extent. D, Dose
dependence of the effect of CPZ on area of GABA-evoked currents
(hatched columns) and of mIPSCs (open
columns). Both at 30 and 100 µM, CPZ affects the
area of mIPSC to a larger extent because of a stronger effect of CPZ on
mIPSC decay kinetics (at 30 µM, CPZ had almost no effect
on decay of evoked currents). In C and D,
the values are normalized to the controls (dotted
lines).
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CPZ affected also the decay of currents evoked by fast GABA pulses. At
10 and 30 µM CPZ, the effect was negligible, but at 100 µM a clear acceleration of the decay kinetics was
observed (Fig. 4B, Table 2) reproducing qualitatively
the effect of CPZ on the mIPSC decaying phase.
What is the mechanism whereby CPZ affects so strongly mIPSCs and
currents evoked by fast GABA applications? A hint to understand this
problem came from the analysis of the effect of CPZ on rise time of
currents evoked by brief pulses of GABA. As shown in Figure 5A, the onset of the current
evoked by GABA (1 mM) in the presence of 100 µM CPZ was clearly slower than in control. On average, in
control conditions, the 10-90% rise time was 1.12 ± 0.08 msec (n = 10), and in the presence of 100 µM
CPZ the rise time was significantly slower (1.82 ± 0.06 msec;
n = 10; p < 0.05). The effect of CPZ
on the rise time of GABA-evoked currents was dose-dependent (Table 2).
The observed decrease in the onset rate of GABA-evoked currents
indicates that CPZ interferes with the activation kinetics of
GABAA receptor. In terms of processes underlying receptor
activation, this means that CPZ affects binding/unbinding rate
constants and/or the transition rates between bound and open states.
For instance, if CPZ reduces the binding rate, the presence of the
agonist in the synaptic cleft could be too short to activate the
receptors to the same extent as in control conditions. However,
prolongation of the receptor exposure to agonist would increase the
chance of forming bound receptors giving rise to larger currents. This hypothesis was tested by applying GABA pulses of different time duration using the fast perfusion system. As shown in Figure
5B, in the presence of 100 µM CPZ, the
amplitude of GABA-evoked currents clearly increased with time of GABA
application (six experiments). On the contrary, in control conditions,
application of GABA for time ranging from 1 to 5 msec did not modify
either the rise time or the current amplitude (Fig. 5C;
seven experiments). These results confirm that indeed CPZ interferes
with the activation kinetics of GABAA receptors and that
such effect is sufficient to decrease GABA-evoked current given that
the agonist application is sufficiently short.
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Figure 5.
CPZ affects the rising phase of currents evoked by
brief pulses of GABA. A, Examples of normalized currents
evoked by brief GABA applications (1 mM GABA for 2 msec) in
control conditions (thick line) and in the presence of
100 µM CPZ (thin line). A clearly slower
rate of current onset is seen in the presence of CPZ. B,
Currents evoked in the presence of 100 µM CPZ by 1 mM GABA pulses of different time duration (1, 2, and 5 msec). The current amplitude and the time-to-peak increased with time
of GABA application. C, Currents evoked by GABA (1 mM) were applied for the same time intervals as in
B (1, 2, and 5 msec) but in the absence of CPZ. Time
duration of GABA pulse within this range had no effect on the time
course of the currents. D, Currents evoked by 300 µM GABA pulses of 1, 2, and 5 msec duration. At this GABA
concentration, the time duration of GABA pulses had qualitatively
similar effect on the rising phase as in the case of 1 mM
GABA in the presence of 100 µM CPZ
(B). The insets above current
traces indicate the time course of GABA application.
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A similar effect was obtained by lowering the concentration of GABA
from 1 to 0.3 mM (Fig. 5D, four experiments). At
such low concentration, binding of GABA (rate = kon · [GABA]; see also "Modelling"
below) becomes rate limiting giving rise to a slower current rise time.
Thus, when the presence of agonist is too short (e.g., 1 msec at 0.3 mM GABA), binding step is not completed, and, consequently,
a smaller percentage of receptors can reach the open state.
As it will be discussed in details later, the rising phase depends
basically both on binding and unbinding rates and on opening and
closing rates of the bound receptor, and, a priori it is difficult to
discriminate between these two mechanisms. However, the fact that the
binding rate depends on the agonist concentration
(kon · [GABA]), offers the opportunity to
distinguish between these two possibilities. If CPZ lowers the
kon rate constant, its effect on rise time would
be equivalent to lowering the concentration of GABA (as in Fig.
4D). Moreover, the effect of CPZ on rise time should
be compensated by applying a saturating dose of GABA (e.g., 10 mM).
Thus, to test whether CPZ affects the binding kinetics, first 1 mM and then 10 mM GABA pulses were applied in
the presence of 100 µM CPZ. As shown in Figure
6A, application of 10 mM GABA strongly accelerated the rising phase with respect
to 1 mM GABA. Moreover, the time-to-peak of currents evoked
by 10 mM GABA in the presence of 100 µM CPZ
(0.81 ± 0.06 msec; n = 6) and in control conditions (0.76 ± 0.05 msec; n = 5) were not
significantly different (p > 0.3; Table 2; Fig.
6B,C). Therefore, the effect of CPZ on rise
time of GABA-induced currents can be completely compensated by the
increase in GABA concentration. In addition, at variance to 1 mM GABA responses (Fig. 5B), when applying 10 mM GABA pulses of various time duration (1, 2, or 5 msec)
in the presence of 100 µM CPZ, no differences either in
amplitude or in time course were seen (Fig. 6D, three
experiments). Interestingly, on average, the amplitude of currents
evoked by 10 mM GABA in the presence of 100 µM CPZ was only slightly smaller than that induced by the same GABA concentration in control conditions (Fig.
6E). This observation indicates that most of the
observed CPZ-induced decrease in the amplitude of the GABA-evoked
current was caused by a decrease in the binding rate.
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Figure 6.
Effect of CPZ on the rising phase of GABA-evoked
currents can be reversed by saturating GABA concentrations. In
contrast, the CPZ-induced acceleration of deactivation current cannot
be compensated by increasing doses of GABA. A, Examples
of currents evoked by 2 msec pulse of 1 mM (thin
line) and 10 mM GABA (thick line) in
the presence of 100 µM CPZ. B, The rising
phases of currents evoked by 10 mM GABA in control
conditions (thick line) and in the presence of 100 µM CPZ (thin line) have indistinguishable
onset kinetics. C, The current traces shown in
B are plotted in a different time scale. A clearly
faster decay kinetics is seen in the presence of 100 µM
CPZ. D, Currents evoked by 10 mM GABA pulses
applied for 1, 2, and 5 msec in the presence of 100 µM
CPZ. Within this range, time duration of GABA pulse had no effect on
the current time course. In A-D, the
insets above current traces indicate the time course of
GABA application. E, Absolute values of mean amplitudes
of currents evoked by GABA pulses (2 msec) in conditions described
above the columns. In particular, the current amplitudes evoked by 10 mM GABA in the presence and in the absence of CPZ were not
significantly different (p > 0.4).
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Desensitization kinetics
In previous studies, Jones and Westbrook (1995, 1996) have
demonstrated that the desensitization process of GABAA
receptors plays an important role in shaping inhibitory synaptic
currents. These authors proposed that sojourns in the desensitized
states preceding channel reopening effectively prolong the synaptic
currents. Such single-channel openings separated by silent periods
(sojourn in the desensitized state) were also observed in our
experiments (Fig. 7A). This
finding indicates that also, in our case, desensitization participates
in shaping mIPSCs and raises the possibility that CPZ could affect
mIPSC decay kinetics by a modulation of the desensitization process. To
test this hypothesis, long pulses of saturating doses of GABA (10 mM) were applied in the presence and in the absence of 100 µM CPZ. In these conditions, the binding reaction is not rate limiting, and the current time course is supposed to depend only
on the opening and closing rates and desensitization kinetics. As shown
in Figure 7, B and C, the currents evoked by long
pulses of GABA (200 msec) could be well fitted by the sum of two
exponentials (Eq. 1, Materials and Methods), indicating the presence of
two desensitization components (in control: fast = 8.7 ± 0.9 msec, Afast = 0.38 ± 0.03, slow = 263 ± 56 msec, Aslow = 0.35 ± 0.04, As = 0.17 ± 0.05, n = 6; in 100 µM CPZ: fast = 9.4 ± 0.9 msec, Afast = 0.59 ± 0.05, slow = 143 ± 38 msec, Aslow = 0.21 ± 0.02, As = 0.16 ± 0.04). The
time constants of the fast components of the current decay were not
statistically different (p > 0.5), indicating
that the onset rate of the fast desensitization was not affected by
CPZ. There was a difference in the percentage of the slow component of
desensitization in controls and in the presence of CPZ
(p < 0.05). However, because the time constant of slow desensitization is as slow as >150 msec (rate of onset < 0.007 msec
1), it is unlikely that this component
would have any significant impact on the currents evoked
by very brief GABA applications. Consequently, the lack of effect on
CPZ on the kinetics of fast desensitization component indicates that
the observed effect of CPZ on the deactivation is not caused by
modification of the desensitization kinetics.
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Figure 7.
The onset of the fast desensitization component is
not affected by CPZ. A, Example of a current evoked by
GABA pulse (1 mM for 2 msec, see inset above
the current trace) in which single-channel activity can be observed.
During the deactivation phase (after removal of GABA), the
single-channel openings are separated by silent periods indicating
sojourns in a desensitized state. B, C,
Examples of normalized current responses to long (200 msec) GABA
applications (10 mM, solid bars) in control
conditions (B) and in the presence of 100 µM CPZ (C). The decaying phases,
during GABA applications, were fitted with Equation 1 (Materials and
Methods). Time constants are specified below the traces,
and the steady-state fractions of currents
(As) are 0.21 and 0.17 for control
and in the presence of 100 µM CPZ, respectively.
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Kinetic modelling of currents evoked by brief GABA pulses
To provide a better quantitative description of the effect of CPZ
on GABAA receptors, a kinetic model was investigated. The model (Fig. 8A)
proposed by Jones and Westbrook (1995) allows to predict all major
properties of currents evoked by brief GABA applications. However, to
adopt the model to our experimental data, the values of the rate
constants were readjusted. The main difference between the prediction
given by Jones and Westbrook's parameters and our experimental data
were the rise time of the responses evoked by brief GABA (1 mM) pulses (~2 msec, Jones and Westbrook's model;
1.12 ± 0.08 msec, our experimental data). Moreover, using their
parameters, an increase in amplitude by ~10% was predicted when
increasing the time of GABA (1 mM) application from 1 to 5 msec. This finding is contrary to our experimental data, as shown in
Figure 5C, variation of application time within this interval did not lead to any significant change either in amplitude or
in the time course of GABA-evoked currents. A good reproduction of the
rising phase kinetics (Fig.
9A,B)
was obtained by increasing the binding rate constant
(kon) and the rate of transition from doubly bound to open state (
2) to the values
indicated in Figure 8B. The decaying phase was well
reproduced by setting the unbinding rate koff
and the rate constants governing the fast desensitization kinetics
(d2, r2) as shown in Figure
8B. In the simulations of responses to fast GABA
applications (1 mM; 1 msec) using the values of
parameters as indicated in Figure 8B, the occupancy of the singly bound open state (AR*) was very small (<2%).
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Figure 8.
Quantitative model of GABAA channel
gating (from Jones and Westbrook, 1995). A, Scheme of
transitions available for the channel. It is assumed that the receptor
R has two independent binding sites for the agonist
A (bound states: AR,
A2R). The channel may reach the
open states (AR*,
A2R*) both from singly and doubly
bound states. The model postulates also the singly and doubly bound
desensitized states (AD,
A2D). B, Values of
the rate constants reproducing the current responses to GABA in control
conditions and in the presence of 100 µM CPZ.
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Figure 9.
Kinetic modelling of current responses to brief
pulses of GABA in control conditions and in the presence of CPZ.
Decrease in the binding rate kon and
increase in the unbinding rate koff
reproduces qualitatively the effects of CPZ. A, Control
responses evoked by 1 msec pulses of GABA at different concentrations.
For these simulations, the following values of rate constants were
assumed: kon = 12 mM/msec,
koff = 0.25 msec1,
1 = 0.2 msec1, 1 = 1.1 msec1, d1 = 0.013 msec1, r1 = 0.00013 msec1, 2 = 8 msec1, 2 = 0.285 msec1, d2 = 1.7 msec1, and r2 = 0.04 msec1. B, Same traces shown in
A in an expanded time scale. The onset of GABA responses
saturates at ~3 mM GABA. C, Modelling of
the effect of CPZ on GABA-evoked currents by decreasing
kon rate constant
(kon = 1.5 msec1, other
kinetic parameters are unchanged, thin line).
Thick line, control (rate constants as indicated for
simulations shown in A and B). The
identical time course to the control (1 mM GABA for 1 msec)
was obtained when applying 8 mM GABA (1 msec) assuming
kon = 1.5 msec1 because
in these conditions kon · [GABA] is
equal to that in control conditions (kon = 12 msec1, [GABA] = 1 mM).
D, The same traces as in C, after
normalization and in an expanded time scale. A clear decrease in the
onset rate as well as a moderate acceleration of the decay is seen when
kon = 1.5 msec1.
However, an increase in GABA concentration (to 8 mM),
reverses both effects. For control
PAR*/Popen = 0.017 and in the case of kon = 1.5 (other
parameters unchanged),
PAR*/Popen = 0.107, where PAR* = maximum open probability of singly
bound open state (AR*) and Popen = total
maximum open probability. E, Modelling the effect of CPZ
by decreasing the binding rate (to kon = 1.5 msec1) and by increasing the unbinding rate (to
koff = 0.5 msec1).
Thick line, Control (1 mM GABA for 1 msec).
Thin line, (kon = 1.5 msec1, koff = 0.5 msec1, other parameters unchanged, 10 mM GABA for 1 msec), in this case, the amplitude is very
close to that in control, but the decay is clearly faster. Thick
gray line, (kon = 1.5 msec1, koff = 0.5 msec1, other parameters unchanged, 1 mM GABA for 1 msec). F, The same traces as
in E but normalized and in an expanded time scale.
Control and response to 10 mM GABA application at
kon = 1.5 msec1 and
koff = 0.5 msec1 have
very similar rise time, but the latter has faster decay. One
micromolar GABA application (1 msec) at
kon = 1.5 msec1 and
koff = 0.5 msec1 has
slower rise time and faster decay than control. Moreover, at
(kon = 1.5 msec1,
koff = 0.5 msec1) 1 mM GABA, response has faster decay than that to 10 mM GABA. The values of
PAR*/Popen
are: 0.017 (control), 0.014 (kon = 1.5 msec1, koff = 0.5 msec1, 10 mM GABA), 0.11 (kon = 1.5 msec1,
koff = 0.5 msec1, 1 mM GABA). In B, D,
F, insets above the curves indicate the
time course of the agonist.
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Kinetic modelling of currents evoked by brief GABA pulses in the
presence of CPZ
The model simulations were also used to explore the mechanisms
underlying the observed effects of CPZ. The main differences between
the responses recorded in the presence of CPZ and control ones were:
(1) slower rise time, (2) faster deactivation kinetics, and (3) lower
amplitude. As already mentioned, a decrease in the rate of onset of the
GABA-evoked current could potentially involve modifications in:
kon, koff,
2 and 2. As shown in Figure
9C,D, lowering kon is
sufficient to predict a decrease both in amplitude (Fig. 9C)
and in the rate of onset (Fig. 9D). Because 2
and 2 are assumed not to depend on agonist
concentration, the following observations argue against the possibility
that the observed effect of CPZ on the current onset is related to
modifications of these rate constants: (1) a decrease in the onset rate
of current evoked by 1 mM GABA, observed in the presence of
100 µM CPZ (Fig. 5A), can be reversed by
increasing GABA concentration (Fig. 6A), (2) the
onset kinetics of currents evoked by a saturating GABA concentration (10 mM) in control conditions and in the presence of 100 µM CPZ are indistinguishable (Fig. 6B),
and (3) the currents evoked by 10 mM GABA in control
conditions and in the presence of 100 µM CPZ have very
similar amplitudes (Fig. 6E). In addition, when modelling the effect of CPZ by decreasing 2 (leaving
kon unchanged), it was impossible to reproduce
the increase in current amplitude (Fig. 5B) with duration of
GABA application (simulation not shown). It is worth emphasizing that
although the rate of binding reaction is determined by the reciprocal
of (kon · [GABA] + koff), the kinetics of this process is
dominated by the binding rate both in control conditions and in the
presence of CPZ (kon · [GABA] 
koff at [GABA] 1 mM, see also
below for koff value). In our model, we mimicked
the effect of 100 µM CPZ on the current onset by lowering
the value of kon to 1-2 mM/msec
(Fig. 8B), leaving the values of 2 and
2 unchanged.
In our experiments we have also observed a very pronounced effect of
CPZ on the decay kinetics of the evoked currents (Figs. 4C,
6C). A decrease in kon, which
accounts for the slower current onset and lower peak open probability,
is insufficient to predict such strong acceleration of the current
decay (Figs. 3, 4, 9C,D). Although, as shown in
Figure 9D, a decrease in the binding rate to
kon = 1.5 mM/msec gives rise to an
acceleration of decay, but when increasing GABA concentration, a time
course identical to control is restored. The last prediction results
from the fact that the rate of binding reaction is determined by the
product kon · [GABA] and, obviously, when
multiplying [GABA] by the same factor (8 in Fig.
9C,D) by which kon was
divided, the model resumes perfectly the control conditions. The
acceleration of the decay kinetics obtained at
kon = 1.5 mM/msec results from an
increased probability of singly liganded open receptors (AR*, see
legend of Fig. 9). Because the rate constant d1
is much smaller than d2, the singly bound
desensitized state (AD) cannot contribute to the decay kinetics to the
same extent as A2D. However, in our experiments, the effect
of CPZ on the decay kinetics was present also at saturating GABA
concentration (10 mM, Fig. 6C), indicating that
other factors than kon must be also involved. In
previous investigations, Jones and Westbrook (1995, 1997) pointed out
that the decay kinetics of the deactivation currents strongly depends on the balance between the unbinding rate koff
and the rate constants governing the fast desensitization
(A2D). The reason for such relation is that the slower is
the unbinding rate, the larger is the probability of "visiting" the
desensitized state that, as already mentioned, leads to a prolongation
of the deactivation currents. Thus, it is likely that the accelerated
deactivation, observed in the presence of CPZ, involves a modification
of the unbinding rate koff and/or of the fast
desensitization kinetics. However, our data show that the onset
kinetics of the fast desensitization is not affected by CPZ. In
addition, as already mentioned, the slower desensitization phase is
unlikely to affect the kinetics of the decay currents and for this
reason this component was omitted in our model.
A good prediction of the effect of CPZ on the deactivation kinetics was
obtained by increasing the value of the unbinding rate
koff. As shown in Figure 9, E and
F, an increase in the unbinding rate from
koff = 0.25 msec1 to 0.5 msec1, leads to a strong acceleration of the decay
kinetics. When setting koff = 0.5 msec1 (kon = 1.5 mM/msec), the faster deactivation kinetics is clearly present both in current evoked by 1 and 10 mM GABA (Fig.
9F). Moreover, as seen in Figure 9F, the
currents evoked by 10 mM GABA in control conditions and in
the "presence of CPZ" (kon = 1.5 mM/msec; koff = 0.5 msec1) have almost identical rising phases. These
model predictions basically reproduce the effects of CPZ on current
responses to fast GABA applications observed in our experiments
(compare Figs. 5A,B,
6B with Fig.
9E,F). Thus, the lack of
effect of CPZ on the fast desensitization component and the above model
simulation provide clear, although indirect, evidence that the
mechanism underlying the acceleration of the deactivation kinetics is
an increase in the unbinding rate koff.
In conclusion, the investigations described above show that the
necessary requirement for a qualitative reproduction of the effect of
CPZs on currents evoked by fast GABA application is a decrease in the
binding and increase in the unbinding rate constants kon and koff, respectively.
Kinetic modelling of mIPSCs in control conditions and in the
presence of CPZ
In the previous sections we have pointed out that the currents
evoked by brief pulses of GABA closely resembled mIPSCs and that the
effects of CPZ on these currents were qualitatively similar. There
were, however, some differences: (1) CPZ had no effect on the rising
phase of the mIPSC but strongly affected the onset of the
evoked currents and (2) at 100 µM concentration, CPZ
decreased the amplitude of the synaptic currents to a larger extent
than that of the evoked ones. It is possible that a source of these discrepancies is that the time course of the agonist applied using our
perfusion system differs from that in the synaptic cleft. Although the
time course of applied GABA concentration is expected to be of nearly
rectangular shape, the time evolution of agonist in the synapse is
different. The simulations as well as experiments (for review, see
Clements, 1996) indicate that in the synapse, the onset of agonist
concentration is very fast (time-to-peak ~101
µsec), and its clearance is characterized predominantly by a time
constant in the range between 5 × 101 and
102 µsec (Clements, 1996; Silver et al., 1996).
Because no perfusion system is able to apply the agonist with such
kinetics, the model simulations offer a unique opportunity to explore
the effects of CPZ on mIPSCs by extrapolating our experimental findings
to the situation in the synapse. To simulate the synaptic response, the
time course of the agonist was modelled by an exponential function:
A · exp(t/), where A is the
maximum value of the agonist concentration and , the time constant.
The time constant was chosen to be 100 µsec because, as it will
be discussed later, a slower time constant of the agonist clearance
(e.g., 200 or 300 µsec) would lead to a clear decrease in the
"mIPSC" onset rate in the presence of CPZ (not seen in the
experiments, Fig. 1D). This value of agonist decay
rate is in agreement with studies on the time course of agonist in the
synapse (Clements, 1996; Silver et al., 1996). The peak concentration
A = 3 mM was chosen to assure the
saturation of mIPSC. For instance, when choosing A = 1 mM, the maximum open probability was
<0.4, and when increasing A, the maximum
Popen increased substantially, reaching
saturation at A 3 mM
(Popen ~0.78 at saturation). Thus, these
simulations indicate that the peak synaptic concentration is at least 3 mM, as it is known that GABA released in the synapse is
sufficient to saturate the amplitude of mIPSC (Mody et al., 1994). As
shown in Figure 10, A and
B, the synaptic currents modelled by "exponential agonist
application" with parameters A = 3 mM and
= 100 µsec had the rise time and decay kinetics almost
indistinguishable from those obtained when simulating responses evoked
by 1 msec pulses of 3 mM GABA. There was only a small
difference in the maximum open probability (<5%, Fig.
10B).
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Figure 10.
Kinetic modelling of mIPSCs in control
conditions and in the presence of CPZ. The synaptic current is modelled
by current response to "exponential application": A
· exp(t/), A = 3 mM, = 0.1 msec. A, B,
Comparison of mIPSC (thick line) to response to GABA
pulse (thin line, 3 mM for 1 msec). The
decaying (A) and rising phases
(B) of the two currents are very similar. The
peak open probability for mIPSC is slightly (<5%) smaller that that
for the response elicited by 1 msec GABA pulse. C,
Comparison of the effect of CPZ on mIPSC and on responses evoked by
GABA pulse. Thick line, Control mIPSC; thick gray
line, mIPSC in the presence of CPZ
(kon = 1.5 msec1,
koff = 0.5 msec1);
thin line, response to GABA (1 mM for 1 msec) in the presence of CPZ. The CPZ treatment diminishes the mIPSC
amplitude to a larger extent than that of current evoked by 1 msec GABA
pulse. D, The same traces as in C after
normalization. CPZ accelerates the decay both of mIPSC and of response
to 1 msec GABA pulse. In the case of mIPSC, the decay acceleration is
slightly larger than that of response to GABA pulse. The values of
PAR*/Popen
are: 0.009 (control mIPSC), 0.25 (mIPSC in the presence of CPZ:
kon = 1.5 msec1,
koff = 0.5 msec1),
0.11 (kon = 1.5 msec1, koff = 0.5 msec1, 1 mM GABA), where
PAR* = maximum open probability of singly bound open state
(AR*) and Popen = total maximum open
probability. E, The same traces as in D
in an expanded time scale. The slowest rate of onset is predicted for
GABA responses (1 mM for 1 msec) in the presence of CPZ.
The rise time of mIPSC is only slightly affected by CPZ.
B, E, Insets above the
traces indicate the time course of agonist in the case of mIPSC
(thick line) and GABA application (thin
line).
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As discussed above, the effect of CPZ was simulated by decreasing the
binding rate kon and by increasing the unbinding
rate koff (as indicated in Fig.
8B). As shown in Figure 10C, the CPZ treatment results in a strong reduction in mIPSC amplitude, notably larger than in the case of effect of CPZ on the evoked currents. This
prediction is in agreement with experimental observation that CPZ (100 µM) diminishes mIPSCs amplitudes to a larger extent than
that of the evoked currents (Fig. 4C). The simulation of the
effect of CPZ on mIPSCs reproduced also the acceleration of the current
decay kinetics (Fig. 10D). Moreover, in agreement
with the experimental data, in the presence of CPZ, the acceleration of
deactivation is stronger for mIPSCs than for the currents evoked by
brief pulses of GABA (Fig. 10D). This difference is a
consequence of a larger proportion of singly bound open states (AR*) in
the case of mIPSCs (0.25 vs 0.11; Fig. 10, legend). Such
larger percentage of singly bound states in mIPSCs results from a
shorter exposure of receptors activated by "synaptic GABA
application" (Fig.
10B,D,E, insets). Moreover, as shown in Figure 10, E and
F, in the case of "synaptic" time course of the agonist,
CPZ affected the rise time kinetics to a much smaller extent when
compared with currents evoked by brief agonist application. However,
when modelling the synaptic current, assuming slower time constant of
agonist decay (e.g., 200 or 300 µsec), the model would predict a
stronger effect of CPZ on the mIPSC rise time (similar to the effect of CPZ on the rise time of current evoked by 1 msec GABA application, data
not shown) contrary to experimental evidence.
Thus, our analysis provides evidence that the differences between the
effect of CPZ on mIPSCs and on the evoked currents are mostly caused by
a different time course of agonist concentration in the two situations.
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DISCUSSION |
The major finding of the present work is that CPZ, a widely used
antipsychotic drug, strongly affects the GABA-mediated inhibitory synaptic transmission by decreasing the amplitude and by accelerating the decay of mIPSCs. We provide evidence that these effects are caused
by CPZ-induced changes in the binding and unbinding kinetics of
GABAA receptors. Interestingly, our data show that the
effects of CPZ on mIPSCs could be obtained at micromolar concentrations similar to those attained in the brain of psychotic patients (May and
Van Putten, 1978).
A noncompetitive block of GABAA receptors by CPZ has been
proposed by Zorumski and Yang (1988). The EC50 for CPZ
reported by these authors (2.6 mM) was much larger than
that obtained in our experiments even when using slower multibarrel
perfusion system (30% of block by 100 µM CPZ indicates
EC50 ~230 µM). The source of this
discrepancy is not clear. The limited speed of drug application used by
Zorumski et al. (1988) (as in the case of the multibarrel system) did
not allow to elucidate the mechanisms of CPZ action. By using the fast
perfusion system, we were able to observe the effects of CPZ with a
much better time resolution, making it possible to get an insight into
the mechanisms underlying the effects of CPZ. The modification of the
kinetics of the mIPSCs was clearly of postsynaptic origin because this
drug had a similar effect on the responses evoked by fast GABA
applications to outside-out patches. The present data as well as the
model simulations indicate that the decrease in the binding rate
kon and the increase in the unbinding rate
koff are sufficient to reproduce qualitatively the postsynaptic effects of CPZ on mIPSCs. In particular, the postulated decrease in kon by CPZ would slow
agonist binding, making this process rate limiting for channel
activation. Consequently, in conditions of very short exposure of
GABAARs to GABA (especially in the case of mIPSC), a lower
percentage of receptors would get bound and reach the open state.
Strong acceleration of mIPSC decay kinetics is attributed mainly to
CPZ-induced increase in the unbinding rate koff.
According to the model (Fig. 8A), the increase in
koff leads to a decrease in the probability of
visiting the desensitized states (not affected by CPZ), shortening thus
the deactivation process (Jones and Westbrook, 1995, 1997). Thus, it
seems that the mIPSC inhibition by CPZ is not a consequence of a direct
block of channel pore by the drug but rather results from an
"upsetting" of kinetics of the receptor, probably caused by an
allosteric modulation by CPZ. An occlusion of channel pore by CPZ, if
present, seems to be negligible because the current amplitudes elicited by saturating GABA concentration in control conditions and in the
presence of CPZ are not significantly different (Fig.
6E). In addition, the observed mIPSC inhibition by
CPZ does not fulfill the criteria of a classical competitive block. In
the case of such antagonism, the receptor before it is activated, must
dissociate the blocker molecule. We could suppose that the CPZ-induced
decrease in the onset rate of GABA responses reflects a delay caused by dissociation of the CPZ molecule from the GABA-binding site. However, in classical mechanism of competitive block, antagonist dissociation rate does not depend on the agonist concentration. Thus, the fact that
the effect of CPZ on rise time kinetics can be compensated by
increasing dose of GABA (Fig. 6A,B)
argues against any significant contribution of competitive
antagonism to the observed mIPSC inhibition by CPZ.
In our simulations we used the model (Fig. 8A)
previously proposed by Jones and Westbrook (1995). However, as
described in Results, the values of the rate constants were changed to
obtain a better fit to our experimental data. The most apparent was the difference between the rate constants determining the rising kinetics (kon, 2) reflecting
faster current onset in our experiments. In the study of Jones and
Westbrook (1995) (see Fig. 2A), the rise time
of the responses to 1 and 3 mM GABA were 3-4 msec and 1-2
msec, respectively, whereas in our experiments these values were 1.12 msec and 0.76 msec. This discrepancy may reflect different receptor
kinetics in the two preparations and possibly a difference in speed of
agonist application.
An interesting prediction of our model is that the effect of CPZ on
mIPSCs and on currents evoked by brief GABA applications is associated
with an increased proportion of monoliganded open states during current
activation. This is a consequence of a slower activation kinetics while
the agonist application remains short. As described in Results, the
model simulations indicate that such increased proportion of
monoliganded open states accelerates the deactivation kinetics because
of a low probability of entering the singly bound desensitized state
(AD). In our experiments, we have observed that CPZ affects the mIPSC
decaying phase to a larger extent than that of the evoked currents [30
µM CPZ caused a strong acceleration of mIPSC decay (Fig.
1), whereas its effect on GABA-evoked currents was negligible (Table
2)]. A possible explanation of this difference could be a larger
proportion of monoliganded open states during activation of mIPSC
caused by much shorter receptor exposure to agonist in the case of the
synaptic current.
CPZ effect reveals fast GABA clearance during mIPSC
The analysis of the effect of CPZ on mIPSCs and on evoked currents
provides evidence that the agonist clearance is very fast (~100
µsec). The key indication was the concomitant observation of a
stronger CPZ effect on amplitude and smaller effect on the rising rate
of mIPSC with respect to the currents evoked by brief GABA pulses. This
indicates that during mIPSC, the presence of GABA is so short that in
the case of slower receptor activation (caused by CPZ) less receptors
activate, but still no effect is seen on the mIPSC onset. The above
indications are nicely confirmed by the model simulations showing that
for slower values of GABA clearance, the effect of CPZ on mIPSCs
should be associated with a decrease in mIPSC rising rate (not observed
in experiments). Thus, the effect of CPZ provides a tool to reveal
kinetics of agonist clearance during GABAergic synaptic transmission.
Physiological implications from modelling mIPSCs
Although the fast perfusion system is an excellent and, so far,
unique tool to mimic the synaptic events, the comparison of responses
evoked using this technique to the synaptic ones should be done with
caution. The main reason for possible discrepancies is that the
synaptic agonist time course is still faster than that obtained with
fast perfusion. For instance, we suggest that the differences in the
effect of CPZ on mIPSCs and on evoked currents are consequences of
faster agonist clearance during mIPSC.
Our data indicate that the peak GABA concentration in the synaptic
cleft is at least 3 mM. This is supported by the
observations that the rise time of currents evoked by fast GABA
applications saturates at ~3 mM and that this maximum
rising rate is very similar to that of mIPSCs (Table 1, Fig. 3).
However, this peak value of [GABA] differs substantially from those
reported previously (Maconochie et al., 1994, 500-1000
µM; Jones and Wesbrook 1995, 527 µM). This
discrepancy most likely reflects differences in the activation rates of
GABA responses. Maconochie et al. (1994) have observed that the on rate
of the evoked currents reaches saturation already at [GABA] = 1 mM, whereas in our experiments at [GABA] = 3 mM. Jones and Westbrook (1995), reported the IPSC rise time
of ~3 msec (rate of rise, 347 sec1) whereas in
our experiments the time-to-peak was 0.68 ± 0.09 msec. It needs
to be emphasized, however, that also in a number of other studies a
submillisecond rise time of IPSC was reported (Edwards et al., 1990;
Maconochie et al., 1994; Bier et al., 1996; Mellor and Randall, 1998;
Williams et al., 1998).
It seems interesting how sensitive the response of the postsynaptic
receptors is to different patterns of agonist time course investigated
in the present work. The effective exposure E (see Materials
and Methods) for the simulation of synaptic current (A = 3 mM, = 100 µsec) is 10 times smaller than that for
the square-like GABA pulse (3 mM for 1 msec) being 0.3 and
3 mM/msec, respectively. However, the charge transfer
caused by the mIPSC is only slightly smaller (<5%) than that evoked
by square GABA pulse. This suggests that receptors, bearing the
properties predicted by our model, are particularly suitable to
efficiently respond to agonist applications characterized by high peak
concentration and very fast time course. Consequently, such receptors
would assure a good performance of synaptic transmission. It is known that high peak agonist concentration is caused by a very small volume
of the synaptic cleft, and that the fast time course is mainly caused
by diffusion, that at
TOP
ABSTRACT
INTRODUCTION
