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The Journal of Neuroscience, December 1, 2000, 20(23):8618-8627
Zinc Inhibits Miniature GABAergic Currents by Allosteric
Modulation of GABAA Receptor Gating
Andrea
Barberis1,
Enrico
Cherubini1, and
Jerzy W.
Mozrzymas1, 2
1 Neuroscience Program and Istituto Nazionale Fisica
della Materia Unit, International School for Advanced Studies (SISSA),
34014 Trieste, Italy, and 2 Department of Biophysics,
Wroclaw University of Medicine, 50-368 Wroclaw, Poland
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ABSTRACT |
Zinc is abundantly present in the CNS, and after nerve
stimulation is thought to be released in sufficient quantity to
modulate the synaptic transmission. Although it is known that this
divalent cation inhibits the GABAergic synaptic currents, the
underlying mechanisms were not fully elucidated. Here we report that
zinc reduced the amplitude, slowed the rise time, and accelerated the decay of mIPSCs in cultured hippocampal neurons. The analysis of
current responses to rapid GABA applications and model simulations indicated that these effects on mIPSCs are caused by zinc modulation of
GABAA receptor gating. In particular, zinc slowed the onset of GABA-evoked currents by decreasing both the binding
(kon) and the transition rate from
closed to open state ( 2). Moreover, slower onset
and recovery from desensitization as well as an increased unbinding
rate (koff) were shown to underlie
the accelerated deactivation kinetics in the presence of zinc.
The nonequilibrium conditions of GABAA receptor activation
were found to strongly affect zinc modulation of this receptor. In
particular, an extremely fast clearance of synaptic GABA is implicated
to be responsible for a stronger zinc effect on mIPSCs than on current
responses to exogenous GABA. Finally, the analysis of currents
evoked by GABA coapplied with zinc indicated that the interaction
between zinc and GABAA receptors was too slow to explain
zinc effects in terms of competitive antagonism. In conclusion, our
results provide evidence that inhibition of mIPSCs by zinc is
attributable to the allosteric modulation of GABAA receptor gating.
Key words:
mIPSCs; GABAA receptors; -alanine; fast
perfusion; nonequilibrium conditions; kinetics analysis; hippocampus; cell cultures
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INTRODUCTION |
Among all transition metals, zinc
ions (Zn2+) are particularly abundant in
the brain, where they can be detected with the Timm's sulfide-silver
staining technique (Timm, 1958 ). Using this method, large amounts of
chelatable zinc have been localized in the cerebral cortex and in the
limbic region (Frederickson, 1989). In the hippocampus, this divalent
cation is stored in synaptic vesicles, present on mossy fiber terminals
(Slomianka, 1992; but see, Lee et al., 2000), where it is colocalized
with the putative zinc transporter ZnT-3 (Wenzel et al., 1997) and with
the neurotransmitter glutamate. Stimulation of mossy fibers induces
co-release of glutamate and zinc into the synaptic cleft (Vogt et al.,
2000) where, according to the stimulation intensity, it can reach a
local concentration up to 300 µM (Assaf and Chung, 1984;
Smart et al., 1994). It has been demonstrated that
Zn2+ downregulates and upregulates NMDA
and AMPA receptor-mediated responses, respectively (Peters at al. 1987;
Westbrook and Mayer, 1987; Rassendren et al., 1990). Zinc has been also
shown to inhibit GABAA receptors (Westbrook and
Mayer, 1987) (for review, see Smart et al., 1994). Although there is no
evidence that zinc is co-released with GABA, it is likely that in the
case of sustained nerve activation, Zn2+
spills over from glutamatergic terminals to neighboring GABAergic synapses, leading to inhibition of GABAA
receptor-mediated responses (for review, see Smart et al., 1994). It
should be stressed however that in this case the tonic concentration of
Zn2+ will be much less than the peak local
concentration at the release site. Several hypotheses have been put
forward to explain inhibition of GABAA receptors
by zinc. It has been proposed that zinc affects GABA-induced currents
by reducing the frequency of channel opening (Legendre and Westbrook,
1991), an effect that could be achieved either by a decreased or by an
increased rate constant of GABA binding or unbinding, respectively.
However, these possibilities have been considered unlikely because of
the lack of effects of zinc on GABAAR
single-channel kinetics (Legendre and Westbrook, 1991; Smart, 1992;
Smart et al., 1994). The possibility that binding of
Zn2+ might allosterically trigger a
transition to a long-lived nonconducting state has been suggested
(Celentano et al., 1991; Smart, 1992; Smart et al., 1994; Gingrich and
Burkat, 1998), but the mechanism of such modulatory effect has not been
fully clarified. Alternatively, Zn2+ could
increase the onset of desensitization, a phenomenon that may be
responsible for the acceleration of GABA-induced current deactivation
(Berger et al., 1998). This mechanism however, does not accord with
Jones and Westbrook's (1995) finding that desensitization prolongs the
deactivation process. It seems thus that the mechanism through which
Zn2+ affects GABAA
receptors remains uncertain. In particular, it is not clear whether a
direct block of the channel pore is involved and to what extent
Zn2+ inhibitory action is a consequence of
an allosteric modulation of GABAA receptor kinetics.
The aim of the present work was to determine the mechanisms
whereby zinc affects miniature IPSCs (mIPSCs) in cultured rat hippocampal neurons. We provide evidence that inhibition of mIPSCs by
Zn2+ is caused by allosteric modulation of
GABAA receptor microscopic gating, including
binding, desensitization, and conformational change from bound closed
to bound open state.
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MATERIALS AND METHODS |
Cell culture. Primary cell culture was prepared as
previously described (Andjus et al., 1997). Briefly, postnatal day 2 (P2)-P4 Wistar rats were decapitated after being anesthetized with an intraperitoneal injection of urethane (2 gm/kg). This procedure is in accordance with the regulation of the Italian Animal Welfare Act
and was approved by the local authority veterinary service. Hippocampi
were dissected free, sliced, and digested with trypsin, mechanically
triturated, centrifuged twice at 40 × g, plated in the
Petri dishes, and cultured for up to 14 d. Experiments were performed on cells cultured for at least 7 d.
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, 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. The intrapipette
solution contained (in mM): CsCl 137, CaCl2 1, MgCl2 2, 1,2-bis(2-aminophenoxy)ethane-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 excitatory postsynaptic events. All the experiments
were performed at room temperature (22-24°C).
The current signals were stored on a video recorder following
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, Cambridge, UK) and stored on
the computer hard disk. The data and acquisition software were kindly
given by Dr. J. Dempster (Strathclyde University, Glasgow, UK).
GABA, -alanine, and zinc were applied to excised patches using an
ultrafast perfusion system based on piezoelectric driven theta-glass
application pipette (Jonas, 1995). The piezoelectric translator was
from Physik Instrumente (Waldbronn, Germany), and theta glass tubing
was from Hilgenberg (Malsfeld, Germany). In the case of high
concentration (30 mM) of GABA, the osmolarity was
compensated by omitting glucose; in the case of high
concentration (100 mM) of -alanine, glucose was not
added, and NaCl was reduced from 137 to 100 mM. The
concentration of chloride was still saturating the GABA receptor
channels. The [Cl] in the pipette was adjusted to maintain the
chloride solution symmetrical. The open tip recordings of the liquid
junction potentials revealed that the 10-90% exchange of solution
occurred within 40-80 µsec. The speed of the solution exchange was
also estimated around the excised patch by the 10-90% onset of the
membrane depolarization induced by application of high (25 mM) potassium saline. In this case the 10-90% rise time value (60-90 µsec) was very close to that found for the open tip recordings.
In experiments aiming to study Zn2+
effects on GABAARs, GABA was applied in the
presence of Zn2+ after pre-equilibrating
the receptors with the same Zn2+
concentration for at least 1 min. In some experiments, GABA and Zn2+ were coapplied without
pre-equilibration of GABAA receptors in Zn2+.
Data analysis. Synaptic currents were analyzed with the
AxoGraph 3.5.5 program (Axon Instruments, Foster City, CA). This
program uses a detection algorithm based on a sliding template. The
template did not induce any bias in the sampling of events because it
was moved along the data trace one point at a time and was optimally scaled to fit the data at each position. The detection criterion was
calculated from the template scaling factor and from how closely the
scaled template fitted the data. The threshold for detection was set at
3.5 times the SD of the baseline noise. Using the same program,
the decay time constant of averaged mIPSCs was taken from biexponential
fit of the decay time. The rise time was estimated as the time needed
for 10-90% increase of the peak current response.
The decaying phase of the GABA-evoked currents was fitted with a
biexponential function in the form:
|
(1)
|
where Afast,
Aslow are the fraction of the fast and
slow component, respectively, 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 = 1. In the case of current
responses elicited by long duration GABA pulses ( 50 msec), the
desensitization onset was described by
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(2)
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where As is the steady state current.
Charge transfer (Q) associated with GABA-induced
current, was calculated as the current integral:
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(3)
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where i(t) is the current and t, time. In
the case of mIPSCs, the charge transfer was calculated by integration
of the averaged trace.
Brief (1-2 msec) paired pulses separated by a variable time interval
were used to test whether or not the entrance of bound receptors into
the desensitized state proceeded after the agonist removal. The
parameter R was calculated according to the formula:
|
(4)
|
where I1 is the first peak
amplitude, Iend is
the current value immediately before the
application of the second pulse, and I2 is the second peak amplitude.
During 1-2 msec pulse the onset of the use-dependent desensitization
is minimal. Thus, in the case of continued entrance into the
desensitized state after the first short agonist pulse, the peak of the
second response (I2) was smaller than
the first one resulting in R < 1.
To determine the recovery from desensitization, the time duration of
the first (conditioning) pulse was set sufficiently long to induce the
use-dependent desensitization, and the second (test) pulse was applied
at variable time interval. The extent of recovery was assessed using
Eq. 4, but Iend in this case
corresponded to the value of the current at the end of the conditioning
pulse and I1,
I2 represented the current peaks
evoked by conditioning and test pulses, respectively. For the reasons
explained in detail in Results, the recovery from desensitization was
determined using this protocol only in the case of -alanine.
The kinetic modeling was performed with the Bioq software kindly
provided by Dr. H. Parnas from the Hebrew University (Jerusalem). The
Bioq software converted the kinetic model (see Fig. 7) 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 was performed by optimizing the values of rate
constants. The procedure for the rate constants optimization was based
on the comparison of the time course of recorded currents and that of
simulated responses. As described in detail in Results, specific experimental protocols were used to estimate different sets of rate constants.
Data are expressed as mean ± SEM, and all the values included in
the statistics represent recordings from separate cells. Statistical
comparisons were made with the use of unpaired t test and
Wilcoxon signed rank test (p < 0.05 was taken
as significant).
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RESULTS |
Zn2+ affects the kinetics of mIPSCs
mIPSCs were recorded from cultured hippocampal neurons in the
whole-cell configuration of the patch-clamp technique at a holding potential of 70 mV and in the presence of TTX (1 µM)
and kynurenic acid (1 mM) to block fast sodium spikes and
ionotropic glutamatergic currents, respectively. mIPSCs were
GABAA-mediated because they were reversibly
blocked by bicuculline (30 µM; data not shown). mIPSCs
had a mean amplitude and frequency of 77.6 ± 11.1 pA and 3.1 ± 0.4 Hz, respectively (n = 11).
Zn2+ (30-300 µM)
reversibly reduced the amplitude of mIPSCs and shifted to the left the
cumulative amplitude distribution in a concentration-dependent manner
(Fig. 1A,D). The mean
mIPSC amplitude reduction induced by Zn2+
(100 µM) was 65 ± 5%, n = 5. Zn2+ at concentrations of 30-100
µM did not significantly
(p > 0.5) alter the frequency of mIPSCs (the
mean frequency reduction was 15 ± 13% and 20 ± 19% in 30 and 100 µM Zn2+,
respectively; see also DeFazio and Hablitz, 1998). At higher (300 µM) Zn2+
concentration a significant (p < 0.001)
reduction of 56 ± 9% was detected. As suggested by the
experiments with exogenous application of GABA (see next paragraph), in
Zn2+ (300 µM), the
extent of GABAA receptor inhibition would be so high that a considerable proportion of mIPSCs would fall into an
undetectable level. This would lead to a decrease in apparent mIPSCs
frequency, thus precluding a proper determination of averaged amplitude
and charge transfer values (data not shown in Fig. 1).
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Figure 1.
Concentration-dependent effect of
Zn2+ on amplitude of mIPSCs and kinetics.
A, Average of 64 mIPSCs in control conditions and in the
presence of Zn2+ (100 µM).
B, The two traces shown in A are
normalized and superimposed (thick line, control;
thin line, Zn2+). C,
Normalized mIPSC onset in control conditions and in the presence of
Zn2+ (100 µM). Each trace is the
average of 64 mIPSCs. D, Cumulative amplitude histograms
of mIPSCs in control conditions (thick line) and in the
presence of Zn2+ (100 µM, thin
line). E, Cumulative rise time histograms of
mIPSCs in control conditions and in the presence of
Zn2+ (100 and 300 µM, respectively).
F, Dose-dependent effect of Zn2+ on
mIPSC amplitude (gray columns) and on total
charge transfer (white columns). Amplitudes and total
charge transfers were normalized to control values. In this and the
following figures, error bars indicate SEM. G,
Each column represents the mean (n = 6-10)
10-90% mIPSC rise time value obtained in control
(black) and in the presence of different
Zn2+ concentrations (white).
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The effect of Zn2+ on mIPSC amplitude was
accompanied by a concentration-dependent decrease in the onset rate and
acceleration of the deactivation kinetics (Fig.
1B,C,E,G). The 10-90% mIPSC rise time increased
from 0.68 ± 0.03 msec in control conditions to 0.91 ± 0.02 msec in Zn2+ (100 µM; n = 11; Fig.
1G). In the absence of Zn2+,
mIPSC decay was fitted with two exponentials characterized by fast and slow of
4.7 ± 0.7 msec and 33.7 ± 3.1 msec (n = 10; see also Edwards et al., 1990; Jones and Westbrook, 1995; Mozrzymas et
al., 1999). In the presence of Zn2+ a
clear acceleration of the decay was observed because of a decrease in
the area and in the time constant of the slower component (Table 1). To assess the net
Zn2+ effect on mIPSCs, the total charge
transfer for different Zn2+ doses was
calculated by integrating mIPSCs area. Thus, the total charge transfer
was significantly (p < 0.001) reduced from
1759.5 ± 248.6 nC (n = 10) in control to
369.5 ± 79.6 nC (n = 6; Fig. 1F) in Zn2+ (100 µM).
Zn2+ affects the kinetics of currents evoked by
ultrafast applications of GABA
One of the major problems in determining the mechanisms of drug
action on synaptic currents is that the concentration and time course
of synaptic neurotransmitter cannot be easily manipulated precluding
standard approaches based on constructions of dose-response relationships. A recent report (Mozrzymas et al., 1999) has indicated that GABA transient in the synaptic cleft is extremely fast (hundreds of microseconds) and that the crucial requirement to properly mimic
synaptic currents is to approach the speed of synaptic agonist changes.
This task can be reasonably achieved by ultrafast agonist application
system (Jonas, 1995; Jones and Westbrook, 1995; Tia et al., 1996) and
in our experiments, the 10-90% exchange of solution around the open
tip of a pipette occurred within 40-80 µsec.
Figure 2, A and B,
shows typical current responses evoked by ultrafast application of a
saturating concentration of GABA. Similarly to mIPSCs, GABA responses
were characterized by a rapid rising phase (Fig. 2C,E)
followed by a biphasic decay, having time constant of 4.1 ± 0.5 and 95.1 ± 7.8 msec (fast and
slow, respectively; n = 13).
As in the case of mIPSCs, Zn2+ induced a
dose-dependent reduction of the amplitude and of charge transfer of
GABA-induced currents (Fig. 2A,D).
Zn2+ slowed the rise time and accelerated
their decay kinetics (Fig. 2B,D; Table 1). In
particular, the faster current decay in
Zn2+ (100 µM) was
associated with a decrease in the area and in the time constant of the
slower component (Table 1).
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Figure 2.
Concentration-dependent effect of
Zn2+ on current responses elicited by ultrafast
brief GABA applications. A, Currents evoked by brief (2 msec) GABA pulses (10 mM, upward deflection in the
top trace), in control conditions, and in the presence
of Zn2+ (100 µM). B,
The two traces in A are normalized and superimposed.
C, Normalized onset of current responses elicited by
GABA (3 mM) in control conditions (thick)
and in the presence of Zn2+ (100 µM,
thin). D, Concentration-dependent effect
of Zn2+ on the amplitude (gray
columns) and on total charge transfer (white
columns) of GABA-evoked currents. Amplitudes and total charge
transfers were normalized to control values. E, Each
column represents the mean (n = 13-26) 10-90%
rise time value of currents elicited by GABA (10 mM) in
control (black) and in the presence of different
Zn2+ concentrations (white).
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Zn2+ slows the onset kinetics of
GABA-induced currents
Similarly to what observed for mIPSCs,
Zn2+ decreased the rate of onset of the
currents evoked by rapid GABA application. As shown in Figure
3, the 10-90% rise time of the current
evoked by a saturating concentrations of GABA (10, 30 mM)
in the presence of Zn2+ (100 µM) was significantly (p < 0.001)
slower than in control (0.26 ± 0.01 msec in control vs 0.9 ± 0.1 msec in Zn2+; n = 14). Zn2+-induced reduction in the rate of
current onset persisted when GABA concentration was increased from 10 to 30 mM (Fig. 3A,B).
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Figure 3.
Zn2+ affects the rate of
current onset by decreasing rate constants of binding and
conformational changes. A, Normalized onset responses to
saturating concentration of GABA (10 and 30 mM) in the
absence (thick lines) and in the presence of
Zn2+ (100 µM, thin
lines). A clear slower rate of current onset was seen in the
presence of Zn2+. Note that this effect was not
reversed when GABA concentration was raised from 10 to 30 mM. B, Each column represents the mean
(n = 14-22) 10-90% rise time of currents evoked
by GABA (10-30 mM) in control conditions and in the
presence of Zn2+ (100 µM).
C, Normalized onset responses to nonsaturating
concentration of GABA (300 and 500 µM) in the absence
(thick lines) and in the presence of
Zn2+ (100 µM, thin
lines). D, Normalized current onset to a
saturating concentration of GABA (3 mM, thick
line). Zn2+ (300 µM) slowed
the current onset that was partially restored by increasing GABA
concentration to 30 mM. E, Each column
represents the mean 10-90% rise time of currents evoked either by
GABA (3 mM) in control (n = 6; black)
or by GABA 3 and 30 mM in the presence of
Zn2+ (300 µM, n = 19; white). F, Currents evoked by GABA
pulses (3 mM) of different time duration (1, 2, 5, and 10 msec) in the presence of Zn2+ (300 µM). The current amplitude and rise time increased with
time of GABA application.
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Activation of GABAA receptors (as any other
ligand-gated channel) consists of at least two steps: binding of the
agonist to the receptor and conformational change from bound closed to
bound open state. Thus, the investigation of
Zn2+ action on the rise time kinetics
requires the assessment of Zn2+ effects on
these two GABAAR activation components. For this
purpose, we have used the Jones and Westbrook (1995) model (see Fig. 7 for the scheme), which after optimization of the rate constants, allowed us to properly express our experimental results in terms of
microscopic gating. As predicted by the model, the only process whose
kinetics depends on GABA concentration is the binding step (binding
rate = kon · [GABA]). Thus,
by changing the concentration of applied GABA, we are able to
manipulate this rate constant making it e.g., either rate limiting (at
low [GABA]) or much faster than the conformational transition
(2). Moreover, the zinc effect on binding and
conformational change (kon and
2) are expected to be distinguished on the
basis of their dependence on [GABA]. Thus, the reduction in the
binding rate caused by a decreased kon
value should be compensated by a respective increase in [GABA] while
reduction of 2 would remain unaffected by
[GABA]. As shown in Figure 3, A and B, the rise
times of currents evoked by 10 and 30 mM GABA in
the presence of 100 µM Zn were not different from each other but were significantly slower than those in control conditions. The constant value of rise time at these GABA
concentrations indicates that, in these conditions, the conformational
step (2) is rate limiting. Consequently, the
slower rise time in the presence of Zn2+
provides evidence that this cation slows the rate constant
2. However, it remains to be elucidated
whether Zn2+ affects also the binding
step. For this purpose we have recorded the current responses to rapid
applications of GABA at concentrations at which binding is rate
limiting. Similarly to previous reports (Jones and Westbrook, 1995;
Mozrzymas et al., 1999) we have found that saturation of the rise time
kinetics was achieved at GABA concentration close to 3 mM. The 10-90% rise time of responses evoked by
low concentrations of GABA (0.3 and 0.5 mM) was
strongly concentration-dependent and several fold slower (2.19 ± 0.07 msec, n = 25 and 1.79 ± 0.11 msec,
n = 15, for 0.3 and 0.5 mM GABA, respectively) than that of currents evoked by saturating [GABA] (compare Fig. 3A,C). In the presence of
Zn2+ (100 µM), the
rise time increased to 8.29 ± 0.7 msec, n = 56 and to 6.62 ± 0.4, n = 28 for 0.3 and 0.5 mM GABA, respectively (Fig. 3C). The
fact that both in control conditions and in the presence of
Zn2+ the rise times at 0.3 and 0.5 mM GABA were several times slower than at
saturating [GABA] clearly indicates that at 0.3 and 0.5 mM GABA the binding step is predominant and
therefore Zn2+ effect on the rise time is
mainly caused by Zn2+-induced decrease in
the binding rate. Altogether, the use of low and high [GABA] allowed
to reveal that the mechanism underlying zinc effects on the rise time
kinetics consists of a decrease of both the binding
(kon) and conformational change
(2) rate constants of
GABAA receptors. In the quantitative
determination of Zn2+ effect on the rate
constants using model simulations, 2 was first
determined at high [GABA], and this rate constant was subsequently included in the analysis of the current rise time kinetics at low
[GABA] in which Zn2+ effect on both
kon and 2 has
to be considered. Although for the responses evoked by 3 mM GABA in the presence of 100 µM Zn2+, the
conformational change step was rate limiting (the rise time of
0.95 ± 0.07 msec was not accelerated by increase in [GABA]; compare with Fig. 3B), a higher
Zn2+ concentration (300 µM) caused an increase in the rise time that could be partially restored by increasing [GABA] to 30 mM (Fig. 3D,E). The interpretation of
these findings is that the increase in
Zn2+ concentration to 300 µM reduces the rate of binding to such extent that at 3 mM GABA, the conformational change step
(2) is not any more rate limiting. The
increase in [GABA] to 30 mM accelerates binding
sufficiently to make the conformational transition rate limiting again.
In these conditions (30 mM GABA and 300 µM Zn2+) the rise
time considerably increased in comparison to the case of 100 µM Zn2+ and 30 mM GABA, indicating that the increase in
Zn2+ concentration further reduced the
rate of conformational transition (2). The
described mechanism whereby Zn2+ modulates
the activation kinetics of GABAARs predicts that
this divalent cation may affect the threshold value of GABA
concentration below which the binding step becomes rate limiting. Such
a prediction is borne out by experiments in which currents induced by
varying pulses of GABA (3 mM) were recorded in
the presence of Zn2+ (300 µM). Because, as mentioned, 3 mM GABA is saturating, variation of pulse
duration from 1 to 10 msec did not affect either the onset kinetics or
the amplitude of GABA control responses (data not shown). As shown in
Figure 3F, in the presence of
Zn2+ (300 µM), the
increase in pulse duration resulted in the increase of current
amplitude and rise time. This is a consequence of the fact that at
Zn2+ 300 µM, the
reduction of kon is so strong that the
binding step becomes rate limiting. In addition, in these conditions,
the rate of binding becomes too slow to complete this process within 1 msec (as it took place in the absence of
Zn2+), and for this reason the
prolongation of pulse increases the probability of binding leading to a
larger response.
Zn2+ affects desensitization
of GABAAR
Because it is known that desensitization plays a crucial role in
shaping the deactivation kinetics of GABA-evoked currents, a series of
experiments have been performed to assess the effects of
Zn2+ on this process. To describe the
desensitization onset kinetics, long pulses of saturating GABA (10 mM) were applied in the presence and absence of
Zn2+. Application of GABA for 300 msec
induced a clearly biphasic desensitization described by time constants
of 4.9 ± 0.7 and 125.9 ± 17.9 msec
(A1 = 34 ± 14%;
A2 = 30 ± 2%;
AS = 35 ± 13%,
n = 10; Fig.
4A-C). Because the
slower component is unlikely to significantly affect the time course of
synaptic currents, further analysis has been limited to the rapid one,
which at pulse duration of 50 msec was predominant
(1 = 4.9 ± 0.7 msec; A = 43 ± 2%; AS = 56 ± 2%;
data not shown). Zn2+ slowed the time
constant of the onset and increased the extent of desensitization in a
concentration-dependent manner (at 100 µM,
1 = 22.9 ± 1.2 msec; A = 56 ± 4%; AS = 44 ± 4%).
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Figure 4.
Zn2+ affects desensitization
kinetics of GABA-evoked currents. A, Normalized currents
evoked by GABA (10 mM, for 300 msec) in the absence
(thick line) and in the presence of
Zn2+ (100 µM, thin
line). B, C, Mean (n = 5-11) time constant () desensitization onset and relative area
(A1) of GABA responses (10 mM) evoked in the absence (black) and in the
presence (white) of different Zn2+
concentrations. D, Paired brief (2 msec) GABA pulses (10 mM) elicited at 5 msec interval in the absence
(thick line) and in the presence of
Zn2+ (100 µM, thin
line). Note that Zn2+ strongly accelerated
the recovery of currents evoked by the second pulse. E,
Normalized recovery of the second peak evoked in the absence
(open circles) or in the presence of
Zn2+ (100 µM, closed
circles). Each point represents the mean (n = 5-9). In the inset, a part of the graph has been
enlarged to show effects of Zn2+ at shorter time
intervals.
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Paired pulse protocol is commonly used to study the recovery from
desensitization kinetics. It needs to be emphasized, however, that in
the case in which the unbinding rate
(koff, see scheme in the Fig. 7) is
comparable or slower than the desensitization onset rate
(d2) and opening rate
(2), the experimentally observable recovery
from desensitization (i.e., transition from the desensitized state
A2D to the activatable state
R) would be delayed by multiple entrances into the
desensitized and open states. Thus, in these conditions, the time
course of the second peak recovery would reflect not solely the
recovery from desensitization but a more complex process including
multiple sojourns in the open and desensitized states. Such functional
link between unbinding and desensitization has been also proposed to
underlie the slow deactivation of GABA-evoked responses (Jones and
Westbrook, 1995). In the case of such multiple entrances, it would be
expected that after the agonist removal, a proportion of bound
receptors would temporarily accumulate in the desensitized state. To
test for such possibility, paired responses to brief (1-2 msec) GABA
pulses, separated by variable time intervals were recorded. During such
a short GABA application (1-2 msec), the onset of the use-dependent
desensitization is small, and therefore the difference between the
first and the second peak (peak1-peak2) is an index of the number of
receptors that accumulated in the desensitized state after the first
GABA pulse. As shown in the representative example of Figure
4D, such double-pulse protocol revealed a strong
accumulation in the desensitized state, especially in the absence of
Zn2+. When increasing the interstimulus
interval, a gradual recovery (calculated using Eq. 4) was observed and,
as shown in the summary plot of Figure 4E,
Zn2+ (100 µM)
strongly accelerated this process. However, according to the proposed
model, it is still unclear whether this effect was attributable to a
slower onset of desensitization, as revealed by the experiments with
long GABA pulses, or to faster unbinding kinetics.
In the attempt to "dissect" the effect of
Zn2+ on desensitization from that on
unbinding kinetics, we have used a weak GABAAR agonist, -alanine, which in comparison to GABA has a much slower binding and a faster unbinding kinetics (Jones and Westbrook, 1997). In
agreement with a previous study (Jones and Westbrook, 1995), paired
pulses (1-2 msec duration) of -alanine (100 mM) did not
reveal any accumulation of desensitization for any interval tested
(i.e., I1  I2; data not shown). This finding
implies that a 1-2 msec pulse of -alanine is too short to induce
any detectable desensitization onset and that this process does not
proceed (at variance with the GABA-evoked currents; Fig.
4D) during the deactivation phase (after removal of
the agonist). This difference in the progress of desensitization onset
during deactivation of GABA- and -alanine-induced currents is most
likely attributable to a more effective unbinding rate in the case of
-alanine, which strongly reduces the probability of multiple
entrances into the desensitized state. The current response to a single
pulse (1 msec) of -alanine (100 mM) was characterized by a very fast, nearly monoexponential decay
(1, 3.2 ± 0.6 msec;
A1, 89 ± 2%;
2, 20.8 ± 3.08 msec;
A2, 11 ± 2%, n = 10; Fig. 5A), indicating
that indeed, such functional coupling between desensitization and
unbinding rate was practically absent. Longer (50 msec) -alanine
pulses induced a desensitization (Fig. 5B) whose onset was
well described by a single exponential ( = 5.9 ± 1.6 msec; A1 = 41 ± 5%;
AS = 59 ± 5%). Similarly to
what was observed for GABA-evoked currents,
Zn2+ (100 µM)
significantly (p < 0.001) slowed the onset and
increased the extent of desensitization of -alanine-induced currents
( = 28.7 ± 0.6 msec; A1 = 59 ± 5%; AS = 41 ± 5%). Because, as explained above, the desensitization onset would be
practically terminated after removal of -alanine, the recovery from
desensitization was studied using a standard approach based on the
application, at different time intervals, of a long (50 msec)
-alanine conditioning pulse (to induce the use-dependent
desensitization) followed by a test pulse. As shown in Figure 5,
B and C, Zn2+(100
µM) strongly slowed the recovery from
desensitization. This effect was exactly the opposite of what was
observed when double short GABA pulses were applied (Fig.
4E). Moreover, whereas in Zn2+ (100 µM) the
recovery from desensitization of -alanine-induced currents was
negligible within the first 20 msec (Fig. 5C), the recovery
of current elicited by the second short GABA pulse was very pronounced
(~50% recovery after 20 msec; Fig. 4, inset). These
findings clearly rule out the possibility that zinc-induced acceleration of the recovery of currents induced by short GABA pulses
(Fig. 4D) is caused by changes in recovery from
desensitization. Both the faster recovery of responses to short GABA
pulses and the faster deactivation kinetics in zinc are compatible with
a decrease in the desensitization onset. It remains, however, still to
be elucidated whether Zn2+affects also the
unbinding rate of GABAARs. As explained in detail below, the model simulations provide indirect evidence that unbinding rate is being increased in the presence of zinc.
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Figure 5.
Zn2+ slows the recovery from
desensitization. Current response elicited by a brief (2 msec) pulse of
-alanine (100 mM). B, Conditioning pulse
of -alanine (100 mM) followed by a test pulse with a
time interval of 50 msec, in control (thick line) and in
the presence of Zn2+ (100 µM,
thin line). C, Normalized recovery from
desensitization of -alanine currents evoked in the absence
(open circles) or in the presence of
Zn2+ (100 µM, closed
circles). Each point represents the mean (n = 7-56).
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Zn2+ does not act as a fast
competitive antagonist
Based on the data presented above, the effects of
Zn2+ on the rising phase of current
responses to rapid GABA applications may arise from a decrease in the
rate constants of binding (kon) and of
conformational change (2). Alternatively, a
competitive block of GABA-binding site by
Zn2+ could account for the slower onset.
In such a case, the activation of GABAARs would
be delayed by the time needed for Zn2+ to
unbind from GABA-binding site. From the difference in the rise time of
currents evoked by saturating GABA, in control conditions and in the
presence of 100 µM
Zn2+ (Fig. 3A), it can be
deduced that the dissociation of zinc should occur very quickly (within
0.5-0.6 msec). To assess the time of interaction between
Zn2+ and GABAARs,
current responses to coapplication of GABA and
Zn2+ were examined. As shown in the Figure
6, A and B, the
time course of currents evoked by short (1-2 msec) pulses of GABA (10 mM) alone or coapplied with
Zn2+ (100 µM) were
indistinguishable, indicating that 1-2 msec is too short for
Zn2+ to exert its effect on
GABAARs. Moreover, when a conditioning pulse of
GABA coapplied with Zn2+ (for 40 msec) was
followed (after 40 msec of wash) by a second short (5 msec) test pulse,
the onset of the latter response was slower than that of the first one
(Fig. 6C,D). The rise time kinetics of the test response was
very similar to that observed when the current was evoked after
pre-equilibrating GABA receptors with Zn2+
(Fig. 3A). This indicates that a 40 msec duration of the
conditioning pulse is sufficient for zinc to affect
GABAARs, and 40 msec of wash with a
Zn2+-free medium is too short for
Zn2+ removal. When applying the same
protocol in the absence of zinc (40 msec conditioning pulse followed
after 40 msec by a test pulse), the rise time of the two pulses were
indistinguishable (data not shown).
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Figure 6.
Onset and decay kinetics of current responses
elicited by coapplication of GABA and Zn2+.
A, Normalized current responses to application of GABA
(10 mM, thick line) and coapplication of
GABA and Zn2+ (100 µM, thin
line). For clarity the two responses have been slightly
separated. Note that the two responses almost overlap.
B, The onsets of the responses shown in A
are illustrated at an expanded time scale. C,
Conditioning pulse (40 msec) of GABA (10 mM) and
Zn2+ (100 µM), followed at 40 msec
interval by a short (5 msec) test pulse of GABA and
Zn2+. The dotted line indicates
perfusion with a Zn2+-free solution.
D, Onset of the conditioning and test responses shown in
C are superimposed and shown with an expanded time
scale. E, Responses to brief (2 msec) GABA pulses (10 mM in the absence of Zn2+), applied
either in control conditions (thick line) or after
pre-equilibration in Zn2+-containing solution.
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A further indication that the kinetics of zinc interaction with
GABAARs is too slow to explain our observations
in terms of competitive antagonism came from experiments in which short
GABA pulses (10 mM without
Zn2+) were applied for 2 msec to
outside-out patches of membranes pre-equilibrated in
Zn2+ (100 µM). As seen in
the Figure 6E, the rise time of such currents (0.9 ± 0.01 msec; p > 0.5; n = 8) did not differ from that obtained in the continuos presence of
Zn2+ (Fig. 2).
Model simulations
To provide a better quantitative description of the
Zn2+ effects on
GABAA receptors, model simulations were used
(Fig. 7F). We used the
kinetic model proposed by Jones and Westbrook (1995) as the minimum
requirement to properly reproduce the basic properties of
GABAARs such as two binding sites (suggested by
Hill slope analysis), saturation of the onset rate at high [GABA],
desensitization process, and slow deactivation resulting from the
functional link between desensitization and unbinding rate. This model
fairly reproduces the behavior of the system when applying various
experimental protocols, and its relative simplicity enabled us to
estimate most of crucial rate constants in strict reference to the
experimental data.
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Figure 7.
Model simulation of Zn2+
effects on GABAA receptors. For simulation of control
recordings the parameters are kon = 8 msec/mM, koff = 0.13 msec 1, 2 = 8 msec1,
 2 = 1 msec1,
d2 = 1.5 msec1,
r2 = 0.15 msec1, and for recordings in
Zn2+, kon = 2 msec/mM, koff = 0.25 msec1, 2 = 1.5 msec1,
2 = 1 msec1,
d2 = 0.5 msec1, and
r2 = 0.04 msec1. The values of the rate
constants for singly bound receptors were adopted from Jones and
Westbrook (1995) and were assumed not to be affected by zinc.
A, Simulation of current responses to paired pulses of
10 mM GABA (2 msec pulse duration at 10 msec time interval)
in control (thick line) and in the presence of
Zn2+ (100 µM, thin
line). B, Simulation of zinc effects on
desensitization onset. Note the Zn2+ decreased the
rate and increased the extent of desensitization. C,
Normalized rise (left) and decay time
(right) of GABA response (10 mM) in the
presence or absence of Zn2+.
Zn2+-induced reduction of the onset rate and
acceleration of the decay is clearly reproduced. D,
Current responses in the presence of Zn2+ evoked by
"synaptic" GABA application [A · exp(t/); A = 5 mM,
= 0.2 msec] and to a 2 msec pulse of 5 mM GABA.
Similar to the experimental results, a larger reduction was observed in
the case of "synaptic" response. E, Simulation of
rise time kinetics of the "synaptic" responses to GABA in the
presence (thin line) and the absence of Zn (thick
line). F, Kinetic model (Jones and Westbrook,
1995). G, Values of the rate constants reproducing the
current responses in the control conditions and in the presence of 100 µM Zn.
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The analysis of the rise time kinetics at saturating GABA
concentrations (3 mM) revealed the rate constant of the
conformational change from the bound closed to the bound open receptor
(2). Once the value of
2 was determined, it was included in the rise time analysis at lower GABA doses, allowing us to precisely
determine the value of the binding rate
kon. The estimation of rate constant values for the onset (d2) and the
recovery (r2) from desensitization were based on the respective experiments in which kinetics of this
process was investigated (Figs. 4, 5). The 2
and koff rate constants have been
optimized to reproduce the decaying phase of the currents evoked by
brief, saturating pulses of GABA. In our simulations the rate constants
for transitions of single bound receptors have been adopted from Jones
and Westbrook (1995) model, but because of their small values, the
probability of occupancy of these states was low.
Subsequently, effects of Zn2+ (100 µM) on current kinetics were included in the analysis,
and the values of kon,
2, d2, and r2 were determined. However, our
experimental evidence was insufficient to assess the effect of
Zn2+ on the unbinding rate
(koff). In particular, it was not
clear whether a faster recovery of second pulse evoked by brief GABA applications (Fig. 4) was solely attributable to a reduced entrance into the desensitized state (decreased
d2) or was also a consequence of
higher unbinding rate. Our model simulations indicate that both
mechanisms are involved. We found that when keeping
koff unchanged (0.1-0.2
msec1) the
extent of recovery of the second peak in the presence of Zn2+ (Fig. 4) could be reproduced by
reducing the d2 rate constant from
1-2 to ~ 0.1-0.2
msec1.
However, such low values of d2
precluded the reproduction of Zn2+-induced
increase in the steady-state fraction of the desensitized state (Fig.
4A). Apparently, the entrance into the desensitized state at such low d2 values was too
slow to permit the accumulation of receptors in this state despite a
strongly reduced recovery rate (r2). A
good quantitative reproduction of both accelerated recovery (like that
shown in Fig. 4) and the increased extent of desensitization in
Zn2+ was obtained when an increase in the
unbinding rate koff was included (Fig.
7B,C).
Altogether, the experimental data and model simulations indicate that
the Zn2+ mechanism on
GABAA receptors includes a decrease in the rate constants determining binding (kon),
conformational transition from closed to open state
(2), desensitization onset
(d2), and recovery
(r2) as well as an increase in the
unbinding rate (koff). Such modulation
of GABAAR gating was sufficient to reproduce
fairly well the reduction in amplitude (Fig. 7A), the
increase in desensitization extent (Fig. 7B), the decrease
in the rate of current onset, and acceleration of decay (Fig.
7C) of GABA-induced currents. Model simulation of the
current responses to brief GABA applications provided further evidence
for an increase in the unbinding rate by
Zn2+. When assuming the control
koff value and desensitization onset d2 sufficiently low (0.1-0.2
msec1) to
reproduce the recovery in double short pulse experiments (Fig. 4), the
slow deactivation component was much longer than that observed in the
present experiments (>50 msec vs ~ 35 msec in the present
experiments). When increasing the value of the unbinding rate to
0.2-0.3
msec1, a
good reproduction of deactivation kinetics was obtained. As mentioned,
the rate constant 2 was deduced from formal
fitting of model predictions to the experimental data. Our analysis did not provide any obvious indication that
Zn2+ affects this rate constant. However,
we cannot exclude that a more detailed analysis (e.g., at the
single-channel level) would reveal such effect.
Although at 30 µM Zn2+ the
amplitude reductions of mIPSCs and of currents evoked by rapid GABA
applications were similar, at 100 µM
Zn2+, the decrease in synaptic current
amplitude was much stronger (compare Figs. 1F,
2D). This discrepancy could be attributable to
differences in synaptic and extrasynaptic receptors (Banks and Pearce,
2000) or to differences in nonequilibrium conditions of receptor
activation in the synapse or in excised patch recordings (Mozrzymas et
al., 1999). Recently, it has been reported that the predominant
component of the time course of agonist clearance in the synaptic cleft
is in the range of hundreds of microseconds, and the concentration of
GABA at the peak of synaptic response is >3 mM
(Mozrzymas et al., 1999). To clarify this issue,
Zn2+ effect was simulated on current
responses to "synaptic" GABA application ([GABA] = A
· exp(t/); A = 5 mM; = 0.2 msec) and to a 2 msec pulse of
GABA (5 mM). As shown in Figure 7D,
the reduction of current evoked by "synaptic" application of GABA
was considerably stronger. When assuming a faster clearance (e.g.,
= 0.1 msec), the difference was even larger (data not shown).
A simple explanation of this finding is that in conditions in which the
binding rate is strongly reduced by Zn2+,
the exposure of receptors to synaptically released GABA becomes too
short to complete the binding step. Clearly, a considerably longer
application of GABA (2 msec pulse) would increase the proportion of
bound receptors giving rise to larger amplitude responses. This finding
emphasizes the importance of nonequilibrium conditions of postsynaptic
receptor activation in determining the pharmacological modulation of
synaptic currents.
The model simulations have been also performed to see whether the
putative time course of synaptic GABA might influence
Zn2+ effect on mIPSC rise time. As seen in
Figure 7E, in agreement with our experimental observations
(Fig. 1), the rise time of normalized responses to "synaptic" GABA
application was significantly increased in the presence of
Zn2+.
| |
DISCUSSION |
The major finding of the present work is that
Zn2+, a divalent cation known to be
co-released during synaptic transmission, affects mIPSCs by allosteric
modulation of GABAA receptors. On the basis of
our kinetic analysis, the reduction of amplitude, the decrease in the
onset rate, and the acceleration of decay of mIPSCs by
Zn2+, can be consistently explained in
terms of modulation of GABAAR microscopic gating.
Similar to our results, it has been reported that, in rat neocortical
pyramidal cells, Zn2+ reduces the decay
time, peak amplitude, and rate of rise of mIPSCs without affecting
their frequency (DeFazio and Hablitz, 1998). Although these authors
claimed that Zn2+ effect was consistent
with a reduction in the affinity of the receptor for GABA possibly via
a "mixed antagonism" as observed for recombinant 1 2
receptors (Gingrich and Burkat, 1998; Krishek et al., 1998), they did
not explore the effect of Zn2+ on
GABAAR kinetics. The use of the ultrafast
perfusion system enabled us to get an insight into the mechanisms of
drug-receptor interaction under nonequilibrium conditions similar to
those presumably occurring in the synapse. However, even if the current
responses to fast GABA application reproduced the major kinetic
characteristics of mIPSCs, in agreement with other reports (Galarreta
and Hestrin, 1997; Jones and Westbrook, 1997; Mozrzymas et al., 1999;
Perrais and Ropert, 1999), synaptic currents were characterized by a
faster decay. The source of this discrepancy could arise either from the faster agonist clearance from the cleft in the case of mIPSCs (Mozrzymas et al., 1999) or from differences between
GABAARs in excised patches and the synaptic ones
(Banks and Pearce, 2000). Moreover, the slower onset kinetics of mIPSCs
observed in control conditions in comparison to that of GABA-evoked
responses (Figs. 1G, 2E) could be
attributed to electronic filtering, asynchronous multivesicular release
from a single site (Auger and Marty, 2000), and/or changes in receptor subtypes.
To our knowledge, the present report is the first one in which a
decrease in binding (kon) and in
conformational change (2) rate constants have
been demonstrated to underlie the effect of Zn2+ on the rising phase of mIPSC and of
GABA-evoked currents. In previous studies such a mechanism has not been
described, probably because slower drug application systems precluded
the resolution of kinetic changes at submillisecond time scale.
Our results provide evidence that the mechanism underlying the effect
of Zn2+ on mIPSCs decay is related to a
slower desensitization kinetics (both onset and recovery) and to the
increase in the unbinding rate. In contrast to the present findings,
Berger et al. (1998) have reported that
Zn2+ enhanced the desensitization onset
and proposed that this effect underlies a faster current deactivation
in zinc. A possible reason for this discrepancy is that Berger et al.
(1998) estimated the effects of Zn2+ on
the desensitization by coapplying GABA and
Zn2+. In these conditions, the
desensitization onset would be "accompanied" by the ongoing
inhibitory Zn2+ effect on
GABAARs.
Our double pulse experiments provide an important methodological
indication that the recovery inferred from the amplitude of the second
response is strongly dependent on the unbinding rate because the slow
unbinding favors multiple entrances into the desensitized state. This
implies that the classical double pulse protocol is an adequate tool to
determine the recovery from desensitization only when the agonist
unbinds sufficiently fast to make negligible the probability of such
multiple entrances.
Whereas the effects of Zn2+ on
kon, 2,
d2, and
r2 rate constants were directly
supported by experimental evidence, the
Zn2+-induced increase in
koff was deduced from model
simulations. In the case of Zn2+-induced
reduction of kon, an increase in
koff would be expected because it is
known that a decrease in the affinity of GABAARs is associated with a decrease in binding and an increase in the unbinding rate constants (Jones et al., 1998). Similarly, in our previous study (Mozrzymas et al., 1999) chlorpromazine-induced reduction of kon was accompanied by an
increase in koff. It appears thus that
when altering the strength of binding, the changes in kon and in
koff are negatively correlated.
The Zn2+-induced reduction of
desensitization onset and increase in its extent is consistent with a
strong slowing of recovery from desensitization. Such slower exit would
favor the accumulation of desensitized receptors leading to a larger
steady-state proportion of receptors in this state. On the basis of
single-channel studies, it has been proposed that
Zn2+ stabilizes the receptor in the closed
state (Smart et al., 1994). It is possible that this observation
reflects, at least in part, prolonged "sojourns" in the
desensitized state caused by the slower receptor exit from this
conformation. Single-channel studies have also shown that
Zn2+ reduces the opening frequency with no
evidence of a flickering block (Legendre and Westbrook, 1991; Smart,
1992; Kilic et al., 1993). As suggested by Krishek et al. (1998) and,
in line with the present experiments, this observation favors the
hypothesis that Zn2+ might interact with
an allosteric modulatory site located on the extracellular domain of
the GABAA receptor.
In our previous study (Mozrzymas et al., 1999) we found that
chlorpromazine (CPZ) strongly reduced the amplitude of mIPSCs without
any clear effect on the rise time kinetics. The explanation of this
finding is that CPZ affects only the binding/unbinding kinetics, and
the synaptic GABA transient is too short to reveal the slower current
onset. In the present study, however, Zn2+
caused a marked increase in the rise time of mIPSCs, and this effect
was well reproduced by model simulations. The explanation for the
different effect of Zn2+ and CPZ on mIPSC
rise time lies in Zn2+-induced reduction
in the rate constant 2 that was unaffected by
CPZ. Thus, the reduction of the binding rate by CPZ strongly diminished
the recruitment of bound receptors during the synaptic GABA transient,
but those receptors that bound GABA proceeded to the open state with
normal (i.e., control) kinetics described by the
2 rate constant. On the contrary, in
Zn2+, the transition from bound closed to
bound open conformation was slowed (smaller 2
value), giving rise to a slower mIPSCs onset in spite of a very
transient synaptic GABA application.
The experiments presented in Figure 6 provide clear evidence that the
kinetics of interaction between Zn2+ and
GABAA receptors is too slow to explain the
observed Zn2+ effects in terms of
competitive antagonisms. The noncompetitive mechanism of
GABAA receptors inhibition is additionally
supported by the fact that at low Zn2+
concentrations ( 30 µM) no evidence for a biphasic rise
time kinetics was observed. Thus, when applying a saturating dose of agonist, the nonoccupied receptors would be activated with normal kinetics, and the activation of occupied ones would be slowed by the
unbinding of the antagonist giving rise to a biphasic onset. This
concept has been used for studying the time course of synaptic transmitter at glutamatergic synapses using quickly dissociating competitive antagonists (Clements et al., 1992).
In the case of sustained neuronal activity, a local increase in
Zn2+ concentration, caused by release from
glutamatergic synapses may affect neighboring GABAergic synapses,
leading to an impairment of GABAergic function. In particular, in
pathological conditions, zinc released from aberrantly sprouted mossy
fibers may cause chronic changes in cell excitability and epileptic
seizures (Buhl et al., 1996). Diffusion of zinc, caused by spill over,
is expected to be much slower than the transient release of GABA during
synaptic transmission. This implies that in the time scale of synaptic events, fluctuations in Zn2+
concentrations may be regarded as tonic ones. Thus, the effects of
Zn2+ on synaptic currents would be similar
to those observed in "pre-equilibration" experiments.
| |
FOOTNOTES |
Received June 12, 2000; revised Sept. 7, 2000; accepted Sept. 15, 2000.
This work was supported by a grant from Ministero
dell'Università e Ricerca Scientifica e Tecnologica to E.C.
J.W.M. was supported by Wroclaw University of Medicine Grant 561 and by
Polish Committee for Scientific Research (KBN) research funds for
Wroclaw University of Medicine, and A.B. was supported by a fellowship from Novartis Pharmaceuticals. The Bioq software with which the kinetic
modeling was performed was kindly given by Dr. H. Parnas from the
Hebrew University (Jerusalem).
Correspondence should be addressed to Enrico Cherubini, International
School for Advanced Studies (SISSA), via Beirut 2-4, 34014 Trieste,
Italy. E-mail: cher{at}sissa.it.
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INTRODUCTION
