The Journal of Neuroscience, September 3, 2003, 23(22):7981-7992
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Modulation of GABAA Receptors by Hydrogen Ions Reveals Synaptic GABA Transient and a Crucial Role of the Desensitization Process
Jerzy W. Mozrzymas,1
Ewa D. 
armowska,1
Maria Pytel,1 and
Katarzyna Mercik1,2
1Department of Biophysics, Wroclaw Medical
University, 50-368 Wroclaw, Poland, and 2Institute of
Physics, Technical University of Wroclaw, 50-370 Wroclaw, Poland
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Abstract
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Protons are the most ubiquitous and very potent modulators of the
biological systems. Hydrogen ions are known to modulate GABAA
receptors (GABAARs), but the mechanism whereby these ions affect
IPSCs and the gating of GABAARs is not clear. In the present study
we examined the effect of protons on miniature IPSCs (mIPSCs) and found that
hydrogen ions strongly affected both their amplitude and time course. To
explore the underlying mechanisms with resolution adequate to the time scale
of synaptic transmission, we recorded current responses to ultrafast GABA
applications at various pH. These experiments revealed that the major effect
of protons on GABAAR gating is a strong enhancement of
desensitization and binding rates at increasing pH. This analysis also
indicated that desensitization rate is the fastest ligand-independent
transition in the GABAAR gating scheme. Although proton effects on
the time course of mIPSCs and current responses to saturating [GABA] were
similar, the pH dependencies of amplitudes were almost opposite. Our
quantitative analysis, based on model simulations, indicated that this
difference resulted from a much shorter receptor exposure to agonist in the
case of mIPSCs. Modeling of IPSCs as current responses to brief exponentially
decaying GABA applications was sufficient to reproduce correctly the pH
dependence of mIPSCs, and optimal fit was obtained for peak [GABA] of 1.5-3
mM and a clearance time constant of 0.075-0.125 msec. Our analysis
indicates that, for these parameters of GABA transient, in control conditions
(pH 7.2) mIPSCs are not saturated.
Key words: hydrogen ions; GABAA receptor; gating; mIPSC; synaptic GABA transient; synaptic receptor saturation; desensitization
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Introduction
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GABA is the major inhibitory neurotransmitter in the adult CNS
(Cherubini and Conti, 2001
),
and the time course of GABAergic IPSCs is a key determinant of GABA-mediated
inhibition. It is known that local proton concentration is regulated by a
number of mechanisms, including enzymes (e.g., carbonic anhydrase), active
transport, and cotransporters as well as passive ion transport (for review,
see Kaila, 1994). As pointed
out by Kaila and coworkers, the permeation of HCO3-
anions through the GABAA receptors (GABAARs) may
influence the pH level in the closest vicinity of the channel pore
(Kaila et al., 1992;
Kaila, 1994;
Voipio et al., 1995). Hydrogen
ions have been found to be a potent modulator of GABAARs.
Pasternack et al. (1996) have
reported that, in rat hippocampal neurons, current responses to high [GABA]
were enhanced when proton concentration was increased, whereas the opposite
was observed for currents elicited by low [GABA]. Qualitatively similar
results have been obtained by Krishek and Smart
(2001) in granule cells and
for GABAARs expressed in Xenopus oocytes and encoded by
poly(A+) mRNA from rat brain
(Robello et al., 2000).
Moreover, the proton effects were found to depend strongly on the
GABAA receptor subtypes (Krishek et al.,
1996,
1998).
The mechanism underlying the modulation of GABAARs by protons
has not been elucidated fully. It is surprising that the current responses to
nearly saturating [GABA] are enhanced strongly by lowering the pH
(Pasternack et al., 1996),
whereas neither the single-channel conductance nor the opening frequency is
clearly affected by hydrogen ions (Krishek
and Smart, 2001). Moreover, the characterization of pH effect on
GABAergic IPSCs in relation to the modulation of GABAA receptor
microscopic gating is essentially lacking. Recent studies have emphasized that
extreme non-equilibrium conditions of synaptic receptor activation resulting
from very fast agonist clearance have a crucial impact on the time course and
pharmacological modulation of synaptic currents
(Puia et al., 1994;
Jones and Westbrook, 1995;
Clements, 1996;
Jones et al., 1998;
Mozrzymas et al., 1999;
Barberis et al., 2000). Thus
the characterization of the receptor gating needs to be performed with a
resolution adequate to the time scale of the synaptic events.
The goal of the present work was to characterize the effects of
extracellular proton concentration on miniature IPSCs (mIPSCs) and to explore
the underlying mechanisms in terms of microscopic gating of GABAA
receptors. We found that protons strongly affected both the amplitude and the
time course of mIPSCs in hippocampal neurons. The effect of hydrogen ions on
GABAA receptors has been studied by analyzing the current responses
to ultrafast GABA applications. Our results indicate that the modulation of
mIPSCs is mainly attributable to an upregulation of desensitization and
binding rates of GABAARs by decreasing proton concentration
(increasing pH). Our quantitative analysis enabled us to estimate the peak
concentration (1.5-3 mM) and time of clearance of the synaptic GABA
(0.075-0.125 msec) and provided evidence that desensitization is the fastest
process in the GABAAR gating scheme.
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Materials and Methods
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Cell culture. Primary cell culture was prepared as described by
Andjus et al. (1997). Briefly,
postnatal day 2 (P2) to P4 Wistar rats were decapitated after being
anesthetized with an intraperitoneal injection of urethane (2 gm/kg). This
procedure is in accordance with the regulations of the Polish Animal Welfare
Act. Hippocampi were dissected from 2- to 4-d-old rats, sliced, treated with
trypsin, mechanically dissociated and centrifuged twice at 40 x
g, plated in the Petri dishes, and cultured. Experiments were
performed on cells between 10 and 15 d in culture.
Electrophysiological recordings. Currents were recorded in the
whole-cell and outside-out mode of the patch-clamp technique, using the EPC-7
amplifier (List Medical, Darmstadt, Germany) at a holding potential
(Vh)of -70 mV. The intrapipette solution contained (in
mM) 137 CsCl, 1 CaCl2, 2 MgCl2, 11 BAPTA, 2
ATP, 10 HEPES, pH 7.2, with CsOH. The composition of the standard external
solution was (in mM) 137 NaCl, 5 KCl, 2 CaCl2, 1
MgCl2, 20 glucose, 10 HEPES, pH 7.2, with NaOH. HEPES was used to
buffer the external solutions with pH in the range 6.8 - 8.0. For external
solutions with pH between 5.0 and 6.8, MES
(C6H13NO4S) was used at concentration of 15
mM. In experiments performed by using external solutions with pH
higher than 8.0 (buffered with 20 mM TRIS), the conditions of cells
quickly deteriorated, giving rise to a progressive increase in leakage current
and eventually to loss of patch. For this reason the upper limit of pH
considered in the present study was set at 8.0. Acidic pH, even at values as
low as 5.0 - 6.0, was much less harmful to the cells than basic ones above
8.0. Recordings at acidic pH (not lower than 6.0) showed a better stability
even than in control conditions (pH 7.2). The whole-cell recordings for the
considered pH range were characterized by good stability for up to 30 min
(records in which >10% rundown occurred were excluded from the analysis).
To reduce the impact of rundown further, we recorded mIPSCs in 5 min sweeps,
alternating controls (pH 7.2) with recordings at various pH values. Within
5-10 min recordings the rundown was negligible.
The characteristics of the time course (e.g., 10-90% rise time, time
constants of desensitization and deactivation...) of current responses to
rapid GABA applications showed little cell-to-cell variability, so the values
of these parameters estimated from different cells were pooled. The analysis
of proton effect on current amplitudes required a comparison of recordings
made on the same patch (see Fig.
2). Stable recordings (<10% of rundown) were available for
10-20 min. Because current responses were recorded every 1-2 min, the
impact of rundown was small. Controls and recordings at various pH were
alternated. The amount of rundown was estimated from control current
amplitudes before and after the test pulse. In the case of detectable rundown,
the amplitude of the test current was compared with the average of control
amplitudes immediately before and after the test recording. Because the pulses
were separated by the same time interval, such a procedure allowed for an
interpolation of the control current amplitude at the moment of the test
recording. Rundown was faster at basic pH (up to 10% rundown within 10
min), but application of the interpolation procedure described above allowed
for the comparison of peak currents at various pH values.
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Figure 2. Changes in extracellular proton concentration strongly affect the
amplitudes and the rise time of current responses evoked by saturating GABA
concentrations. A, B, Typical current responses to 10
mM GABA recorded from the same patch at pH 7.2 (thick line) and at
pH 8.0 (thin line). C, D, Examples of responses to 10
mM (at pH 7.2, thick line) and to 30 mM (at pH 6.0, thin
line) recorded from the same patch. E, Dependence of relative current
amplitude (divided by the value of amplitude recorded at pH 7.2 from the same
cell) on pH. F, Normalized currents recorded at pH 5.0 and 7.2.
G, Dependence of averaged 10-90% rise times of currents on pH value.
The time course of applied GABA is depicted by the inset above the current
traces. Each average was calculated for responses recorded from at least 12
patches. Asterisks indicate significant difference with respect to the control
values.
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All experiments were performed at room temperature, 22-24°C. mIPSCs
were recorded in the whole-cell configuration in the presence of tetrodotoxin
(TTX; 1 µM) and kynurenic acid (1 mM). In the
whole-cell mode the series resistance (Rs) was in
the range 4 - 8 M
, and 50-70% of Rs
compensation was accomplished.
The current signals were low-pass filtered at 10 kHz with a Butter-worth
filter and sampled at 50-100 kHz, using the analog-to-digital converter CED
micro1401 (Cambridge Electronic Design, Cambridge, UK), and were stored on the
computer hard disk. The acquisition and analysis software were kindly given by
Dr. J. Dempster (Strathclyde University, Glasgow, UK).
GABA was applied to excised patches via the ultrafast perfusion system,
based on a piezoelectric-driven theta-glass application pipette
(Jonas, 1995). The
piezoelectric translator was from Physik Instrumente (preloaded HVPZT
translator, 80 µm; Waldbronn, Germany) and the theta-glass tubing from
Hilgenberg (Malsfeld, Germany). The open tip recordings of the liquid junction
potentials revealed that a complete exchange of solution occurred within 40 -
60 µsec. A minimum duration of drug application was 1 msec (when
shorter pulses were applied, oscillations often appeared). At very low GABA
concentrations (0.2-5 µM) the current responses were too small
in excised patches and therefore were recorded in the whole-cell configuration
with a multibarrel system RSC-200 (Bio-Logic, Grenoble, France). With the use
of this system the exchange of solution surrounding an open tip occurred
within 10-20 msec (in the whole-cell mode). To assess the exchange time around
a neuron, we have recorded the current responses to high potassium saline and
to saturating [GABA] in the whole-cell configuration. In both cases the onset
of response was characterized by 10-90% rise time between 15 and 25 msec.
Because at these GABA concentrations the rise time was considerably slower
than 100 msec, such application speed was sufficient to describe the time
course of these responses.
Analysis. The decaying phase of the currents was fit with a
function in the form:
 | (Eq. 1) |
where Ai is the fraction of respective
components, As is the steady-state current, and
i is the time constant. For normalized currents,
Ai + As = 1.
Deactivation time course was well fit with a sum of two exponentials
(n = 2) and As = 0. The desensitization
onset was fit with either one or two exponentials and
As > 0.
The recovery process in the double-pulse protocol (see
Fig. 4 F-I) was
estimated by using the following formula:
 | (Eq. 2) |
where R is the percentage of recovery, I1 the
first peak amplitude, Iend the current value immediately
before the application of the second pulse, and I2 the
second peak amplitude.
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Figure 4. Decrease in pH strongly affects the desensitization process and the
recovery of response to the second pulse in the double-pulse experiment.
A-C, Typical normalized current responses evoked by long
pulses of saturating GABA concentrations applied to the same cell. D,
E, pH dependence of averaged values of the time constant of
desensitization onset and steady-state to peak ratio, respectively. The values
of parameters presented in D and E were obtained by fitting
a sum of one exponential and a constant value to the decaying phase of the
current response (Eq. 1). Asterisks in D and E indicate
significant difference with respect to the control values.
F-H, Typical current responses to pairs of short pulses (2
msec) of saturating GABA concentrations applied to the same cell. Insets above
the current traces indicate the time course of applied agonist. I,
Dependence of recovery on time duration of gap interval between pulses at
different pH values. The value of the recovery parameter was calculated by
using Equation 3. The values of recovery parameter recorded at pH values
indicated in the graph were significantly different from those measured in
control conditions (pH = 7.2) for any considered gap interval longer than 5
msec. At pH 6.0 (A, F) 30 mM GABA was used to ensure
saturation, whereas at pH 7.2 (B, G) and pH 8.0 (C, H) 10
mM GABA was applied. Each average was calculated for responses
recorded from at least nine patches.
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The kinetic modeling was performed with the Bioq software kindly provided
by Dr. H. Parnas (Hebrew University, Jerusalem, Israel). The Bioq software
converted the kinetic model (see Fig. 6
A) 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
"synaptic GABA application" in the exponential form: A
· exp(-t/), where A is the peak concentration
and is the time constant of agonist clearance. The solution of such
equations yielded the time courses of occupancies of all of the states
included in the model. The fitting algorithm was based on a choice of the
entire set of the rate constants that best reproduced the time course of
currents recorded in all of the protocols that were used. To this end, changes
in respective rate constants were introduced manually, and the parameters
describing the time course (10-90% rise-time, steady-state to peak, time
constants of desensitization, deactivation...) for model predictions and
experimental traces were compared.
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Figure 6. Simulations of current responses elicited by ultrafast GABA applications.
The traces represent the total occupancy of open states
(AR* and A2R*).
A, Jones and Westbrook's model
(1995). B-D,
Simulated current responses to brief (2 msec) applications of saturating GABA
concentrations for the rate constants optimized to reproduce the
GABAA receptor gating at pH values of 6.0, 7.2, and 8.0,
respectively (see Table 1).
E, pH dependence of amplitudes of simulated current responses to
saturating GABA concentrations (compare with
Fig. 2). F, G, Time
courses of simulated deactivation phases of currents evoked by saturating GABA
concentrations at pH 6.0, 7.2 (thick line), and 8.0. In F, a strong
proton effect on the fast deactivation component and in G (expanded
time scale) on the slow component is seen (compare with
Fig. 3). H, pH
dependence of percentages of fast component (A1, filled
bars) and of slow component (A2, open bars). The simulated
pH effect of proton concentration on the deactivation kinetics qualitatively
reproduces the experimental observations on mIPSCs (see
Fig. 1) and on current
responses to exogenous GABA applications (see
Fig. 3). I, Simulation
of pH effect on the rising phase of normalized currents elicited by saturating
GABA concentrations. Note that acidic pH slows down and basic pH accelerates
the current onset with respect to the control current (pH 7.2, thick line).
This qualitatively reproduces the experimental data (see
Fig. 2). J, Simulation
of the effect of hydrogen ions on the kinetics of the desensitization onset.
Normalized responses are shown for pH 6.0, 7.2 (thick line), and 8.0.
Simulations well reproduce the theory that a decrease in proton concentration
accelerates the time constant of desensitization onset and reduces the
steady-state to peak ratio (compare with
Fig. 4). K, Simulation
of current responses evoked by pairs of short pulses. Slower recovery of the
second pulse results mainly from an increase in the rate of entry
(d2) into desensitized state at decreasing proton
concentration. L, Simulation pH dependence of the rising phase of
currents evoked by a nonsaturating GABA concentration (300 µM).
Normalized traces are shown for pH 6.0, 7.2 (thick line), and 8.0 (compare
with Fig. 5). In graphs shown
in B-D, F, G, I-L, the insets above the traces represent the time
course of agonist applied at a saturating concentration.
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Table 1. Values of rate constants optimized to reproduce the time course of
current responses to rapid GABA applications at different pH
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Figure 1. Hydrogen ions affect the amplitudes, frequency, and time course of mIPSCs.
A, Examples of mIPSCs recorded at different pH values (indicated on
the right side of the traces) and at holding potential of -70 mV. B,
Typical averaged mIPSC recorded at -70 mV and at pH 7.2. C, pH
dependence of mIPSC frequency. The frequencies are given relative to the
control value (at pH 7.2). D, Dependence of mean amplitude of mIPSC
on pH. Note that at pH 6.5 the mean amplitude is larger than in the control
conditions (pH 7.2). E, Typical cumulative histograms for mIPSC
amplitudes recorded at pH values 6.0, 6.5, and 8.0. The thick line in each
graph represents the cumulative histogram for amplitudes recorded at pH 7.2 on
the same cell. F, Averaged rise time of mIPSCs at pH 6.0, 7.2, and
8.0. G, H, Averaged values of the fast
(fast) and slow (slow) components of the
decaying phase of the mIPSCs, respectively. The values of the rate constants
were obtained from the fit of a sum of two exponentials (see Eq. 1).
I, Percentages of fast A1 (filled bars) and slow
A2 (open bars) components of the decaying phase of mIPSCs
(see Eq. 1). J, pH dependence of charge transfer mediated by averaged
mIPSC. The values of charge transfer were calculated relative to the control
(pH 7.2) value. In C, D and F-J, each mean value was
calculated from recordings from at least six cells. Asterisks indicate
significant difference with respect to the control values.
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Figure 5. Hydrogen ions affect the time course of current responses to nonsaturating
GABA concentrations. A, Typical normalized current responses to 100
µM GABA in control conditions (pH 7.2, thick line) and at basic
pH 8.0 (thin line). B, Typical normalized responses to 300
µM GABA in control conditions (pH 7.2, thick line) and at basic
pH 8.0 (thin line). C, pH dependence of averaged 10-90% rise times of
current responses to 100 µM (filled bars) and 300
µM (open bars) GABA. D, Typical current responses to
0.2, 1, and 5 µM GABA at pH 6.0 (left), pH 7.2 (middle, thick
lines), and pH 8.0 (right). Insets above the current traces indicate the
application time of the agonist. E, Relative amplitudes (normalized
to the amplitudes of currents recorded from the same cell at pH 7.2) of
currents evoked by 0.2, 1, and 5 µM at pH 6.0 (filled bars) and
at pH 8.0 (open bars). F, pH dependence of 10-90% rise times of
currents elicited by 1 and 5 µM GABA. Each average was
calculated for responses recorded from at least eight patches. Asterisks
indicate significant difference with respect to the control values.
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Data are expressed as the mean ± SEM. The amplitudes of both
synaptic currents (see Fig. 1)
and of current responses to rapid GABA applications were measured at various
pH, and a comparison was made to the peaks of control currents (at pH 7.2)
measured on the same cell or excised patch. Thus for analysis of amplitudes
the Student's paired t test was used. For other parameters such as
rise time (see Figs. 1
F, 2 F,
G), time constants, and respective fractions of slow and fast
components of deactivation (see Figs.
1,
3,
4) the data were pooled, and
the Student's un-paired t test was used.
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Results
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Changes in extracellular proton concentration affect the mIPSCs
mIPSCs were recorded in the whole-cell configuration at a membrane voltage
of -70 mV (Fig. 1A,B).
The amplitudes of mIPSCs showed large cell-to-cell variability; in control
conditions (pH 7.2) the averaged peak value was 43.24 ± 2.57 pA
(n = 39). At control and acidic pH (6.0-7.2) the averaged frequency
of mIPSCs showed little variability (at pH 7.2, 1.21 ± 0.09 Hz;
n = 18), but at lower proton concentrations (pH 7.6 and 8.0) it was
increased significantly (Fig.
1C). The effect of hydrogen ions on mIPSC amplitude was
examined within the pH range 5.0 - 8.0, and amplitudes at each pH were
calculated relative to the control value (pH 7.2) recorded on the same cell.
Strongly acidic pH (5.0 - 6.0) caused a pronounced reduction, whereas at basic
pH values the mIPSC amplitude was affected only weakly
(Fig. 1D,E). At pH
5.0, in two of six cells that were tested, a complete inhibition of mIPSCs was
observed. Interestingly, at pH 6.5 the peak of mIPSCs was increased with
respect to control (Fig.
1D). As shown in
Figure 1E, these
effects of hydrogen ions on mIPSC amplitudes are associated with clear shifts
in the cumulative amplitude histograms.
The analysis of the time course of the mIPSCs was performed on averaged
current traces (Fig.
1B). In control conditions (pH 7.2) the averaged mIPSC
rise time was 0.65 ± 0.06 msec (n = 9). The mIPSC onset phase
accelerated with increasing pH (Fig.
1F), but this effect was significant (p <
0.05) only at strongly acidic pH values (at pH 6.0, 0.84 ± 0.07;
n = 6). The decaying phase of mIPSCs was clearly biphasic and at pH
7.2 could be described with a sum of exponentials (fast = 6.85
± 0.59 msec, A1 = 0.56 ± 0.012,
slow = 51.08 ± 2.35 msec, A2 = 0.44
± 0.12; n = 26). Increase in pH affected the decay of the
mIPSCs by decreasing the value of rapid time constant (fast;
Fig. 1G) and by
increasing the slow one (slow;
Fig. 1H). Moreover,
when pH was increased, the fractions of the fast and slow components
(A1 and A2) monotonically increased
and decreased, respectively (Fig.
1I). At pH 7.2 the averaged charge transfer mediated by
mIPSCs was 1.21 ± 0.14 pC (n = 9), and this parameter showed a
pH dependence (Fig.
1J) that was qualitatively similar to that observed for
mIPSC amplitudes (Fig.
1D).
The effect of hydrogen ions on the kinetics of currents evoked by
ultrafast applications of GABA
To describe the effects of hydrogen ions on GABAA receptor
gating in the time scale corresponding to that of the synaptic events, we
studied the current responses to ultrafast GABA applications.
Amplitudes of current responses to saturating GABA concentrations
strongly depend on proton concentration
At sufficiently high (saturating) GABA concentrations the binding step
becomes much faster than any ligand-independent transition, and both the
current amplitude (proportional to the occupancy of open states) and the onset
rate reach their maximum values. Pasternack et al.
(1996) have reported that
acidic pH upregulated the amplitudes of current responses to nearly saturating
[GABA] (500 µM). However, the perfusion system used by these
authors was relatively slow, and it cannot be excluded that receptors
partially predesensitized before reaching the peak. To clarify this issue, we
recorded current responses to ultrafast applications of saturating [GABA]
(10-30 mM) at pH ranging from 5.0 to 8.0. To ensure that the
agonist concentration was saturating at each pH value, we varied [GABA]
between 10 and 30 mM. At pH higher or equal to 7.2, 10
mM was saturating, and at pH lower than 7.2, 30 mM was
used to ensure the saturating conditions. For instance, at pH 5.0 the 10-90%
rise times of responses to 30 and 10 mM GABA were 0.69 ±
0.06 msec (n = 11) and 1.22 ± 0.14 msec (n = 5),
respectively, indicating that 10 mM was insufficient to saturate
the response at this proton concentration. As recently shown by Mercik et al.
(2002), 30 mM GABA
induced a self-block of current responses at pH 7.2. However, at lower pH
(6.5, 6.0, 5.0) this effect was not observed (at 30 mM GABA; data
not shown), which is consistent with one of the conclusions of the present
work that acidic pH reduces the affinity of GABAAR (see below). At
pH 7.2 the averaged amplitude of currents evoked by 10 mM GABA was
257.2 ± 32.4 pA (n = 47). As shown in
Figure 2A-E, the
amplitude of these responses showed a strong and monotonic dependence on
proton concentration, decreasing approximately onefold per pH unit. As
previously mentioned, this finding is puzzling particularly in the light of
the lack of proton effect on both single-channel conductance and frequency
(Krishek et al., 2001). Moreover, the effect of proton concentration on
amplitudes of mIPSCs was almost opposite
(Fig. 1D). Thus to
explore the mechanism underlying these effects of hydrogen ions further, we
used more experimental protocols.
The rise time kinetics of current responses to saturating GABA is
modulated by protons
The current onset rate was assessed as 10-90% rise time of current
response. As shown in Figure 2, F
and G, acidification of external solution markedly slowed
down the rising phase kinetics, whereas at basic pH a slight acceleration was
present. At first glance, this result could suggest that an increase in proton
concentration slows down the transition rate from the bound closed to the
bound open state (
2; model in
Fig. 6A), but it is
also possible that the transition to quickly desensitized state could affect
the current rise time (see Model simulations).
Effect of proton concentration on the deactivation kinetics
After agonist removal the current response does not fall immediately to
zero but shows a relaxation defined as the deactivation process that, for
GABAAR-mediated responses, may exceed hundreds of milliseconds (see
Jones and Westbrook, 1995;
Mozrzymas et al., 1999). Jones
and Westbrook (1995) have
demonstrated that the mechanism underlying such slow decaying phase is a slow
unbinding rate that favors multiple sojourns in the open and desensitized
states. Moreover, because the presence of agonist in the synapse is very short
(at most, hundreds of microseconds;
Clements, 1996;
Mozrzymas et al., 1999), the
decaying phase of IPSCs represents mainly the deactivation process. The
deactivation kinetics thus may provide important information on proportions
among unbinding, opening, and desensitization rates. In control conditions (pH
7.2) the deactivation was clearly biphasic (fast = 2.47
± 0.17 msec, A1 = 0.725 ± 0.11,
slow = 110 ± 7.5 msec, A2 = 0.275
± 0.06; n = 27). The fast deactivation component
(fast) was similar to that found, for example, by Berger et
al. [2.2 msec (1998)], Zhu and
Vicini [3.6 msec(1997)], and
Perrais and Ropert [3.1 msec
(1999)] but differed
substantially from that reported by, for example, Jones and Westbrook [18 msec
(1995)] as well as Banks and
Pearce [13.9 msec (2000)]. This
diversity may reflect a variety of receptor types and differences in
experimental conditions. The slow time constant slow was
clearly larger than that in the case of mIPSCs (compare Figs.
1H,3D),
and this difference may be attributed to different receptor properties
(Banks and Pearce, 2000). A
markedly faster rapid phase of decay (fast) in the case of
current responses (Figs.
1G,
3E) is likely to
result additionally from a much smaller electrotonic filtering by the excised
patch of neuronal membrane than in the case of synaptic current recordings.
Similarly to mIPSCs, the slow decay component increased and the fast one
decreased with increasing pH (Fig.
3D,E). Moreover, the fractions of fast and slow time
constants increased and decreased, respectively, with pH
(Fig. 3F). At pH 7.2
the averaged charge transfer mediated by current responses to short
applications of saturating [GABA] was 8.34 ± 2.4 pC (n = 9),
and this parameter showed a monotonic decrease at increasing pH
(Fig. 3G).
Hydrogen ions affect the desensitization of GABAA
receptors
Long applications of saturating GABA concentration were used to study the
kinetics of the desensitization process
(Fig. 4A-E). When 300
msec pulses were applied at pH 7.2, the desensitization onset was clearly
biphasic and characterized by the time constants (fast = 2.41
± 0.17 msec, A1 = 0.76 ± 0.11,
slow = 126 ± 7.5 msec, A2 = 0.14
± 0.04, As = 0.1 ± 0.01; n = 16).
The slow phase of desensitization becomes clearly visible during long
applications of agonist (> 100 msec), whereas the synaptic clearance, as
previously mentioned, is believed to occur within hundreds of microseconds.
Moreover, because of faster rate into the rapidly desensitizing state, the
role of this conformation in shaping the deactivation kinetics appears to be
predominant. This is supported by the proper reproduction of the deactivation
time course, using the kinetic model including only the fast desensitizing
state (Jones and Westbrook,
1995; Mozrzymas et al.,
1999; Barberis et al.,
2000) (see also Model simulations). Thus the analysis of the
desensitization onset has been limited to the fast component that at 50 msec
pulse duration was predominant (at pH 7.2, fast = 2.78
± 0.17 msec, A1 = 0.8085 ± 0.11,
As = 0.1915 ± 0.02; n = 27). Increase in
pH caused a marked acceleration of the desensitization onset
(Fig. 4A-D) and
reduction of the steady-state to peak ratio
(Fig. 4A-C,E). These
effects were associated with a decrease in amplitude as presented in
Figure 2A-E. The
desensitization onset at acidic pH was studied by the application of long
pulses of 30 mM GABA (instead of 10 mM) to ensure the
saturation conditions.
Pairs of short (2-3 msec) pulses of saturating [GABA] separated by a
variable gap were used to assess the recovery of the second pulse amplitude.
Basic pH slowed down, whereas acidic pH accelerated the recovery process
(Fig. 4F-I). This
effect potentially could involve at least three factors: increase in the
desensitization rate (d2), decrease in resensitization
rate (r2), and decrease in the unbinding rate
(koff) when proton concentration is decreased in the
extracellular medium. The effect of pH on these processes will be discussed in
detail in Model simulations.
Hydrogen ions modulate the binding rate of GABA to the binding site
on GABAA receptor
At GABA concentrations below saturating ones, the activation of
GABAA receptor slows down in a dose-dependent manner. This reflects
the fact that, for nonsaturating [GABA], activation kinetics of
GABAAR depends on GABA binding rate. Thus the analysis of the onset
rate of current responses to nonsaturating GABA concentrations may provide us
with crucial information on kon rate constant. The rate of
binding is assumed to be proportional to GABA concentration
(kon · [GABA], where kon
is the binding rate constant), and therefore the velocity of binding process
can be regulated by setting appropriate GABA concentration. To check for the
effect of protons on the binding rate, we recorded current responses to
nonsaturating [GABA] (Fig. 5).
As shown in Figure
5A-C, basic pH clearly accelerated whereas acidic pH
slowed down the current onset rate. The effect of acidic pH was particularly
strong; whereas at pH 7.2 the 10-90% rise time of current response to 300
µM GABA was 1.03 ± 0.05 msec (n = 20), at pH 6.0
and pH 5.0 it was 3.08 ± 0.54 msec (n = 16) and 5.71 ±
0.87 msec (n = 8), respectively. The rise time of currents evoked by
100 µM GABA at pH 7.2 was 1.78 ± 0.2 msec (n =
16), and its pH dependence was qualitatively similar as in the case of
currents evoked by 300 µM GABA
(Fig. 5C). Although
the rise time of current responses potentially can be shaped by the
desensitization process also, in the range of [GABA] 100-300 µM
such strong hydrogen ion effect is unlikely to be predominant because of a
modification of desensitization (see also Model simulations).
The effect of proton concentration on the amplitudes of currents evoked by
100 and 300 µM GABA was qualitatively similar to that observed
for saturating [GABA] (Fig.
2A-E). At 100-300 µM GABA most of receptors
reach the fully bound state, A2R; therefore,
similarly as for responses to saturating [GABA], the peak occupancy of the
open state A2R* depends mainly on the
balance between the opening 2 and desensitization rate
d2 (see Model simulations below). However, at very low
[GABA], binding is expected to be critical for recruitment of receptors into
the open state; therefore, the up/downregulation of the binding rate
kon by basic/acidic pH would be expected to produce an
increase/decrease in amplitude. To test this hypothesis, we recorded current
responses to 0.2, 1, and 5 µM GABA in control conditions (pH
7.2) as well as at pH 6.0 and pH 8.0. As explained in Materials and Methods,
these experiments were performed in the whole-cell configuration because at
such low [GABA] the currents in excised patches were too small. At pH 6.0 the
responses to 0.2 µM GABA were at the baseline noise level, but
at higher pH clear current responses appeared and an increase in amplitude
with decreasing proton concentration was evident
(Fig. 5D; at pH 7.2,
54 ± 18 pA; n = 8). Concentration of 1 µM GABA
was sufficient to evoke detectable response at pH 6.0, and again the currents
clearly increased with pH (Fig.
5D,E; at pH 7.2, 244 ± 27 pA; n = 7). For
responses evoked by 5 µM GABA, acidic pH clearly decreased the
current amplitude with respect to control (pH 7.2, 1108 ± 68 pA;
n = 9), whereas at basic pH the amplitude was comparable to that in
control conditions (Fig.
5D,E). For responses elicited by 1-5 µM
GABA (at 0.2 µM GABA the signal-to-noise ratio was insufficient
to assess the rise time reliably), the rising phase of current accelerated
with pH (Fig. 5F).
These data further indicate that the binding rate is accelerated with
decreasing proton concentration. Our results on current responses to very low
[GABA] are qualitatively compatible with observations of Pasternack et al.
(1996), who reported that
increasing pH enhanced the amplitudes of currents evoked by 5 µM
GABA in acutely isolated rat pyramidal neurons.
Model simulations
To provide a better quantitative description of proton effect on
GABAA receptor gating, we used simulations based on the Jones and
Westbrook model (1995)
(Fig. 6A). This scheme
have been demonstrated to fulfill the criteria for a minimum requirement
model, allowing us to reproduce the basic kinetic properties of
GABAA receptors properly, such as the presence of two binding
sites, saturation of onset rate at high [GABA], desensitization onset, and
slow deactivation because of functional coupling between slow unbinding and
desensitization (Jones and Westbrook,
1995; Jones et al.,
1998; Mozrzymas et al.,
1999; Barberis et al.,
2000).
Simulations of current responses to ultrafast GABA applications
The analysis of the current responses to ultrafast GABA applications
provided important information on possible mechanisms underlying the effect of
hydrogen ions on GABAA receptor gating. However, because the
occupancy of any conformation depends on all of the transition rates and
occupancies of all other states, it is difficult to assess any selected rate
constant based on the use of any single experimental protocol. Therefore, the
optimization procedure was performed until the entire set of the rate
constants allowed us to reproduce optimally the time course of currents
recorded with all of the protocols that were used (Figs.
2,
3,
4,
5). The most important
experimental observations were that an increase in pH caused (1) a decrease in
amplitude of currents evoked by saturating [GABA]
(Fig. 2A-E), whereas
the opposite effect was observed at low [GABA]
(Fig. 5D,E), (2) an
increase in the rate and extent of desensitization
(Fig. 4A-E), and (3)
an increase in the onset rate of responses evoked by nonsaturating [GABA]
(Fig. 5).
In general, the effect of proton concentration not only might affect the
rate constants but also induce a rearrangement of the kinetic scheme. In such
a case one would expect that macroscopic characteristics of currents (e.g.,
desensitization or rise time) would show mixed kinetics described by time
constants typical for control (pH 7.2) and other modes with weights depending
on pH. However, the analysis of the current rising phase, desensitization, and
deactivation indicated gradual proton-induced changes. Similarly, a change in
receptor properties in a discrete manner (mode switch) would be expected to
increase the number of macroscopic kinetic components that, as mentioned, was
not observed. On the other hand, it cannot be excluded that progressive
protonation/deprotonation of GABAAR macromolecules induces a number
of mode-like changes. Such multimodal transitions when a large (and
heterogeneous) channel population is observed could give rise to a
progressive, gradual change in current kinetics. This suggests that
proton-induced mode switching would rely on modification of the
GABAAR microscopic gating by hydrogen ions.
To quantify our experimental observations, we have made an attempt to
reproduce the proton-induced effects by gradual variations of the respective
rate constants. Our experiments suggested that a rise in pH could induce an
increase in d2, 2, and
kon and a decrease in r2. Initially,
for control conditions the set of the rate constants assessed by Barberis et
al. (2000) was used. As
expected, a reduction of d2 produced a slower
desensitization onset and an increase in amplitude of currents evoked by
saturating [GABA]. However, when d2 was reduced, the
current amplitude could be enhanced by at most
[d2/(2+d2), i.e., for
2 = 8 msec-1 and d2 = 1.5
msec-1 (Barberis et al.,
2000)] by 16%, which is much less than in the experiment
(Fig. 2A-E). A similar
difficulty was encountered when the rate constants from other studies were
considered (Jones and Westbrook,
1995; Maric et al.,
1999; Banks and Pearce,
2000; Burkat et al.,
2001) because all of them postulate that the opening
2 is much faster than the desensitization rate
d2. However, when (for pH 7.2) 2 = 3
msec-1 and d2 = 12 msec-1 are set, a
decrease or increase in d2 was sufficient to reproduce the
pH dependence of current amplitudes properly (compare Figs.
6B-E,
2A-E). Such
qualitative change in d2 and 2 with
respect to the previous models also made it necessary to reassess other rate
constants to mimic the currents recorded with other protocols (Figs.
3,
4). In control conditions an
optimal reproduction of deactivation and desensitization kinetics required
setting koff = 1 msec-1 and
r2 = 0.07 msec-1. The rate constants
(
1, 1, d1,
r1) describing transitions for the singly bound open
(AR*) and desensitized (AD) states were estimated
for the same experimental model in a separate study
(Mozrzymas et al., 2003). In
model simulations of current responses to ultrafast GABA applications (Figs.
2,
3,
4,
5) the occupancy of singly
bound states was negligible, and we made a simplifying assumption that the
kinetics of these states does not depend on proton concentration.
As shown in Figure 6, the
sets of the rate constants (Table
1) allowed us to reproduce properly the pH dependence of currents
recorded with various experimental protocols. The major changes were required
in desensitization (d2) and binding
(kon), and small modifications were made in unbinding
(koff) and resensitization rates (r2).
In addition to binding and desensitization rates, the closing rate
2 also had to be altered to reproduce strong pH dependencies
of rapid components of both deactivation
(Fig. 3) and desensitization
(compare Figs. 4,
6F-J). These
modifications allowed us to reproduce the pH-induced changes in amplitudes
(compare Figs. 6B-E,
2E), deactivation
kinetics (compare Figs.
6F-H,
3), rate and extent of
desensitization (compare Figs.
6J,
4A-E), recovery in the
paired pulse protocol (compare Figs.
6K,
4F-I), and rising
phase of currents elicited by low [GABA] (compare Figs.
6L,
5A-C). The recovery
process in the double-pulse experiments
(Fig. 4F-I) is a
complex phenomenon that may depend on resensitization r2,
unbinding koff, desensitization d2,
and opening/closing rates 2/2. Our model
simulations indicate that the observed pH dependence of recovery in this
protocol results mainly from a decrease in the desensitization rate
d2 and, to a much smaller extent, from an increase in
resensitization r2
(Fig. 6K,
Table 1). The present analysis
provided evidence for a somehow surprising conclusion that the fastest
ligand-independent process in the GABAA receptor gating scheme is
not the opening (as assumed in most of models) but desensitization.
The analysis of the rise time kinetics of current responses to saturating
[GABA] revealed that acidic pH slows down the rising phase, whereas basic pH
had only a slight effect (Fig.
2F,G). A slower current onset at acidic pH could suggest
a reduction of the transition rate from the bound closed to the bound open
state (2). However, a direct association of a slower rise
time with a decrease in 2 would be correct only in the case
in which the opening (2) would be much faster than the
desensitization rate (d2), which, according to the results
presented above, seems not to be the case. Moreover, for the bifurcating
reactions (Eq. 3; because d2 >>
r2 and 2 >> 2,
this scheme is expected to give a good approximation for the initial phase of
the current onset for saturating [GABA]) both the onset of current response
(occupancy of AR*) and the entry into the desensitized
state (AD) proceed with the time constant =
1/(+d):
 | (Eq. 3) |
Thus for d2 >> r2 the rising
phase kinetics is shaped predominantly by the desensitization rate
d2 (not by 2), and a decrease in
d2 (at acidic pH) is expected to slow down the rising
phase of currents (Fig.
6I) similar to what was observed in our experiments
(Fig. 2F,G). Basic pH
had only a slight effect on the onset rate of currents evoked by saturating
[GABA] (Fig. 2F,G),
whereas the model simulations predict a more pronounced acceleration of the
current onset (Fig.
6I). This difference results most likely from limits in
the velocity of drug application with our perfusion system.
In the light of investigations based on an analysis of Equation 3, the
pH-induced alterations in the rise time of currents elicited by nonsaturating
GABA concentrations (100-300 µM;
Fig. 5A-C) could be
caused by changes in desensitization kinetics. However, because binding at
this [GABA] is considerably slower than both desensitization
(d2) and opening (2), the kinetics of
receptor activation at these GABA concentrations is shaped predominantly by
the binding rate.
The experiments in which currents were evoked by very low GABA
concentrations (Fig.
5D-F) provided further indication that the binding rate
is upregulated by decreasing concentrations of hydrogen ions. At these
concentrations only a minority of receptors reaches the fully bound state
(especially at 0.2 and 1 µMGABA); therefore, the current
amplitude is expected to depend on the binding rate. The model simulations
well reproduce the experimental findings (for 0.2 µM GABA, the
occupancies of open state were 0.00011, 0.00019, and 0.00035 at pH 6.0, 7.2,
and 8.0, respectively; for 1 µM GABA the amplitudes were
0.00061, 0.0013, and 0.0021 at pH 6.0, 7.2, and 8.0, respectively). Thus our
analysis provides a simple explanation for apparently opposite effect of
hydrogen ions on amplitudes of responses evoked by very low and saturating
[GABA]. At very low agonist concentrations the upregulation of the affinity
increases the chance for the receptor to activate, whereas at high [GABA] (at
which binding is close to being complete) the current amplitude is set by the
balance between entrance into the open
(A2R*) and desensitized state
(A2D). It is expected that at sufficiently high
[GABA] an enhanced recruitment into the bound states caused by an increase in
kon at basic pH will be counterbalanced by an increased
entrance into the desensitized state. Such a trend is reflected by the
observation that the current amplitudes evoked by 5 µM GABA are
comparable in control conditions (pH 7.2) and at basic pH 8
(Fig. 5D,E), whereas,
for example, at 100 or 300 µM GABA the basic pH strongly reduces
current amplitude.
Simulation of proton effects on synaptic currents
The model simulations of current responses evoked by ultrafast agonist
applications provided key information on the effect of hydrogen ions on the
GABAA receptors. However, although protons exert a similar effect
on decaying phases of IPSCs and current responses (Figs.
1,
3), there is a striking
disagreement in the effect on the amplitudes of these currents (compare Figs.
1D,
2E). A possible
explanation for this discrepancy is that the conditions of receptor activation
are different in the two situations. Indeed, it has been demonstrated that the
differences between synaptic agonist transient (very fast exponential-like
decay) and that applied with ultrafast perfusion (square-like) may give rise
to different drug effects on mIPSCs and on current responses to rapid GABA
applications (Mozrzymas et al.,
1999; Barberis et al.,
2000). To elucidate to what extent such difference in the agonist
waveform could account for different effects of protons on these currents, we
used model simulations (with models described in
Fig. 6A,
Table 1) in which synaptic
agonist transient was modeled as an exponentially decaying function:
A · exp(-t/), where A is the peak value
and is the time constant of the agonist clearance. was varied
between 50 and 1000 µsec, and A was considered in the range
between 1 and 5 mM. In Figure
7A, simulated current responses to such
"synaptic" GABA applications are shown for various pH and for
A = 1.5 mM and = 125 µsec. These synaptic current
responses well reproduced the observation that, when proton concentration is
increased, the fast component of the decaying phase (fast)
decreased and the slow one (slow) increased (compare Figs.
7B,C,
1G,H). The 
2 statistics were used to select the values of A and
that best reproduced the pH dependence of the mIPSC amplitudes
(Fig. 7D,E). A good
reproduction of experimental data (Fig.
1) was obtained for A in the range 1.5-3 mM
and within 0.075-0.125 msec. For the following pairs of A,
, we obtained the highest fit quality: 3 mM, 0.075 msec; 2
mM, 0.1 msec; and 1.5 mM, 0.125 msec. The reproduction
of the pH dependence of the modeled mIPSCs was critically dependent on the
time constant ; when this parameter was increased above 150-200 µsec,
the fit quickly deteriorated. For instance, for = 300 and 1000 µsec
(A = 1.5 mM) the quantitative reproduction of mIPSC pH
dependence was poor, but the property that peak current shows a maximum at
acidic pH was still present (compare Figs.
7E,
1D). For the above
mentioned values of A and , the model simulation allowed us to
reproduce well the inhibition of synaptic currents at high proton
concentration, an increase in amplitude at approximately pH 6.5, and a minor
effect on mIPSC amplitude at basic pH (Fig.
7D). Particularly striking was the opposite effect of
strongly acidic pH values on amplitudes of mIPSCs
(Fig. 1) and of current
responses (Fig. 2). Our
analysis provides an explanation for this apparent discrepancy. When
kon is reduced, the receptor activation slows down, and
the exposure to synaptic agonist transient becomes too short to activate the
same number of channels as in control conditions. The apparent lack of effect
on mIPSC amplitudes at basic pH seems to result from a mutual compensation of
two factors: enhancement of receptor binding rate and increased entrance into
the desensitized state A2D.
View larger version (49K):
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|
Figure 7. Model simulation of synaptic currents. Synaptic currents were simulated as
current responses to exponentially decaying synaptic agonist transient
[A · exp(-t/), where A is the peak concentration
and is the time constant of agonist clearance]. A, Synaptic
currents simulated for models with rate constants optimized to reproduce the
GABAA receptor gating at various pH values (see
Table 1) and assuming the peak
agonist concentration of A = 1.5 mM and a time constant of
agonist clearance of = 0.125 msec. B, C, Normalized
responses to synaptic GABA transient (A = 1.5 mM, =
0.125 msec) are shown in two different time scales for pH 6.0, 7.2 (thick
line), and 8.0. The effect of proton concentration on the fast and slow
component of the decaying phase qualitatively reproduces the experimental
observations (compare with Fig.
1). D, pH dependence of the amplitudes of synaptic
currents simulated for agonist transient described by A = 1.5
mM and = 0.125 msec. This prediction well reproduces the pH
dependence of mIPSC amplitudes (see
Fig.1). E, Simulation
of the IPSCs for A = 1.5 mM and =0.3 msec (open
bars) and 1 msec (filled bars). Note that in both cases the amplitude pH
dependence shows a peak similar to that of synaptic currents (see
Fig. 1 D). F,
Optimization of A and parameters to best reproduce the pH
dependence of mIPSCs. Standard 2 statistics were used for a
comparison of pH dependence of mIPSC amplitudes (see
Fig. 1) and amplitudes of
simulated responses to synaptic GABA application. The values of A and
parameters were varied to minimize the value of 2
statistics. The highest fit quality (lowest 2 statistics
values) was obtained for the following (A, ) parameters: 3
mM, 0.075 msec; 2 mM, 0.1 msec; and 1.5 mM,
0.125 msec.
|
|
|
Discussion
|
|---|
The major finding of the present work is a demonstration that protons
affect the mIPSCs by modulating desensitization and the affinity of
GABAA receptors.
Our analysis indicates that, at physiological pH, saturating [GABA]
activate only a minority of receptors (19%) while remaining ones enter the
desensitized conformation. This hypothesis is supported by the fact that a
decrease in pH enhances responses to saturating [GABA] by several-fold whereas
single-channel conductance is not affected
(Krishek and Smart, 2001). Our
assessment of maximum open probability differs from those reported by other
authors (in the range 0.5-0.8; Newland et
al., 1991; Jones and
Westbrook, 1995; Nusser et al.,
1997,
2001;
Mozrzymas et al., 1999;
Perrais and Ropert, 1999;
Barberis et al., 2000). The
source of this discrepancy is not clear. A non-equilibrium analysis at the
single-channel level will be required to elucidate this issue.
Further arguments for crucial role of desensitization
As mentioned, desensitization plays an important role in shaping the
GABAergic currents. Besides its impact on deactivation, the predictions of the
model of Jones and Westbrook
(1995) as well as of other
investigators (Gingrich et al.,
1995; Haas and Macdonald,
1999) indicate that desensitization may affect current amplitude.
In the present work we further confirm this prediction and provide evidence
that this effect is much stronger than what was predicted by previous models.
In addition, we show that desensitization affects the onset of currents
elicited by saturating [GABA]. We propose that such strong desensitization
impact on GABAAR kinetics reflects the fact that this process is
the fastest ligand-independent transition (in control conditions). This is
supported by a strong pH dependence of current amplitudes and desensitization
kinetics (Figs. 2,
4; see Model simulations). An
alternative explanation for the pH dependence of current amplitude could be
alteration of the opening rate 2. However, such a mechanism
would lead to an accelerated onset of currents at acidic pH, contrary to
experimental observation (Fig.
2F).
Effect of protons on GABAAR affinity shapes the responses
to low [GABA]
The analysis of the onset rate of currents
(Fig. 5) evoked by
nonsaturating [GABA] together with model simulations provided key evidence
that the binding rate is enhanced by an increase in pH. This was confirmed
further by pH dependence of current amplitude evoked by very low [GABA]
(Fig. 5D-F).
Similarly, Pasternack et al.
(1996) have observed that
responses to low [GABA] are increased, whereas those evoked by high [GABA] are
decreased when pH is increased and have attributed this finding to the
presence of two receptor populations. Although in neurons such heterogeneity
is quite likely, our data indicate a different explanation. As pointed out in
Model simulations, at low [GABA] an increase in amplitude with pH is
attributable to an increase in the binding rate, whereas at high [GABA] a
decrease in amplitude reflects preferential entrance into the desensitized
state (d2 >> 2).
Modulation of GABAA receptor by protons reveals synaptic
GABA transient
Our analysis indicates that a striking difference in the amplitude pH
dependencies of mIPSCs and current responses (Figs.
1,
2) results mainly from
different agonist time courses in the two situations. Thus the hydrogen ions
can be used as a convenient modifier of GABAAR gating to probe the
synaptic GABA transient. The time constant of agonist clearance estimated in
the present study (0.075-0.125 msec) was similar to that inferred in our
previous work in which chlorpromazine was used as a modifier of gating
(Mozrzymas et al., 1999). In
the present study we were able additionally to assess the peak GABA
concentration (1.5-3 mM) and to conclude that at physiological pH
the mIPSCs are not saturating (when A is increased from 2.5 to 10
mM, the amplitude of IPSC reaches saturation and increases by
50%). Thus our data reinforce the view that GABAergic mIPSCs are not
saturated (Frerking et al.,
1995; Frerking and Wilson,
1996; Perrais and Ropert,
1999; Hajos et al.,
2000; Nusser et al.,
2001). Moreover, the proposed mechanism implies that
acidification/alkalization moves the activation conditions farther/closer from
saturation.
Although it is possible that protons could affect mIPSCs by modulating
synaptic GABA transient, it is unlikely that they could modify the diffusion
coefficient (and therefore clearance) of GABA. Our data and model simulations
(Figs. 6,
7) are consistent with the pH
dependence of mIPSCs resulting from proton modulation of GABAA
receptor gating with negligible effect on synaptic GABA transient. A
contribution from proton effect on releasing mechanism, however, cannot be
ruled out based only on electrophysiological data. Neurochemical approaches
would help to elucidate this issue. The mechanism of presynaptic proton effect
leading to the increase in mIPSC frequency is unknown and will be investigated
in a separate study.
Physiological significance
The present study provides further evidence that GABAA receptors
are highly sensitive to protons. We show that variation in the range of a few
tenths of pH unit gives rise to significant modulation of the GABAA
receptor (e.g., Figs. 2,
3,
4). It is known that such range
of variation may occur in physiological (e.g., because of
HCO3- transport) (for review, see
Kaila, 1994) and in
pathological conditions such as epilepsy, ischemia, and hypoxia
(Chesler, 1990;
Chesler and Kaila, 1992). It
is worth noting that, even if basic pH weakly affects mIPSC amplitudes
(Fig. 1), the fact that
receptors get closer to saturation may alter the susceptibility of mIPSCs to
modulation by other drugs. In addition, the enhancement of GABAA
receptor affinity at basic pH is expected to increase the shunting inhibition
by ambient GABA.
Validity of experimental approach and model
In the present study the main body of evidence is derived from experiments
on the outside-out patches that contain an unknown mixture of synaptic and
extrasynaptic receptors. Moreover, the intracellular soluble messengers are
lost after patch excision. However, it may be expected that the effects of
protons on synaptic receptors and on those in patches excised from the same
neurons are qualitatively similar. The fact that for both mIPSCs and current
responses the rising and decaying phases showed similar kinetics and pH
dependence (Figs. 1,
2,
3) supports this
hypothesis.
In the present work we used a relatively simple model
(Fig. 6A). In
particular, fully bound slow desensitized states were not considered because
they are believed to play a minor role in shaping the synaptic currents. On
the other hand, the fact that similar kinetics of current responses could be
reproduced by using considerably different sets of the rate constants
(Barberis et al., 2000; this
study) indicates model degeneration that can be reduced by including new
experimental data. Key evidence enabling us to correct the rate constants was
strong pH dependence of responses to saturating [GABA].
Previously, we have described the effects of chlorpromazine and zinc on
microscopic gating of GABAAR
(Mozrzymas et al., 1999;
Barberis et al., 2000). With
the use of the present model, the effects of these drugs also could be well
reproduced by manipulating the same rate constants as the model structure
remains the same. In addition, it is possible that protons might affect the
cooperativity of GABAARs binding sites, which recently was
described in different preparations
(Lavoie et al., 1997;
McClellan and Twyman, 1999;
Mozrzymas et al., 2003).
However, most of proton effects are manifested at saturating [GABA] at which
cooperativity (and binding in general) is not crucial.
Connections between singly and doubly bound open and desensitized states
(Twyman et al., 1990;
Jones et al., 1998) (classical
model for AChR: Katz and Thesleff,
1957; Cachelin and Colquhoun,
1989) may affect the receptor kinetics. Moreover, recent studies
(Chang et al., 2002;
Scheller and Forman, 2002)
have suggested an important role of connections between unbound and bound open
and desensitized conformations. However, recordings performed by Chang et al.
(2002) had the time resolution
of seconds and therefore cannot be referred directly to the synaptic currents.
Scheller and Forman (2002)
found that connections between unbound and bound open and desensitized states
were necessary for considered mutated receptors, whereas the wild type of
channel was described satisfactorily by assuming a minor role of these
transitions. This finding is compatible with analysis of Jones et al.
(1998), who found that, in
native receptors, rate constants for connections between singly and doubly
bound desensitized states were orders of magnitude smaller than those
connecting the closed states. It is worth noting that strong pH dependencies
of amplitudes (Fig. 2) and
de
Top
Abstract
Introduction
