Department of Pharmacology, University of North Texas Health Science Center at Fort Worth, Fort Worth, Texas 76107
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ABSTRACT |
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Huang, Ren-Qi and
Glenn H. Dillon.
Effect of Extracellular pH on GABA-Activated Current in Rat
Recombinant Receptors and Thin Hypothalamic Slices.
J. Neurophysiol. 82: 1233-1243, 1999.
We studied the effects
of extracellular pH (pHo) on -aminobutyric acid
(GABA)-mediated Cl
current in rat hypothalamic neurons
and recombinant type-A GABA (GABAA) receptors stably
expressed in human embryonic kidney cells (HEK 293), using whole cell
and outside-out patch-clamp recordings. In
3
2
2s receptors,
acidic pH decreased, whereas alkaline pH increased the response to GABA
in a reversible and concentration-dependent manner. Acidification
shifted the GABA concentration-response curve to the right,
significantly increasing the EC50 for GABA without
appreciably changing the slope or maximal current induced by GABA. We
obtained similar effects of pH in
1
2
2 receptors and in
GABA-activated currents recorded from thin hypothalamic brain slices.
In outside-out patches recorded from
3
2
2 recombinant receptors, membrane patches were exposed to 5 µM GABA at control (7.3), acidic (6.4), or alkaline (8.4) pH. GABA activated main and
subconductance states of 24 and 16 pS, respectively, in
3
2
2 receptors. Alkaline pHo increased channel opening frequency
and decreased the duration of the long closed state, resulting in an
increase in open probability (from 0.0801 ± 0.015 in pH 7.3 to
0.138 ± 0.02 in pH 8.4). Exposure of the channels to acidic pHo had the opposite effects on open probability (decreased
to 0.006 ± 0.0001). Taken together, our results indicate that the function of GABAA receptors is modulated by extracellular
pH. The proton effect is similar in recombinant and native receptors and is dependent on GABA concentration. In addition, the effect appears
to be independent of the
-subunit isoform, and is due to the ability
of H+ to alter the frequency of channel opening. Our
findings indicate that GABAergic signaling in the CNS may be
significantly altered during conditions that increase or decrease pH.
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INTRODUCTION |
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Changes in brain pH are known to occur during
physiological and pathophysiological conditions (Kaila and
Ransom, 1998; Takahashi and Copenhagen 1995
).
When central pH does change, neuronal function may be adversely
affected. The mechanism(s) by which these changes in neuronal function
occur during acidosis and alkalosis have not been fully determined.
Some factors known to be influenced by pH levels, and thus possibly
important in altering neuronal function, include free radical formation
(Waterfall et al. 1996
), ATP metabolism (Trafton
et al. 1996
), astrocytic L-glutamate uptake (Bender et al. 1997
) and protein denaturation
(Kalimo et al. 1981
).
An additional mechanism for acidosis- or alkalosis-induced changes in
neuronal function appears to be changes in the activity of ion
channels. Extracellular acidification strongly decreases the opening of
voltage-gated Na+, K+, and
Ca2+ channels (Daumas and Andersen
1993; Tombaugh and Somjen 1996
). This effect has
also been observed in some ligand-gated ion channels. For instance,
N-methyl-D-aspartate (NMDA) receptor-activated
channels are markedly inhibited by increases in hydrogen ion
concentration (Chen et al. 1998
; Tang et al.
1990
). Decreasing pH also inhibits glycine- and
acetylcholine-gated channels (Landau et al. 1981
; Tang et al. 1990
). However, reports of the effects of
hydrogen ion on GABA-gated chloride channels have been contradictory.
Acidification of extracellular media has been shown to inhibit
(Smart 1992
; Zhai et al. 1998
), stimulate
(Pasternack et al. 1992
; Robello et al.
1994
), or have no significant effect (Tang et al.
1990
; Vyklický et al. 1993
) on the
activity of these channels. These varying responses may in part be due
to differences in experimental conditions, or to expression of
different GABAA receptor subunits in the
preparations studied (Krishek et al. 1996
).
Regardless of the effects attributed to pH in these previous
investigations, the mechanism of its action at the single-channel level
has not been determined. In the present study, we have characterized the pH sensitivity of GABA-activated Cl current
recorded from both recombinant and native GABAA
receptors. Our results indicate that H+ inhibits
the activity of GABAA receptors recorded from
both preparations. H+ inhibits the activity of
these channels by decreasing the opening frequency of single GABA-gated
channels, without affecting single-channel conductance or mean open
time. In addition, the effect of H+ does not
appear to be influenced by the
-subunit isoform present in the receptor.
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METHODS |
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Cell preparation
CLONED GABAA RECEPTORS.
HEK 293 cells stably expressing configurations of rat
GABAA receptors were generously supplied by
Pharmacia-Upjohn (Kalamazoo, MI). A detailed description of the
preparation of HEK 293 cells stably expressing
GABAA receptors has been published previously (Hamilton et al. 1993). Briefly, the cells were
transfected with plasmids containing cDNA for various
GABAA receptor subunits and a plasmid encoding
G418 resistance. After 2 wk of selection in 1 mg/ml G418, resistant
cells were assayed by Northern blotting for the ability to synthesize
GABAA receptor mRNAs. In the present report, we
studied HEK 293 cells stably expressing either
1
2
2S or
3
2
2S (subsequently denoted as
1
2
2 and
3
2
2,
respectively) configurations of the receptors.
HYPOTHALAMIC NEURONS.
Effects of pH on GABAA receptors of thin
posterior hypothalamic brain slices were also analyzed. Long Evans rats
(Harlan Sprague Dawley, Indianapolis, IN), postnatal day
0-14 (either sex) were rapidly decapitated. Chemical anesthesia
was not used because of its well-known influence on
GABAA receptors (Franks and Lieb 1994). Rat pups of this age can be anesthetized using
hypothermia (Cunningham and McKay 1993
), and thus all
pups were anesthetized in this fashion. All procedures were conducted
in accordance with institutional and federal guidelines. All stages of
brain dissection and tissue slicing were conducted in ice-cold
(~4°C) artificial cerebral-spinal fluid (ACSF) of the following
composition (in mM): 124 NaCl, 5.0 KCl, 1.3 MgSO4, 26 NaHCO3, 1.24 KH2PO4, 2.4 CaCl2, and 10 glucose, pH ~7.4 after
equilibration with a 95% O2-5%
CO2 gas mixture (carbogen). Thin hypothalamic slices (200 µm) were cut with a vibratome (VSL, World Precision Instruments); slices were submerged in ACSF (22-25°C) aerated with the carbogen gas mixture. Slices were transferred to a recording chamber (~2 ml)
and superfused (7-10 ml/min, 22-25°C) with saline. To minimize synaptic influences on neurons under investigation, experiments were
conducted in a synaptic blockade medium consisting of the following (in
mM): 128 NaCl, 3.0 KCl, 11.4 MgCl2, 10 HEPES, 2.0 CaCl2, and 10 glucose, pH 7.3. In some studies examining
current-voltage relationships, TTX (20 nM) was added to minimize
potential influences of synaptic transmission.
Electrophysiology
RECOMBINANT RECEPTORS.
Whole cell and outside-out patch recordings were made at room
temperature (22-25°C). Except during acquisition of current-voltage relationships, cells were voltage clamped at 60 mV. Patch pipettes of
borosilicate glass (1B150F, World Precision Instruments, Sarasota, FL)
were pulled (Flaming/Brown, P-87/PC, Sutter Instrument, Novato, CA) to
a tip resistance of 1-2.5 M
for whole cell recordings, and 8-12
M
for outside-out single-channel recordings. The pipette solution
contained (in mM) 140 CsCl, 10 EGTA, 10 HEPES, and 4 Mg-ATP; pH 7.2. As
we reported previously, high ATP and EGTA was included in the
intracellular solution to prevent current rundown during a prolonged
period of whole cell recording (Huang and Dillon 1998
).
Coverslips containing cultured cells were placed in a small chamber
(~1.5 ml) on the stage of an inverted light microscope (Olympus
IMT-2) and superfused continuously (5-8 ml/min) with the following
external solution containing (in mM) 125 NaCl, 5.5 KCl, 0.8 MgCl2, 3.0 CaCl2, 20 HEPES,
and 25 D-glucose, pH 7.3. GABA-induced
Cl
currents from the whole cell or outside-out
configuration of the patch-clamp technique were obtained using an
Axoclamp 200A amplifier (Axon Instruments, Foster City, CA) equipped
with a CV-4 headstage. For whole cell recording, GABA-induced
Cl
currents were low-pass filtered at 5 kHz,
monitored on an oscilloscope and a chart recorder (Gould TA240), and
stored on a computer (pClamp 6.0, Axon Instruments) for subsequent
analysis. Series resistance compensation (60-80%) was applied at the
amplifier. To monitor the possibility that access resistance changed
over time or during different experimental conditions, at the
initiation of each recording we measured and stored on our digital
oscilloscope the current response to a 5-mV voltage pulse. This stored
trace was continually referenced throughout the recording. If a change
in access resistance was observed throughout the recording period, the
patch was aborted and the data were not included in the analysis. For
single-channel recordings, the currents were filtered with a low-pass
bessel filter (80 dB/decade) at cutoff frequency of 1-2 kHz, and
simultaneously recorded on a video cassette recording system (Sony
SLV-420) via a digital data recorder (VR-10B CRC, Instrutech, Great
Neck, NY).
BRAIN SLICE.
The rapid perforated patch recording technique was used to record
hypothalamic neurons. Briefly, amphotericin B (0.26 mM) taken from
stock solution (60 mg/ml in dimethyl sulfoxide) was added to the
pipette solution listed above. The pipette resistance was 7-8 M.
Full perforation with a low access resistance (~8 M
) was achieved
typically within 1 min and lasted ~3 h. All other recording
conditions were the same as described for recombinant receptor preparations.
EXPERIMENTAL PROTOCOL.
pH of external solutions was altered by addition of NaOH or HCl and
routinely checked before and during experiments. The osmolality of the
control external medium (pH 7.3) was 330 ± 9.1 mosM and was not
changed in basic or acidic solutions (333 ± 7.3 and 348 ± 5.8 mosM, respectively; Micro Osmometer, Precision Systems, Natick,
MA). For all single-channel recordings and whole cell recordings in HEK
293 cells and brain slices, GABA was prepared in the extracellular
solution and was applied from independent reservoirs by gravity flow
for 10-20 s to cells or membrane patches using a Y-shaped tube
positioned within 100 µm of the cells or the membrane patch. With
this system, the 10-90% rise time of the junction potential at the
open tip was 12-51 ms. Receptors were typically activated with roughly
the EC25 GABA concentration; this concentration
was chosen because minimal desensitization, which may confound
interpretation of results, was elicited. Once a control GABA response
was determined, the effect of pH on the response was examined. To
assess the pH effect, cells were first bathed in media that was set to
the test pH, then GABA, dissolved in the same test pH solution, was
applied to the cells. Because in the whole cell recordings, external
solution of low pH elicited, as in other studies (Bevan and
Yeats 1991; Krishek et al. 1996
; Zhai et
al. 1998
), a transient whole cell inward current, GABA application at various pH test values was made after the transient current had recovered and stabilized. GABA applications were separated by at least 2-min intervals to ensure both adequate wash out of GABA
from the bath and recovery of receptors from desensitization, if present.
Data analysis
WHOLE CELL RECORDINGS.
All data were recorded on a chart recorder and stored on a computer for
subsequent off-line analysis (pClamp 6.0, Axon Instruments). GABA
concentration-response profiles were fitted to the following logistic
equation: I/Imax= 1/[1 + (EC50/[GABA])n], where
I and Imax represent the
normalized GABA-induced current at a given concentration and the
maximum current induced by a saturating [GABA],
EC50 is the half-maximal effective GABA
concentration, and n is the slope factor. The time constant
for current decay was used to analyze the effect of protons on receptor
desensitization. The time constant was obtained by fitting an
exponential function to time course-current decay profiles induced by
saturating [GABA] (Clampfit, pClamp, 6.0, Axon Instruments). Time
constants for current decay during application of low [GABA] were
slow and difficult to determine accurately, so percentage of
desensitization (%D) was determined during these conditions.
Percentage of desensitization produced by a low [GABA] application
was calculated according to the formula %D = 100 × (IGABApeak IGABA10s)/IGABApeak,
where IGABApeak is the current at the
peak of the GABA response and IGABA10s
is the current remaining after 10 s of continuous GABA application.
SINGLE-CHANNEL RECORDINGS.
Single-channel currents were replayed from tape and digitized at 53-kHz
sampling frequency. Single-channel current amplitudes and durations
were determined by computer using Fetchan and pStat (pClamp 6.0 software). Channel openings and closings were detected with the 50%
threshold crossing method [based on the main (24 pS) conductance
state]. Openings briefer than 1 ms (approximately equal to the system
dead time) were not detectable. Out of eight patches we studied,
subconductance levels were observed in two patches and accounted for
<10% of the total openings. Although subconductance events were
excluded from dwell analysis by deletion from the events lists,
additional analysis showed that their inclusion did not affect mean
duration of open or closed states. Only patches demonstrating
infrequent multiple openings (no more than 2 simultaneous openings
apparent) were used for kinetic analysis. To reduce errors due to
multichannel patches, we excluded overlaps of these infrequent simultaneous openings in the analysis of closed state dwell data. The
presence of multiple openings would decrease the apparent duration of
longer close components but would have no effect on the open state
properties. Duration histograms were constructed as described by
Sigworth and Sine (1987) and fitted by a maximum likelihood method. The number of exponential functions required to fit
the distribution was increased until additional components did not
significantly improve the fit. Open channel probability (Po) was calculated as
Po = time in open channel state/(total time × number of channels in patch).
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RESULTS |
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Effect of extracellular pH on GABA-activated currents in recombinant receptors
To examine whether different receptor configurations might vary in
their pH sensitivity, we studied the effects of pH on the GABA response
in 1
2
2 and
3
2
2 receptors, configurations with distinct distributions in the CNS. Based on our initial
characterization of the recombinant receptors, 20 and 5 µM GABA are
approximately EC25 concentrations for
3
2
2 and
1
2
2 receptors, respectively. Application of 5 and 20 µM GABA to
1
2
2 and
3
2
2 receptors, respectively, typically elicited little or no receptor desensitization. This is evident by the maintenance of GABA-activated current amplitude throughout the GABA application. In assessing the effect of pH on the
EC25 GABA response, we varied the pH of the
external medium between 8.4 and 6.4. The modulation of GABA-activated
current by extracellular pH is illustrated in Fig.
1. The amplitude of currents was greatly
increased when pH was increased from 7.3 to 7.9 and 8.4, and was
markedly attenuated when pH was decreased from 7.3 to 7.0 and 6.4 (Fig.
1A). No adaptation to the proton effect was observed during
up to 15 min of prolonged perfusion with acidic or alkaline medium
(data not shown). Furthermore, there was no significant change in
access resistance induced by changes in extracellular pH. On average,
compared with the control at pH 7.3 (designated 100%), the GABA
current was enhanced at pH 8.4 to 295 ± 34% and to 265 ± 38% of control in
1
2
2 (n = 5) and
3
2
2 (n = 10) receptors, respectively.
Acidification had the opposite effect; an external pH of 6.4 reduced
GABA current amplitude to 58 ± 6.3% and 41 ± 7.1% of
control in
1
2
2 (n = 5) and
3
2
2
(n = 8) receptors, respectively. Proton inhibition of
current activated by an EC25 GABA concentration
was observed in all cells and was both rapid and completely reversible.
Figure 1B shows the average sensitivity of GABA-activated
current to protons over the range of pH 6.4-8.4. Fitting of the data
to the logistic equation yielded a transition point of pH 7.7 for both receptor configurations. The effect of protons was not significantly different in
1
2
2 and
3
2
2 receptors.
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To test whether protons decreased GABA-activated current amplitude by
altering the Cl ion driving force, the effect
of protons on the reversal potential of GABA-activated current was
examined in
1
2
2 and
3
2
2 GABAA receptors. Figure 2 shows the
current-voltage relationship for GABA-activated current at pH 7.3, 7.9, and 7.0. In
1
2
2 receptors, the reversal potential was 1.5 ± 1.4 (SE) mV at pH 7.3,
0.7 ± 2.1 mV at pH 7.9, and
0.6 ± 1.8 mV at pH 7.0 (n = 5), respectively. These values are not significantly different (P > 0.05, paired t-test). In
3
2
2 receptors, the
reversal potential was more positive than that recorded from
1
2
2 receptors, although similarly unaffected by pH [8.1 ± 2.7 mV at pH 7.3, 10.5 ± 2.7 mV at pH 7.9 and 6.0 ± 2.2 mV at pH 7.0 (n = 5) respectively]. It appears, therefore, that the acidic medium does not alter the
Cl
pump mechanism.
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Protons might produce their inhibition of GABA-activated current by
decreasing the affinity of the receptors for GABA, or decreasing the
efficacy of GABA at the receptor, or both. To distinguish among these
possibilities, current was activated by various concentrations of GABA
at different pH values. Concentration-response curves for
GABA-activated currents at pH 7.0, 7.3, and 7.9 were determined for
both 1
2
2 and for
3
2
2 receptors. As reported in Fig. 3, the EC50 values
for these concentration-response curves were 15.9, 14, and 6.3 µM at
pH 7.0, 7.3, and 7.9
1
2
2 receptors, respectively. In
3
2
2 receptors, the EC50 values were 65, 53, and 25 µM at pH 7.0, 7.3, and 7.9, respectively. The
EC50 value was decreased significantly with the
increase in pH (ANOVA, P < 0.05, n = 6-20) in both the
1
2
2 and
3
2
2 receptor
configurations. In contrast, the maximal GABA-activated currents and
the Hill coefficient values were not significantly influenced by
changes in pH.
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A possible mechanism by which protons might inhibit GABA-activated
current is to increase receptor desensitization. To test this
possibility, the percentage desensitization (%D) of current decay (for
low [GABA]) or time constant of current decay (high [GABA], see
METHODS) were determined during different pH conditions. As
summarized in Table 1, the rate of
current decay induced by GABA slowed with decreasing pH. The
desensitization of 1
2
2 and
3
2
2
GABAA receptor current was more pronounced at
alkaline pH levels at both high and low GABA concentrations
(t-test, P < 0.05). Thus it appears that
protons also have an inhibitory influence on desensitization of
GABAA receptors.
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To gain further insight into the functional effects of protons on
GABA-activated currents, we recorded single-channel currents at control
(pH 7.3), acidic (pH 6.4), and alkaline (pH 8.4) conditions, using
excised outside-out patches. Because the effects of pH on 1
2
2
and
3
2
2 receptors were indistinguishable at the whole cell
level, we studied kinetics of single-channel activity in
3
2
2
receptors only.
The characteristics of single GABA-activated channels in
recombinant 3
2
2 receptors are shown in Figs.
4-6
and Table 2. No spontaneous
single-channel activity was observed in HEK cells expressing
3
2
2 receptors, in the absence of GABA. Application to an
excised patch of 5 µM GABA elicited bursts of channel openings of two
distinct amplitudes, indicating the presence of at least two
conductance states (Fig. 4). The conductance states (24 and 14 pS) were
similar to those recorded previously in
1
2
2 receptors (Dillon et al. 1995
). In addition, also as in
1
2
2 receptors, the larger conductance state (Fig.
4B, *) was recorded more frequently than the small
conductance state (Fig. 4B, **). Our observations are
consistent with a previous report indicating the subconductance state
accounts for <10% of total current in the cloned receptors (Angelotti and Macdonald 1993
). Thus formal analyses
were performed on only the large conductance state. Histograms for 5 µM GABA-induced channel openings were best fit with two-exponential
functions, indicating the presence of two open states with mean
durations of 6.0 and 22.6 ms. Channel open frequency during control
conditions was 7.9 ± 2.1 s
1. Closed
dwell-time distributions for single-channel currents were binned
logarithmically and fitted to a logarithmic scale (Fig. 6B).
Exponential fitting of pooled data from all patches (n = 8) indicated three closed states with mean durations of 4.0, 43, and
857.9 ms.
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Increases or decreases in extracellular pH significantly altered
several single-channel characteristics of 3
2
2 receptors. Whereas during whole cell recording a significant transient inward current was elicited by exposure of cells to acidic pH medium, we
observed no proton-induced activation of channel activity during single-channel recordings (data not shown). Elevating pH from 7.3 to
8.4 markedly and reversibly increased the frequency of GABA-gated
channel openings (Fig. 4A). Acidic treatment (pH 6.4) had an
opposite effect (Fig. 4B). As illustrated in a
representative patch (Fig. 5), the current-voltage relationship for the
main conductance state was unaffected by increasing pH. Mean current amplitude was
1.41 ± 0.06 pA in control conditions,
1.41 ± 0.90 pA at pH 6.4 and
1.37 ± 0.03 pA at pH 8.4. However, the
frequency of channel openings was increased by basic (from 7.9 ± 2.1 to 19.1 ± 4.7 · s
1,
P < 0.05) and decreased by acidic conditions (to
1.6 ± 0.3 · s
1, P < 0.05 from control). A summary of effects of alkalinization on open
and closed states is given in Table 2. Basic conditions had no effect
on mean open states or their relative area. The predominant effect of
alkalinization was a reduction in the duration of the long closed state
(Fig. 6B, Table 2). Acidosis elicited opposite effects on
duration of the long closed state, and also had no effect on open
durations. Due to the fact that relatively few openings were observed
during acidic conditions, statistical assessments could not be
determined. The overall effects of pH changes resulted in an increase
in open probability in basic conditions (from 0.080 ± 0.015 at pH
7.4 to 0.138 ± 0.02, P < 0.05) and a decrease in
open probability in acidic conditions (to 0.006 ± 0.001, P < 0.05).
Effect of protons on GABA-activated current in hypothalamic neurons
Hypothalamic neurons have been shown previously to express several
of the subunits studied in the present investigation (Fenelon et
al. 1995; Wisden et al. 1992
). To assess whether
the response to pH of GABAA receptors is
different in recombinant and native preparations, the effect of protons
on hypothalamic GABAA receptors was studied.
Whole cell currents were recorded from posterior hypothalamic neurons
using amphotericin B perforated patch recordings. Figure
7A demonstrates that exposure
to acidic medium produced a reversible inhibition of GABA-activated
current whereas alkalization enhanced the GABA response. The proton
effect was not associated with any change of reversal potential (Fig.
7C). Figure 7B shows the average sensitivity of
GABA-activated current to protons over the range of pH 6.4-8.4. The
current activated by 20 µM GABA at pH 6.4 was reduced to 60.1 ± 5.9% of the control (P < 0.01, n = 4), whereas pH 8.4 enhanced the response to 221.8 ± 46% of
control (P < 0.05, n = 5). Thus the
effects of hydrogen ion on neuronal GABAA
receptors are similar to those observed in recombinant
1
2
2 and
3
2
2 GABAA receptors.
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DISCUSSION |
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The present study, conducted on both recombinant and native rat GABAA receptors, shows that physiological changes in H+ strongly modulate activity of these receptors. Alkalization enhanced, whereas acidification suppressed GABA-activated currents in a rapid and reversible manner, without affecting the maximal currents induced by GABA. Single-channel recordings indicate that this effect is due to changes in channel opening frequency.
Proton effect on GABA-activated responses
Extracellular acidification exerts an inhibitory influence
on receptors for glutamate (Tang et al. 1990), glycine
(Tang et al. 1990
), and acetylcholine (Landau et
al. 1981
), as well as a number of voltage-gated channels
(Daumas and Andersen 1993
; Tombaugh and Somjen
1996
). However, the scanty data on GABA-gated chloride channels
have been complex and contradictory. Previous reports suggest that GABA
responses are inhibited (Smart 1992
; Zhai et al.
1998
), stimulated (Pasternack et al. 1992
;
Robello et al. 1994
), or unaffected (Tang et al.
1990
; Vyklický et al. 1993
) by pH. One
factor that may contribute to the reportedly divergent effects of pH on
GABAA receptors is differences in GABA application. For example, bath application of GABA results in much
slower channel activation, and thus only effects of a potential modulator on steady-state current can be resolved; effects on peak
current amplitude cannot be determined. This situation was noted in the
study of Krishek et al. (1996)
, as they observed that
peak current as well as current decay were increased at alkaline pH in
mouse
1
1 receptors rapidly activated in HEK 293 cells. Analysis
of the same receptor expressed in Xenopus oocytes and activated via bath application led to the conclusion that it was inhibited by basic conditions, because only the steady-state currents could be resolved. Thus at least some of the apparent discrepancies of
results between studies can be satisfactorily explained by differences
in GABA application kinetics.
An additional factor that may be involved in the reported disparate
effects of protons on GABAA receptors is subunit
composition. Krishek et al. (1996) reported that murine
1
1 receptors expressed in Xenopus oocytes are
stimulated by protons, whereas
1
1
2s receptors were insensitive
to pH. The
-subunit also affected the observed response to pH,
because cells expressing
1
1
2s
subunits were inhibited by
acidic or basic pH. In the present study, the qualitatively similar
H+ sensitivity of rat
1
2
2 and
3
2
2 receptors suggests the proton effect is only modestly
influenced by the
-subunit isoform present in the receptor.
Furthermore, our preliminary studies on human recombinant
1
2
2
GABAA receptors show that extracellular acidosis (pH 6.4) inhibits the response to 5 µM GABA to 66.25 ± 6.9% of the control (pH 7.3); alkalosis (pH 8.4) enhanced the GABA response to
140 ± 14% (n = 5, P < 0.05).
This is similar to the effect we observed in rat
GABAA receptors and suggests that no notable difference in H+ sensitivity exists between rat
and human
1
2
2 GABAA receptors.
Another factor that should be taken into account is the use of
anesthesia in some previous studies on the effects of pH on neuronal
GABAA receptors (Krishek et al.
1996; Pasternack et al. 1996
; Robello et
al. 1994
). Anesthetics are well known to affect GABAA receptor function (Franks and Lieb
1994
) and thus may alter their responsiveness to protons. In
addition, it is possible that uptake mechanisms for GABA are also
affected by pH. Thus, because postsynaptic effects of GABA can be
modulated by its uptake (Curtis et al. 1976
), results
from some in vivo studies may reflect effects of pH on presynaptic and
postsynaptic events simultaneously. Our results, which were obtained
using a rapid agonist application system and with no anesthesia
present, are consistent with those reported by Zhai et al.
(1998)
. They found that extracellular H+
decreased peak GABA current and slowed desensitization of rat dorsal
root ganglia neurons, without changing efficacy.
Site and mechanism of proton modulation
Because the effects of pH on the
GABAA receptor occur across the physiological
range, the nature of the influence of pH on this receptor is of obvious
interest. At physiological pH, GABA is an electrically neutral
zwitterion (pI = 7.32) (Greenstein and Winitz
1961), carrying a positive charge at its amino group, and a
negative charge at its acid group. Thus changes in the charge of the
molecule itself may be responsible for the influence of pH. A recent
model by Krishek et al. (1996)
proposes that, over the
pH range studied in the present investigation, >90% of the GABA
molecule remains in the zwitterionic form. The validity of this pH
model has not been established, however, so the hypothesis that some
effect of pH is due to changes in the GABA molecule itself cannot be
completely dismissed. An additional potential effect on the GABA
molecule itself, which cannot be ruled out unequivocally, is a possible
pH-induced change in its hydration shell. This could theoretically
change the interaction of GABA with its binding domain, and hence alter
function. However, because hydration of a molecule is
predominantly influenced by its charge (Roberts and
Sherman 1993
), and if one accepts the contention that
effect of pH on the charge of the molecule is minimal, it is
unlikely that the hydration shell is significantly altered.
It is possible, however, that the effect of H+ is
not due to alteration of the GABA molecule, but instead due to
protonation of the GABAA receptor or some
associated regulatory protein. In support of the former view is recent
work from Wegelius et al. (1996, 1997
).
They have shown that the rat
1 GABA receptor is down-modulated by
protons across the pH range of 6.4-8.4. Human
1 GABA receptors, on
the other hand, are only sensitive to shifts in pH more acidic than
7.4. Mutational analysis demonstrated that this difference in
H+ sensitivity is due to the presence of distinct
amino acids in the extracellular domains of the two receptors.
Additional support for the hypothesis that pH alters the receptor
itself comes from Kashiwagi et al. (1996)
, who found
that an aspartate residue in the extracellular loop of the NMDA
receptor controls sensitivity to protons. It is unknown, however, if
these residues form part of a putative binding site for
H+, or are involved in the subsequent functional
effect that is elicited by H+.
One must also consider the possibility that the influence of pH on the
GABAA receptor is mediated via secondary changes
in intracellular pH, and subsequent effects on one of the several intracellular enzymes known to modulate the receptor (see Moss and Smart 1996). This seems unlikely for several reasons.
First, in the present study we found that H+
modulation of the GABA response was present in excised outside-out patches, suggesting that an intact cellular environment (including regulatory enzymes) is not required for the effect. Second, Zhai et al. (1998)
found that in rat primary sensory neurons,
inhibition of GABA responses by protons was not significantly different
when the patch pipette solution was buffered at pH 7.4 or 6.5. Finally, perfusing HEK cells with solution at pH 5.4 and 9.4 during whole cell
recordings induced negligible changes in intracellular pH (Krishek et al. 1996
). Thus the evidence does not
support an intracellular site of action for proton inhibition of the
GABAA receptor.
Results from the present report demonstrate that the acidosis-induced
inhibition of GABA function is due to a decrease in affinity of GABA
for its receptor. We observed a similar, twofold decrease in affinity
when pH was decreased from 7.3 to 7.0 in both 1
2
2 and
3
2
2 receptors. No effect on maximal current amplitude was
observed. Our results are consistent with a recent study on rat primary
sensory neurons, in which protons significantly increased
EC50 for GABA without changing maximal GABA
efficacy (Zhai et al. 1998
). Our results differ,
however, from those of Krishek et al. (1996)
, and, to
some extent, those of Pasternack et al. (1996)
. As
discussed above, these differences likely reflect receptor subunit
composition and experimental procedures. Concerning the influence of pH
on receptor desensitization, both our results and those of Zhai
et al. (1998)
demonstrate that H+ slows desensitization.
Whether the proton-induced rightward shift in the GABA
concentration-response curve is due to competitive inhibition for the GABA binding site or allosteric modulation of the receptor has not been
determined. Drawing on the recent knowledge gained from effect of
H+ on the 1 GABA receptor, an allosteric
mechanism seems probable. This proposal is based on the fact that the
amino acids shown to influence pH sensitivity (Wegelius et al.
1997
) are distinct from those believed to be involved in
formation of the GABA binding site (Amin and Weiss
1994
), suggesting that H+ is acting at a
site distinct from that where GABA binds. However, considering the
distinct structure and pharmacology of the
1 GABA receptor when
compared with the other GABA receptor subunits, this analogy may not be justified.
The present study is, to our knowledge, the first to report the effect
of protons on single GABA-activated channels. Although the
GABAA receptor is anion selective, cations such
as Zn2+ can enter into the channel lumen
(Horenstein and Akabas 1998). Thus it is probable that
H+ can similarly penetrate the channel and
possibly act as an open channel blocker. We demonstrate that alteration
of extracellular pH produced no effect on single-channel conductance,
or the mean duration of channel openings. The major effect of protons
was a decrease in open probability, resulting from an increase in the
duration of the long closed states and their relative contribution to
the total closed time distribution. These results at the single-channel level are inconsistent with protons acting as an open channel blocker,
which would generally either decrease the apparent conductance (for
fast dissociating blockers) or channel open time constants (for medium
or slow dissociating blockers). These single-channel effects are
generally similar to those induced by the benzodiazepine inverse
agonist DMCM (methyl-6, 7-dimethoxyl-4-ethyl-
-
carbp;ome-3-carboxylate) (Rogers et al. 1994
). In
addition, DMCM, which allosterically interacts with the
GABAA receptor, also causes a rightward shift in
the GABA concentration-response curve (Kemp et al.
1987
). Thus our data at the single-channel level, as well as
the observed changes in GABA affinity, are consistent with an
extracellular site of action of H+, likely
mediated through an allosteric interaction. The amino acid residue(s)
that are influenced by pH remain to be determined.
The effect of pH on the GABAA receptor has
important implications, because it is known that pH may change under
both physiological and pathophysiological conditions. Elevated neuronal
activity can induce local decreases in pH of several tenths of a pH
unit (Chesler 1990). These shifts in pH may be due to
metabolic increases in CO2 and/or lactic acid
(Chesler and Kaila 1992
). Extracellular alkaline shifts
of up to 0.3 pH units may also occur at the onset of neural activity
(Chesler and Chan 1988
; Sykova and Svoboda 1990
), although these alkaline shifts are typically more
transient than the decreases in pH, which may last several minutes.
Interestingly, flux of bicarbonate ion through the
GABAA receptor itself may contribute to
extracellular alkalinization (Kaila et al. 1990
; Kaila and Voipio 1987
).
Pathophysiological conditions, such as seizures and cerebral ischemia,
elicit more dramatic decreases in extracellular pH. Seizures can cause
extracellular pH to fall up to 0.5 pH units (Siesjo et al.
1985), whereas a drop of 0.7 to 1 pH units can occur during
forebrain ischemia (von Hanwehr et al. 1986
) or hypoxia (Cowan and Martin 1995
). Under these conditions,
GABA-mediated inhibition of neuronal activity would be severely
depressed and may contribute to neuronal damage that accompanies these
pathophysiological events.
In summary, the results presented here indicate that protons inhibit
activity of recombinant and native GABAA receptors. The effect is GABA concentration dependent, and is similar in 1
2
2 and
3
2
2 receptors. In addition, we have demonstrated that this effect of protons is due to a decrease in single-channel open frequency. Because the
1
2
2 receptor configuration is
ubiquitous in the CNS, a significant component of GABAergic
transmission may be inhibited during cerebral acidosis.
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ACKNOWLEDGMENTS |
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We thank Dr. Donald Carter for supplying the cell lines used in this study and C. Bell-Horner for technical assistance.
This work was supported by Texas Advanced Research Program Grant 009768-027 and National Institute of Environmental Health Sciences Grant ES-07904.
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FOOTNOTES |
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Address for reprint requests: G. H. Dillon, Dept. of Pharmacology, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 4 November 1998; accepted in final form 8 April 1999.
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REFERENCES |
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