Effect of Extracellular pH on GABA-Activated Current in Rat Recombinant Receptors and Thin Hypothalamic Slices

Ren-Qi Huang and Glenn H. Dillon

Department of Pharmacology, University of North Texas Health Science Center at Fort Worth, Fort Worth, Texas 76107


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 gamma -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 alpha 3beta 2gamma 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 alpha 1beta 2gamma 2 receptors and in GABA-activated currents recorded from thin hypothalamic brain slices. In outside-out patches recorded from alpha 3beta 2gamma 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 alpha 3beta 2gamma 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 alpha -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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -subunit isoform present in the receptor.


    METHODS
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INTRODUCTION
METHODS
RESULTS
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REFERENCES

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 alpha 1beta 2gamma 2S or alpha 3beta 2gamma 2S (subsequently denoted as alpha 1beta 2gamma 2 and alpha 3beta 2gamma 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.

Individual hypothalamic neurons within the slice were visualized using an upright, fixed stage microscope (Nikon Optiphot-2UD) equipped with standard Hoffman modulation contrast (HMC) optics and a video camera system (Hammamatsu model XC-77 CCD video camera module, C2400 CCD camera control, Tandy video monitor).

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 MOmega for whole cell recordings, and 8-12 MOmega 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 MOmega . Full perforation with a low access resistance (~8 MOmega ) 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).

All data are presented as means ± SE. Student's t-test (paired or unpaired) or one-way ANOVA was used to determine statistical significance (P < 0.05).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 1beta 2gamma 2 and alpha 3beta 2gamma 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 alpha 3beta 2gamma 2 and alpha 1beta 2gamma 2 receptors, respectively. Application of 5 and 20 µM GABA to alpha 1beta 2gamma 2 and alpha 3beta 2gamma 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 alpha 1beta 2gamma 2 (n = 5) and alpha 3beta 2gamma 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 alpha 1beta 2gamma 2 (n = 5) and alpha 3beta 2gamma 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 alpha 1beta 2gamma 2 and alpha 3beta 2gamma 2 receptors.



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Fig. 1. Effect of protons on GABA-activated ion currents in recombinant rat alpha 1beta 2gamma 2 and alpha 3beta 2gamma 2 GABAA receptors. A: representative records of current activated by 20 µM GABA in alpha 3beta 2gamma 2 subunit configuration at pH 7.3, 6.4, 7.0, 7.9, and 8.4 and recovery at pH 7.3. Records are sequential current traces (from left to right) obtained from the same cell voltage clamped at -60 mV. Noting the effect is proton dependent (*) and repeatable (**). B: summary of relative responses induced by GABA (5 µM in alpha 1beta 2gamma 2 and 20 µM in alpha 3beta 2gamma 2) plotted as a function of pH (expressed as pH X/pH 7.3). Each point is the average current from 5-10 cells at a holding potential of -60 mV. The curve shown is the best fit of the data to the logistic equation described in METHODS. Fitting the data to the logistic equation yielded a transition point of pH 7.7 for both recombinant receptors.

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 alpha 1beta 2gamma 2 and alpha 3beta 2gamma 2 GABAA receptors. Figure 2 shows the current-voltage relationship for GABA-activated current at pH 7.3, 7.9, and 7.0. In alpha 1beta 2gamma 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 alpha 3beta 2gamma 2 receptors, the reversal potential was more positive than that recorded from alpha 1beta 2gamma 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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Fig. 2. Current-voltage relations (I-V curve) during application of 5 µM GABA in alpha 1beta 2gamma 2 (A) and 20 µM GABA in alpha 3beta 2gamma 2 (B) at pH 7.0, 7.3, and 7.9. All the currents are normalized to the current amplitude recorded at a holding potential of -60 mV at the control pH (7.3). The reversal potential was not significantly altered by pH in either receptor (paired t-test, P > 0.05).

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 alpha 1beta 2gamma 2 and for alpha 3beta 2gamma 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 alpha 1beta 2gamma 2 receptors, respectively. In alpha 3beta 2gamma 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 alpha 1beta 2gamma 2 and alpha 3beta 2gamma 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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Fig. 3. Concentration-response relation for alpha 1beta 2gamma 2 (B) and alpha 3beta 2gamma 2 (A and C) receptors at pH 7.0, 7.3, and 7.9. Records were carried out at a holding potential of -60 mV. A: records obtained in alpha 3beta 2gamma 2 receptors demonstrating currents activated by the application of 20 µM GABA (left set of traces) and 500 µM (right set of traces) at pH 7.3, 7.0, and 7.9 in the same cells. Note that low pH inhibited the response to lower [GABA] but did not affect the response to saturating [GABA]. B and C: graph plotting the relative amplitude of GABA-activated current at pH 7.0, 7.3, and 7.9 as a function of GABA concentration in alpha 1beta 2gamma 2 (B) and alpha 3beta 2gamma 2 (C). Amplitude is normalized to the current activated by maximum GABA at pH 7.3. Each data point is the average current from 6-20 cells. Curves shown are the best fits of the data to the logistic equation. Changing the external pH shifted the GABA concentration-response curve without significantly affecting the slope or efficacy.

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 alpha 1beta 2gamma 2 and alpha 3beta 2gamma 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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Table 1. Effect of pH on desensitization of GABA-activated current in alpha 1beta 2gamma 2 and alpha 3beta 2gamma 2 recombinant GABAA receptors

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 alpha 1beta 2gamma 2 and alpha 3beta 2gamma 2 receptors were indistinguishable at the whole cell level, we studied kinetics of single-channel activity in alpha 3beta 2gamma 2 receptors only.

The characteristics of single GABA-activated channels in recombinant alpha 3beta 2gamma 2 receptors are shown in Figs. 4-6 and Table 2. No spontaneous single-channel activity was observed in HEK cells expressing alpha 3beta 2gamma 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 alpha 1beta 2gamma 2 receptors (Dillon et al. 1995). In addition, also as in alpha 1beta 2gamma 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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Fig. 4. Representative single-channel currents elicited by 5 µM GABA for 20 s from cell expressing alpha 3beta 2gamma 2 subunits at control (pH 7.3), alkaline (pH 8.4, A) or acidic (pH 6.4, B) medium, followed by recovery. Recordings were performed on outside-out patches at a holding potential of -60 mV. A: opening frequency increased, accompanied by a 2nd channel opening on the patch membrane at pH 8.4. B: single-channel recording traces showing a decrease in opening frequency at pH 6.4. * Main conductance state; ** subconductance state. Responses illustrated here were obtained from 2 different outside-out patches.



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Fig. 5. Current-voltage relationship of single-channel currents from a representative patch exposed to 5 µM GABA at pH 7.3 and 8.4. A: representative traces of single-channel recordings at different pipette potentials at control (pH 7.3) and alkaline medium (pH 8.4). B: reversal potential and slope of I-V plot were not significantly affected by changing pH. Slopes were calculated separately by regression analysis for the ranges -60 to 0 mV and 0 to +60 mV. Data were best fitted by linear regression (r at least 0.98 for all fits). Slope conductances for this channel were 28 pS at the negative potential range at pH 7.3 and 8.4, and 21 and 20 pS, at pH 7.3 and 8.4, respectively, at the positive potential range.



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Fig. 6. Effect of protons on open (A) and closed states (B) of recombinant alpha 3beta 2gamma 2 GABAA receptors. Distributions were normalized and overlaid to display relative frequency distributions for comparison. A: normalized linear-binned open duration frequency distribution histogram for GABA (5 µM) at pH 6.4, 7.3, and 8.4. Open and distributions were placed into 1-ms bins over a range of 1-70 ms. Histograms for open duration were best fitted with the sum of 2 exponential functions for experimental conditions with time constants (and relative areas) of 6.6 ms (0.798), 23.8 (0.202) at pH 8.4, 6 ms (0.619), 22.6 ms (0.381) at pH 7.3, and 5.2 ms (0.663), 22.6 ms (0.337) at pH 6.4. Each histogram is derived from pooled data obtained from 4 (at pH 8.4 or 6.4) and 8 (at control) outside-out patch recordings. B: logarithmic-binned frequency histograms of closed duration were best fitted with sums of 3 exponential functions for experimental conditions. Single-channel closed events were binned on a logarithmic scale using 10 bins per decade resolution. During extracellular alkalization, time constants (and relative areas) obtained from same patches (n = 4) are shifted from 3.6 ms (0.680), 30.6 ms (0.196), and 867 ms (0.124) at control condition (pH 7.3) to 3.6 ms (0.630), 21.9 ms (0.278), and 548.7 ms (0.092). During extracellular acidosis, the parameters obtained from same patches (n = 4) are changed from 4.2 ms (0.684), 49.2 ms (0.242), and 877.6 ms (0.074) at control to 5.2 ms (0.632), 213.9 ms (0.229), and 2,762 ms (0.139) at pH 6.4. Curves in A and B were drawn according to the fits.


                              
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Table 2. Effect on pH on single-channel properties of alpha 3beta 2gamma 2 recombinant GABAA receptors

Increases or decreases in extracellular pH significantly altered several single-channel characteristics of alpha 3beta 2gamma 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 alpha 1beta 2gamma 2 and alpha 3beta 2gamma 2 GABAA receptors.



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Fig. 7. Representative recordings illustrating the effect of pH on GABA-activated currents recorded from rat caudal hypothalamic neurons. A: perforated patch recording traces of whole cell currents activated by 20 µM GABA at control (pH 7.3), alkaline (pH 8.4), or acidic (pH 6.4) medium, and recovery at pH 7.3. Neurons were held at -60 mV and perfused with synaptic blockade medium to minimize synaptic input. B: graph of relative responses induced by 20 µM GABA plotted as a function of pH. Relative currents are expressed as pH X/PH 7.3. Each data point is the average current from 4-6 cells. C: typical whole cell I-V relationships during application of 20 µM GABA at pH 6.4, 7.4, and 8.4. Effects of pH in hypothalamic neurons were similar to those observed in recombinant receptors.


    DISCUSSION
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ABSTRACT
INTRODUCTION
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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 alpha 1beta 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 alpha 1beta 1 receptors expressed in Xenopus oocytes are stimulated by protons, whereas alpha 1beta 1gamma 2s receptors were insensitive to pH. The delta -subunit also affected the observed response to pH, because cells expressing alpha 1beta 1gamma 2sdelta subunits were inhibited by acidic or basic pH. In the present study, the qualitatively similar H+ sensitivity of rat alpha 1beta 2gamma 2 and alpha 3beta 2gamma 2 receptors suggests the proton effect is only modestly influenced by the alpha -subunit isoform present in the receptor. Furthermore, our preliminary studies on human recombinant alpha 1beta 2gamma 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 alpha 1beta 2gamma 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 rho 1 GABA receptor is down-modulated by protons across the pH range of 6.4-8.4. Human rho 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 alpha 1beta 2gamma 2 and alpha 3beta 2gamma 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 rho 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 rho 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-beta - 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 alpha 1beta 2gamma 2 and alpha 3beta 2gamma 2 receptors. In addition, we have demonstrated that this effect of protons is due to a decrease in single-channel open frequency. Because the alpha 1beta 2gamma 2 receptor configuration is ubiquitous in the CNS, a significant component of GABAergic transmission may be inhibited during cerebral acidosis.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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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0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society