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GABAA Receptor Channel Gating and Desensitization
Departments of 1Neurology, 2Molecular Physiology and Biophysics, and 3Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37212
Submitted 22 March 2004; accepted in final form 13 May 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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GABAA receptor currents are phasic and desensitizing, whereas 
GABAA receptor currents are tonic and have no fast desensitization. receptors are subsynaptic and mediate phasic inhibition, whereas receptors are extra- or perisynaptic and mediate tonic inhibition. Given the different roles of these GABAA receptor isoforms and the fact that GABAA receptors are allosterically regulated by extracellular pH in a subunit-dependent manner, we compared the effects of changing pH on rat or 2L subunit–containing GABAA receptor currents. Human embryonic kidney cells (HEK293T) were transfected with cDNAs encoding rat 1, 3, 2L, or GABAA receptor subunits in several binary and ternary combinations, and whole cell and single channel patch-clamp recordings were obtained. Lowering pH substantially enhanced 13 receptor currents. This effect was significantly more pronounced for ternary 13 receptors, whereas ternary 132L receptors were relatively insensitive to lowered pH. Lowering pH did not affect the extent of desensitization of 13 and 132L receptor currents, but significantly increased the extent of desensitization of 13 receptor currents. Lowering pH prolonged deactivation of 13 and 13 receptor currents and enhanced the "steady-state" currents of 13 receptors evoked by long-duration (28 s) GABA applications. Lowering pH significantly increased mean open duration of 13 steady-state single channel currents due to introduction of a longer-duration open state, suggesting that low pH enhances 13 receptor steady-state currents by modifying GABAA receptor gating properties. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Macdonald and Olsen 1994; Olsen and Macdonald 2002). Multiple GABAA receptor subtypes from several subunit families have been cloned, including 1–6, 1–3, 1–3, , 
, 
, and 
(Hevers and Luddens 1998; Mehta and Ticku 1999; Olsen and Macdonald 2002). Native GABAA receptors may be composed primarily of and isoforms (McKernan and Whiting 1996). The isoforms, found mainly in GABAergic synapses, are thought to mediate phasic inhibition, whereas the isoforms selectively target extra- or perisynaptic membranes, where they have been proposed to exert tonic inhibition (Bai et al. 2001; Isaacson 2000; Lerma et al. 1986; Nusser et al. 1998; Saxena and Macdonald 1994; Stell et al. 2003; Wei et al. 2003). GABAA receptors are allosterically modulated by a variety of compounds in a subunit-dependent manner (Pritchett et al. 1989; Sundstrom-Poromaa et al. 2002; Thompson et al. 1999; Wafford et al. 1994; Wallner et al. 2003). It has been recently reported that and GABAA receptors are differentially modulated by neurosteroids and barbiturates (Feng et al. 2002; Wohlfarth et al. 2002), suggesting that phasic inhibition and tonic inhibition may make different contributions to the inhibitory responses in physiological or pathophysiological conditions.
Alteration of extracellular proton concentration (pH) has been shown to occur in pathophysiological conditions such as seizures and to influence neuronal excitability (Chesler 1990; Siesjo et al. 1996; Traynelis and Cull-Candy 1990; Xiong and Stringer 2000). There is substantial evidence suggesting that extracellular pH modulated the function of native and recombinant GABAA receptors (Krishek and Smart 2001; Krishek et al. 1996; Mozrzymas et al. 2003; Pasternack et al. 1996; Robello et al. 1994, 2000; Wilkins et al. 2002; Zhai et al. 1998). Low pHs enhanced 11, 12, and 11 GABAA receptor currents (Krishek et al. 1996; Wilkins et al. 2002). It is unclear how much a subunit contributes to the low pH-evoked enhancement of GABAA receptors, and the effects of pH on the kinetic properties of these receptors are still unknown.
In this study, we observed that, relative to the enhancement of 13 receptor peak currents, incorporation of a 2L subunit into 13 receptors significantly decreased and incorporation of a subunit significantly increased the enhancement of GABAA receptor peak currents by lowered pH. The desensitization of 13 receptors was significantly increased by lowered pH. Lowering pH also significantly enhanced "steady-state" 13 receptor currents evoked by a saturating GABA concentration. Analysis of steady-state single channel currents revealed that lowering pH introduced a longer-duration open state into 13 receptors. The mean open duration was significantly increased by lowered pH, suggesting that the enhancement of lowered pH on 13 receptor steady-state currents is at least partly achieved by increasing mean open duration.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Human embryonic kidney cells (HEK293T, a gift from Dr. P. Connely, COR Therapeutics, San Francisco, CA) were cultured using Dulbecco's Modified Eagle Medium (DMEM; Invitrogen, Grand Island, NY), supplemented with 10% fetal bovine serum (Invitrogen) in an incubator at 37°C with 5% CO2-95% air. The cells were prepared at the density of 400,000/dish 1 day prior to the transfection. The cDNA encoding rat 1 and 3 with or without a 2L or a subunit was co-precipitated with calcium phosphate and transfected into HEK293T cells by being shocked with 15% glycerol (Fisher and Macdonald 1997a). Two micrograms of each subunit cDNA and 2 µg of pHook (Invitrogen, Carlsbad, CA) were used for each transfection. It has been reported that transfections with an excess of 2S subunit were required to form ternary 2S receptors (Boileau et al. 2003). However, receptors transfected with 1, 3, and 2L cDNA in a 1:1:1 ratio share similar kinetic properties to those transfected in a 1:1:10 ratio (Hinkle and Macdonald 2003). The transfected cells were separated using an immunomagnetic bead selection method (Greenfield et al. 1997) and were recorded 24 h later.
Whole cell and single channel recordings
Whole cell and single channel currents were recorded at room temperature using patch-clamp technique from the transfected HEK cells bathed in the external solution composed of (in mM) 142 NaCl, 1 CaCl2, 6 MgCl2, 8 KCl, 10 glucose, and 10 HEPES. The osmolality was adjusted to 325–328 mOsm. The pH of the solution was buffered to 7.4 or was altered to desired pH (5.4–9.4) using NaOH. The electrodes for whole cell recordings were pulled from the thin-wall borosilicate glass tubing (World Precision Instruments, Sarasota, FL), and those for single channel recordings were pulled from the thick-wall borosilicate glass tubing (World Precision Instruments) on a P-2000 Quartz Micropipette Puller (Sutter Instrument, Novato, CA). All the electrodes were fire polished on an MF-830 Micro Forge (Narishige, Tokyo, Japan). The resistances of the whole cell recording electrodes were 0.9–1.6 M
, and those of the single channel recording electrodes were 8–16 M after being filled with an internal solution consisting of (in mM) 153 KCl, 1 MgCl2, 10 HEPES, 5 EGTA, and 2 MgATP (pH 7.3, 301–303 mOsm). Currents were recorded using an Axopatch 200A amplifier (Axon Instruments, Foster City, CA) and Digidata 1322A data acquisition system (Axon Instruments). Cells were voltage clamped at –20 mV for cells transfected with 1, 3, and 2L subunits and at –50 mV for cells transfected with 1, 3, and subunits, due to the smaller amplitudes of 13 receptor currents (Feng et al. 2002; Saxena and Macdonald 1994; Wohlfarth et al. 2002). No voltage-dependent effects were observed between –20 and –50 mV for either of these isoforms. Series resistance was not compensated. Series resistance errors may result in an underestimation of peak current, and the underestimation is larger for larger whole cell currents. The average peak current amplitude was about 4 nA for 132L receptors and 0.9 nA for 13 receptors, and currents with amplitudes larger than 8 nA were not included in kinetic and enhancement analyses. The electrode resistances used in this study were low (
1 M). The voltage drop across the series resistance in this current range does not significantly affect the extent of desensitization (Bianchi and Macdonald 2002).
GABA (Sigma Chemical, St. Louis, MO) was prepared as stock solutions and was diluted to desired concentrations with external solution. GABA was applied by gravity using an ultra-fast delivery devise consisting of multi-barrel tubes connected to a perfusion fast-step system (Warner Instruments, Hamden, CT). The 10–90% rise times for solution exchanges were typically 0.4–1.0 ms when the liquid junction currents were measured by switching the normal and diluted external solutions across an open electrode tip. The intervals between two consecutive applications were 
45 s to avoid accumulation of desensitization if present. The duration of GABA application to cells was 4 or 28 s. Single channel microscopic currents were recorded in excised outside-out patches during steady-state conditions (minutes of exposure to GABA). Voltage was consistently clamped at –75 mV during single channel recordings.
Data analysis
The whole cell and single channel currents were analyzed off-line. The whole cell currents were low-pass filtered at 1–2 kHz and analyzed using Clampfit 8.1 (Axon Instruments). The concentration-response curves were generated by normalizing to 300 µM GABA at pH 7.4 and fitting using a logistic equation with a variable slope: I = Imax/(1 + 10(LogEC50 – Logdrug) x Hill slope). I represents the current evoked by a given concentration of GABA, and Imax denotes the maximal response induced by a saturating concentration of GABA. EC50 was defined as the GABA concentration at which 50% of maximal response was evoked. The amplitudes of peak currents were measured from the base line to the peak, and the amplitudes of the steady-state currents were calculated from the end of the GABA application to the baseline after recovery. The enhancement of GABA currents by lowered pH was calculated as percentage of GABA control. The extent of desensitization was measured as percentage of current reduction after 4- or 28-s GABA application. The rate of deactivation was analyzed by fitting the deactivation currents with one or two exponential components using standard exponential Levenberg-Marquardt method. The exponential function was expressed in the form of 
an
n, where a is the relative amplitude, is the time constant, and n is the number of exponential components. A weighted was calculated based on the following formula: a1 x 1/(a1 + a2) + a2 x 2/(a1 + a2). a1 and a2 were the relative amplitudes, and 1 and 2 were corresponding time constants of the fast and slow components. Single channel data were acquired at 50-µs intervals, low-pass filtered at 2 kHz and analyzed using Fetchan 6.0 (Axon Instruments). Kinetic open states were analyzed using Interval 5 (Dr. Barry S. Pallotta, University of North Carolina, Chapel Hill, NC). Events with intervals <1.5 times of system dead time (100 µs) were plotted but not included in the fitting. All the data were reported as mean ± SE. Paired Student's t-test was performed to compare the changes before and after lowering pH. Unpaired Student's t-test was used to compare different treatment groups. Statistical significance was considered at P < 0.05.
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RESULTS |
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13 receptor currents than 132L receptor currents
The effects of lowered pH on 132L and 13 receptor GABA concentration-response curves were studied by application of GABA at different pHs. GABA evoked slightly greater 132L receptor currents at pH 6.4 than at pH 7.4 (Fig. 1A). The currents evoked by GABA at pH 7.4 and at pH 6.4 were normalized to 300 µM GABA at pH 7.4. Application of different GABA concentrations at low pH (6.4) to 132L receptors produced a small upward shift of the concentration-response curve. The current at 1 mM GABA (pH 6.4) was 109.6 ± 3.2% (n = 6), which was significantly greater than 1 mM GABA at pH 7.4 (P < 0.01). The EC50 was 6.9 ± 2.4 µM for GABA at pH 6.4, which was not significantly different from that for GABA at pH 7.4 (7.6 ± 1.7 µM; Fig. 1B).
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132L receptor currents, 13 receptor currents at pH 6.4 were much greater than those at pH 7.4 (Fig. 1C). Application of a series of GABA concentrations at low pH (6.4) to 13 receptors resulted in an upward shifted concentration-response curve at high GABA concentrations compared with that evoked by GABA at pH 7.4. The current at 1 mM GABA (pH 6.4) was 144.5 ± 5.4% (n = 6), which was significantly greater than that at pH 7.4 (P < 0.001). The current at 1 mM GABA (pH 6.4) for 13 receptors was significantly greater than that for 132L receptors (P < 0.001). For 13 receptors, the EC50 for GABA at pH 6.4 was 8.2 ± 0.6 µM, which was significantly greater than that for GABA at pH 7.4 (4.6 ± 0.7 µM; P < 0.01; Fig. 1D).
Lowering pH altered enhancement, desensitization, and deactivation of 13, 132L, and 13 receptor currents
To further study the effects of lowering pH on GABAA receptor kinetics, cells were lifted from the recording dish bottom and solutions with lowered pH (5.4) were preapplied for 4 s followed by rapid application of a saturating concentration of GABA at lowered pH (Bianchi and Macdonald 2002). Rapid lowering of extracellular pH induced small currents from HEK293T cells, consistent with previous report (Krishek et al. 1996). These currents were not due to activation of GABAA receptors because they were recorded in both transfected and untransfected cells (data not shown). The preapplication technique completely bathes the cell in low pH solution, and preincubation at low pH allows for discrimination of the small pH-induced currents from the allosteric modulation of GABAA currents induced by lowered pH. Therefore this permitted use of lower pHs (pH 5.4) to explore pH-dependent allosteric effects, which have been reported previously (Krishek et al. 1996). Lowering pH to 5.4 enhanced the GABA (1 mM)-evoked currents for 13 receptors (228.5 ± 27.8%, n = 5), consistent with previous reports for isoforms (Krishek et al. 1996; Wilkins et al. 2002) (Fig. 2, B and D). Lowering pH to 5.4 only slightly enhanced ternary 132L receptor currents (111.6 ± 6.3%, n = 7; Fig. 2, A and D). For ternary 13 receptors, lowering pH to 5.4 produced a considerably larger current enhancement (443.8 ± 66.0%, n = 10; Fig. 2, C and D). The enhancement of saturating GABA currents evoked by lowered pH was significantly greater for 13 receptors than for 132L receptors (P < 0.001). The enhancement of 13 currents was significantly greater than for 13 currents (P < 0.05). Compared with 132L currents, lowering pH to 5.4 produced a significantly greater enhancement of 13 currents (P < 0.001; Fig. 2D).
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70%) for both 13 (n = 5) and 132L (n = 7) receptors. Lowering pH to 5.4 did not significantly alter the extent of desensitization for these receptors (Fig. 2, A, B, and E). In agreement with previous studies (Feng et al. 2002; Haas and Macdonald 1999), application of 1 mM GABA at pH 7.4 for 4 s evoked minimal desensitization of 13 receptor currents (5%; n = 7). However, the extent of desensitization was significantly increased to 32.9 ± 7.9% after lowering pH (P < 0.01; Fig. 2, C and E).
Lowering pH to 5.4 did not significantly change the rate of deactivation of 132L currents (Fig. 2, A and F). The mean weighted deactivation time constant was significantly increased from 200.2 ± 42.9 to 368.0 ± 69.4 ms for 13 receptors by lowering pH (P < 0.01; Fig. 2, B and F). Also, lowering pH significantly increased the mean weighted deactivation time constant for 13 receptors from 63.4 ± 8.0 to 163.7 ± 39.1 ms (n = 7; P < 0.05; Fig. 2, C and F).
Reduction of pH enhanced steady-state 13 receptor currents
The effect of lowering pH on the steady-state currents of 13 receptors was examined by applying saturating GABA for 28 s and determining the residual current at the end of the GABA application. Low pH (5.4) treatment of 13 receptors increased the steady-state currents and the rate and amount of desensitization (Fig. 3A). The mean amplitude of steady-state currents was significantly increased from 316.7 ± 121.7 to 560.0 ± 149.8 pA (P < 0.05; Fig. 3B), and the mean desensitization was significantly increased from 29.3 ± 8.2 to 59.5 ± 14.6% after lowering pH (n = 3; P < 0.05; Fig. 3C).
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13 receptor steady-state single channel currents
The mechanistic basis for enhancement of steady-state currents of 13 receptors by lowered pH was investigated using single channel recordings. In the presence of 1 mM GABA at pH 7.4, 13 receptor single channel currents exhibited brief openings (Fig. 4, A1 and A2), and the distribution of open states was best described by two exponential functions (Fig. 4A3). These observations are consistent with previous reports (Feng et al. 2002; Fisher and Macdonald 1997b; Haas and Macdonald 1999; Wohlfarth et al. 2002). With high concentration of protons (pH 5.4), the channel activity evoked by 1 mM GABA was characterized by brief openings mixed with some longer-duration openings (Fig. 4, B1 and B2). The distribution of the open states was fitted best with three exponential functions (Fig. 4B3). Similar to a previous report (Huang and Dillon 1999), no obvious channel activity was observed at pH 5.4 alone when recorded at –75 mV. Single channel analysis showed that the mean open duration was significantly increased from 0.40 ± 0.04 ms at pH 7.4 (n = 6) to 0.60 ± 0.07 ms at pH 5.4 (n = 7; P < 0.05; Fig. 5A). The mean time constant of neither the first component (1) nor the second component (2) was significantly altered by lowering pH. However, lowering pH introduced a longer-duration open state with a time constant (3) of 2.46 ± 0.40 ms (Fig. 5B). Low pH treatment significantly decreased the relative area of the first component (A1) from 80.1 ± 4.2 to 47.7 ± 5.8% (P < 0.01) but significantly increased the relative area of the second component (A2) from 20.0 ± 4.2 to 45.2 ± 4.6% (P < 0.01). The relative area of the longer-duration open state introduced by lowered pH was 7.1 ± 2.6% (Fig. 5C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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132L and 13 recombinant GABAA receptor currents and explored the unique modulation by low pH of 13 receptor macroscopic and microscopic currents. We showed that modulation of the desensitization and deactivation of recombinant receptors by low pH is subunit-dependent. Low pH induced a significantly greater enhancement of ternary 13 receptor currents than 13 receptors, suggesting that incorporation of a subunit into 13 isoform significantly increases proton sensitivity. Low pH significantly increased the steady-state 13 receptor currents by increasing the mean open duration of single channel current.
Extracellular pH differentially modulated 132L and 13 receptor currents
In this study, we observed that lowering pH from 7.4 to 6.4 significantly shifted the GABA concentration-response curve upward for 132L receptors and produced a prominent upward and rightward shift of the GABA concentration-response curve for 13 receptors at high GABA concentrations. The differential effect of lowering pH on the GABA concentration-response patterns of 132L and 13 receptors suggests that protons exert different modulatory effects on these receptor isoforms. Theoretically, lowering pH altered protonation of the GABA molecule itself (pKa is 4.03 for –COOH and 10.0 for –NH2, from Dawson et al. 1986). However, it was reported that the level of GABA molecule protonation would not vary by >8% within the pH range used in this study (Krishek et al. 1996). Therefore it is very unlikely that the current enhancement and alterations of kinetic properties evoked by lowered pHs were induced by protonation of GABA molecules. An enhancement of 2 subunit–containing receptors by low pH has not been previously reported. Previous studies reported that 112S receptors were not responsive to pH changes and that 122S receptors were inhibited by lowered pHs (Huang and Dillon 1999; Krishek et al. 1996). These discrepancies with 132L receptors in this study may be due to the differences in subunits or splice variants of 2 subunit. Compared with 132L receptors, lowering pH substantially upward shifted the 13 receptor GABA concentration-response curve at high GABA concentrations, in agreement with a reported observation for 11 receptors (Krishek et al. 1996). The current and previous studies suggest that protons exert unique modulation of subunit-containing GABAA receptors. Interestingly, a variety of compounds have been shown to positively modulate subunit–containing GABAA receptors (Feng et al. 2002; Lees and Edwards 1998; Sundstrom-Poromaa et al. 2002; Thompson et al. 2002; Wallner et al. 2003; Wohlfarth et al. 2002).
A simple parallel shift of the GABA concentration-response curve in the presence of lowered pH was not obtained for either 132L or 13 receptors, suggesting that protons did not have a competitive interaction with GABA. While this observation has been reported previously (Krishek et al. 1996; Pasternack et al. 1996; Robello et al. 1994; Wilkins et al. 2002), contradictory results have also been reported (Huang and Dillon 1999; Huang et al. 2002). The reasons for these inconsistent data remain uncertain, but may be partially related to differences in subunit composition (Huang and Dillon 1999).
Incorporation of a 2L or a subunit into 13 receptors altered the enhancement, desensitization, and deactivation of GABA currents by lowered pH
Lowering pH has been documented to enhance 11, 12, and 11 GABAA receptor currents (Krishek et al. 1996; Wilkins et al. 2002). This study expands on those findings by showing that the enhancement of ternary 13 receptor currents by lowered pH is significantly greater than that of binary 13 receptors, suggesting that incorporation of a subunit significantly increased the sensitivity of these receptors to protons. Reducing pH resulted only in a slight enhancement of ternary 132L receptor currents, which was significantly less than the enhancement of 13 receptor currents. Thus incorporation of a 2L subunit significantly reduced their sensitivity to protons. The mechanism for these differential alterations is unclear. One explanation may lie in that incorporation of a subunit exposed additional proton binding sites or changed binding affinity, whereas incorporation of a 2L subunit did the opposite. However, we also observed a similar differential modulation of 132L and 13 receptors by neurosteroids and barbiturates (Feng et al. 2002; Wohlfarth et al. 2002). These structurally different compounds are proposed to bind to different sites on GABAA receptors (Celentano et al. 1991; Macdonald and Olsen 1994). Therefore it is more likely that incorporation of a or a 2L subunit altered a "common modulatory pathway." The present and previous studies consistently found that GABA at a saturating concentration evoked larger currents from ternary 132L receptors than 13 receptors and that the mean open duration of 132L receptor single channel currents was greater than that of 13 receptor single channel currents (Feng et al. 2002; Saxena and Macdonald 1994; Wohlfarth et al. 2002), suggesting that 13 receptors are low efficacy receptors compared with 132L receptors (Bianchi and Macdonald 2003).
The desensitization of 132L and 13 receptors was not significantly affected by lowered pH. However, reduction of pH significantly increased 13 receptor desensitization. These findings suggest that protons modulate the desensitization of GABAA receptors in a subunit-dependent manner. A reduction in extracellular pH has been reported to either have no influence on desensitization (Pasternack et al. 1996; Robello et al. 1994) or decrease desensitization (Mozrzymas et al. 2003; Zhai et al. 1998) of GABAA receptors on native neurons. These inconsistent data may be due to the presence of multiple population of GABAA receptor isoforms on these neurons. In addition, the effects of different or subunit on desensitization are relatively unknown, as is the effect of post-translational modification, such as protein phosphorylation.
Lowering pH did not significantly modify the deactivation rate of 132L receptors but did significantly prolong the deactivation of 13 and 13 receptors. The data for 13 receptors were consistent with the idea that prolongation of deactivation is coupled with increased desensitization (Bianchi et al. 2001; Haas and Macdonald 1999; Jones and Westbrook 1995). However, prolongation of deactivation of 13 receptors was not coupled with increased desensitization. This "uncoupling" of deactivation and desensitization has been observed previously (Bianchi and Macdonald 2001; Bianchi et al. 2001; Feng et al. 2002).
Reduction of pH enhanced steady-state currents of 13 receptors
Since lowering pH increased the extent of desensitization of 13 receptors, and extent of desensitization would affect the steady state currents, long-duration (28 s) GABA pulses were applied to examine the residual currents approaching the steady state. Theoretically, prolonged application of GABAA receptor agonist may result in chloride redistribution and varying series resistance error. However, a previous study showed that long-duration GABA application did not significantly change the chloride redistribution and that series resistance did not significantly affect the time course or extent of desensitization (Bianchi and Macdonald 2002). Long-duration exposure of the cells to low pHs may affect the intracellular pH. However, it was reported that exposure of the HEK cells to Krebs solution at pH 5.4 for minutes induced negligible alterations of intracellular pH (Krishek et al. 1996). In addition, limited acidification of the intracellular environment may not affect GABAA receptor function (Pasternack et al. 1992; Zhai et al. 1998). Therefore we interpreted the 28-s application data in the same way as the 4-s application. We found that lowering pH significantly increased the steady-state currents of 13 receptors. Previous studies reported that reducing pH increased steady-state GABAA receptor currents from cerebellar granule cells (Krishek and Smart 2001; Robello et al. 1994). These observations are consistent with the finding in this study since the subunit is predominantly present on cerebellar granule cells (Laurie et al. 1992).
Reduction of pH enhanced 13 receptor steady-state currents by modifying gating properties
How did increasing proton concentration enhance 13 receptor steady-state currents? To answer this question, we recorded the single channel currents before and after lowering pH in a steady-state condition (exposure of the cells to GABA for minutes). GABA (1 mM) with a high proton concentration significantly increased the mean open duration mainly by introducing an additional longer open state, suggesting that protons fundamentally modified gating of the 13 receptors. Previously, we have shown that neurosteroids and barbiturates introduced an additional open state into 13 receptors. Both neurosteroids and barbiturates at high concentrations could directly activate 13 receptors and reveal a third open state (Feng et al. 2002; Wohlfarth et al. 2002). These previous studies indicate that these structurally different compounds did not create the third open state but exposed it when co-applied with saturating GABA. Co-application of a neurosteroid or barbiturate with GABA generated a greater relative area of the third open state than application of a neurosteroid or barbiturate alone (Feng et al. 2002; Wohlfarth et al. 2002). Therefore a portion of the third open state relative area was induced by a "pure" modulatory effect of the neurosteroid or barbiturate on GABAA receptors. Our data in this study indicated that low pHs evoked small currents by activating receptors other than GABAA receptors, and extracellular solution at low pH (5.4) alone failed to generate analyzable microscopic currents, suggesting that protons interact with GABAA receptors in a "pure" modulatory manner. In line with this study, a "pure" modulatory effect of protons on glycine receptors has been reported recently (Li et al. 2003). The present finding supports the proposal that saturating GABA can only access the third open state of 13 receptors if a conformational change occurs in the presence of modulators. The reasons why saturating GABA alone could not access the third open state of 13 receptors are unknown. One possibility is that GABA is a partial agonist for 13 receptors so that even the saturating GABA only partially activates the receptors, leaving some "modulatory potential" for modulators (Bianchi and Macdonald 2003). This hypothesis seems plausible since it has been reported that GABA evoked smaller responses than the GABA analog 4,5,6,7-tetrahydroisoxazole[4,5-c]pyridine-3-ol (THIP) for subunit–containing GABAA receptors (Adkins et al. 2001; Brown et al. 2002).
Implication of modulation of subunit–containing GABAA receptors by extracellular pHs
Different GABAA receptor subunits have been shown to be targeted to different locations on neuronal membrane surface. Most subunits are targeted to the synaptic membrane, where they are thought to mediate phasic inhibition. In contrast, subunits are targeted to extra- or perisynaptic membranes and may be involved in mediating tonic inhibition (Bai et al. 2001; Nusser et al. 1998; Saxena and Macdonald 1994; Stell et al. 2003; Wei et al. 2003). In this study, we showed that lowering pH produced greater enhancement of 13 receptor currents than 132L receptor currents, suggesting that protons play an important role in regulating GABAA receptor tonic inhibition. Protons have also been shown to inhibit the function of NMDA receptors (Traynelis and Cull-Candy 1990). These observations suggest that pH is an important regulator of excitatory and inhibitory function in the brain. Cellular excitability may be regulated by protons under normal conditions since extracellular pH alterations occur during physiological neurotransmission (Chen and Chesler 1992; Kaila 1994) and are at least partly attributable to a conductive net efflux of bicarbonate anions through GABAA receptor channels (Kaila 1994; Kaila et al. 1990). A decrease in extracellular pH (
0.3–1 pH unit) has been documented to occur in a variety of pathophysiological conditions, such as seizures (Chesler 1990; Cowan and Martin 1995; Xiong and Stringer 2000). Increased N-methyl-D-aspartate (NMDA) receptor excitation and reduced GABAA receptor inhibition are observed in experimental models of epilepsy, as well as human epilepsy (Akaike and Himori 2002; Bianchi et al. 2002; Bowser et al. 2002; Feng and Faingold 2000; Feng et al. 2001; Kapur and Macdonald 1997; Wallace et al. 2001). The findings that protons enhance the function of subunit–containing GABAA receptors and inhibit the function of NMDA receptors suggest that protons may be an endogenous protective factor against seizures in some areas of the brain, further supporting the idea that protons are an important modulator of seizure activity (Chesler 1990; Chesler and Kaila 1992; Kaila 1994; Ransom 2000).
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. L. Macdonald, Dept. of Neurology, Vanderbilt Univ. Medical Center, 6140 Medical Research Bldg. III, 465 21st Ave., South, Nashville, TN 37232-8552 (E-mail: robert.macdonald{at}vanderbilt.edu).
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, mean GABA response at pH 6.4; 
, mean GABA response at pH 7.4. Error bars represent SE.
