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¹ Department of Pharmacology, University College London, Medical Sciences Building, Gower Street, London WC1E 6BT, UK

	Abstract

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Regulation of GABA_A receptors by extracellular pH exhibits a dependence on the receptor subunit composition. To date, the molecular mechanism responsible for the modulation of GABA_A receptors at alkaline pH has remained elusive. We report here that the GABA-activated current can be potentiated at pH 8.4 for both ß and ß subunit-containing receptors, but only at GABA concentrations below the EC₄₀. Site-specific mutagenesis revealed that a single lysine residue, K279 in the ß subunit TM2–TM3 linker, was critically important for alkaline pH to modulate the function of both 1ß2 and 1ß22 receptors. The ability of low concentrations of GABA to reveal different pH titration profiles for GABA_A receptors was also examined at acidic pH. At pH 6.4, GABA activation of ß receptors was enhanced at low GABA concentrations. This effect was ablated by the mutation H267A in the ß subunit. Decreasing the pH further to 5.4 inhibited GABA responses via ß receptors, whereas those responses recorded from ß receptors were potentiated. Inserting homologous ß subunit residues into the 2 subunit to recreate, in ß receptors, the proton modulatory profile of ß receptors, established that in the presence of ß2^H267, the mutation 2^T294K was necessary to potentiate the GABA response at pH 5.4. This residue, T294, is homologous to K279 in the ß subunit and suggests that a lysine at this position is an important residue for mediating the allosteric effects of both acidic and alkaline pH changes, rather than forming a direct site for protonation within the GABA_A receptor.

(Received 18 April 2005; accepted after revision 3 June 2005; first published online 9 June 2005)
Corresponding author T.G. Smart: Department of Pharmacology, University College London, Medical Sciences Building, Gower Street, London WC1E 6BT, UK. Email: t.smart{at}ucl.ac.uk

	Introduction

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The regulation of GABA_A receptors by numerous endogenous factors, including phosphorylation, redox reagents and ions normally present in vivo (e.g. H⁺ and Zn²⁺) may be important for their physiological function (Kaila, 1994; Smart et al. 1994; Sieghart, 1995; Rabow et al. 1996; Moss & Smart, 2001). Generally, fast synaptic inhibition proceeds via GABA_A receptors predominantly composed of ß subunits (McKernan & Whiting, 1996). In comparison, extrasynaptic GABA_A receptors underlie continuous or tonic inhibition (Brickley et al. 1995; Brickley et al. 1996; Brickley et al. 1999; Mody, 2001) and their subunit composition is likely to vary depending on the type of neurone. Typical examples will include not only ß isoforms, but also receptors comprising 4, 5 or 6 and subunits, as well as the potential for ß isoforms (Brickley et al. 2001; Mody, 2001).

Protons can exert various effects on GABA_A receptor function depending upon their subunit composition (Krishek et al. 1996). Lowering external pH (to 6.4 or 5.4) potentiates GABA-activated currents recorded from ß, ß and ß GABA_A receptors (Krishek et al. 1996). For the ß isoform, this modulation has been largely attributed to the protonation of a single histidine residue, located in the ion channel lining contributed by the ß subunit (H267) (Wilkins et al. 2002). However, the alkaline pH sensitivity profiles of recombinant GABA_A receptors do appear to vary with their function either augmented, slightly inhibited, or remaining largely unaffected at pH 7.9–8.4 (Krishek et al. 1996; Huang & Dillon, 1999). The reasons for this variation are unclear. Although differences in experimental design may be relevant, such variability is also evident in the pH profiles of native neuronal GABA_A receptors. Here, differences in subunit composition and/or varying proportions of receptor subpopulations offer more likely explanations. For example, alkaline pH inhibited responses to GABA in cerebellar granule cells (Robello et al. 1994) and sympathetic neurones (Smart, 1992; Krishek et al. 1996), whilst in hypothalamic neurones, responses were potentiated (Huang & Dillon, 1999). In acutely solated hippocampal pyramidal neurones, pH modulation was dependent upon the GABA concentration, with responses induced by low and high GABA concentrations being potentiated and inhibited, respectively, by alkaline pH (Pasternack et al. 1996). Furthermore, during neuronal development, the alkaline pH sensitivity of GABA-activated currents recorded from cultured cerebellar granule cells up to 11 days in vitro changed from initial potentiation to relative insensitivity thereafter (Krishek & Smart, 2001). The role of alkaline pH transients in GABA receptor function is still to be defined but it may be important in regulating neuronal excitability. Extracellular alkaline and acidic transients, of up to 0.2 pH unit, have been observed during synaptic transmission, as well as after the activation of GABA_A or glutamate receptors (Chen & Chesler, 1991; Chen & Chesler, 1992; Kaila et al. 1992). Physiologically, during synaptic transmission, bicarbonate ions are thought to flow through GABA_A receptors creating an extracellular alkaline environment for those receptors (Kaila, 1994), whereas extreme extracellular acidosis is more likely to occur in pathophysiological conditions such as ischaemia, brain trauma and hypothermia with deviations of 1 pH unit from physiological conditions possible (Kraig et al. 1987; Hoffman et al. 1996; Hoffman et al. 1999; Anderson & Meyer, 2002).

The present study examined the molecular mechanism by which alkaline pH affected GABA-activated currents and its apparent dependence on GABA concentration. Using site-directed mutagenesis, a single residue was identified which appeared to have a prime role in the modulation of the GABA_A receptor in alkaline pH. In addition, by examining the differential modulation of ß and ß GABA_A receptors in acidic pH we have identified key residues that are involved in the modulation of GABA_A receptors at both alkaline and acidic pH.

	Methods

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cDNA constructs and site-specific mutagenesis

Murine GABA_A receptor 1, ß2 and 2s subunit cDNAs were cloned into the vector pRK5. Site-specific mutagenesis was performed using the QUICKCHANGE (Stratagene) primer-directed polymerase chain reaction method and cDNAs were prepared using the Plasmid Maxi Kit (Qiagen, Crawley, UK). The precision of the point mutations and integrity of the entire coding sequence was assessed using the BigDye ready reaction mix (PerkinElmer/Applied Biosystems) with an ABI 310 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA).

Cell culture and transfection

Human embryonic kidney (HEK) cells were cultured in 10 cm dishes at 37°C in 95% air–5% CO₂ in a growth medium consisting of Dulbecco's modified Eagle's medium (DMEM), 10% fetal calf serum (FCS), 2 mM glutamine, 100 units ml^–1 penicillin G and 100 mg ml^–1 streptomycin. Exponentially growing HEK cells attaining 70% confluence were washed with 5 ml Hanks' balanced salt solution (HBSS) and harvested using 2 ml of 0.5 mg ml^–1 trypsin and 0.2 mg ml^–1 EDTA. Trypsin activity was quenched by adding 10 ml of the growth medium. Cells were centrifuged at 84 g for 2 min and resuspended in 10 ml of DMEM with FCS. Cells were plated onto 22 mm glass poly-L-lysine-coated coverslips in 35 mm culture dishes and allowed to adhere at 37°C in a 95% air–5% CO₂ incubator for at least 1 h.

Prior to DNA transfection, the cells were washed with 2 ml of HBSS and maintained in 1.5 ml of growth medium. For each 35 mm dish of cells, 4 µl of DNA solution for the receptor subunits and the reporter, GFP (total, approximately 4 µg), was mixed gently with 20 µl of 340 mM CaCl₂ solution and 24 µl of double-strength HBSS (containing, 280 mM NaCl, 2.8 mM Na₂HPO₄, 50 mM Hepes, pH 7.2 with 1 N NaOH) and left to stand for at least 20 min for the DNA precipitate to form. The DNA suspension was then applied to the dish (48 µl per dish), which was incubated at 37°C in a 95% air–5% CO₂ overnight. The medium was replaced with growth medium after 18 h and the cells used for electrophysiology for a further 24–48 h.

Patch clamp electrophysiology

Membrane currents were recorded from single HEK cells using the whole-cell patch clamp configuration in conjunction with an Axopatch 1C amplifier. Patch pipettes (resistances 3–5 M) were pulled from thin-walled borosilicate glass and filled with a solution containing (mM): 120 KCl, 1 MgCl₂, 11 EGTA, 30 KOH, 10 Hepes, 1 CaCl₂, 2 adenosine triphosphate and 12 creatine phosphate; pH 7.11. The cells were continuously perfused with Krebs solution containing (mM): 140 NaCl, 4.7 KCl, 1.2 MgCl₂, 2.52 CaCl₂, 11 glucose and either 5 Hepes or 5 Mes (Hepes was used for Krebs solutions in the pH range 7.4–8.4; Mes was used for the pH range, 5.4–6.9). The Krebs solution's pH was adjusted to 5.4–8.4 with 1 or 5 N NaOH. Membrane currents were filtered at 5 kHz (–3 dB, 6th pole Bessel, 36 dB per octave) and stored on a Viglen pentium III computer for analysis with Clampex 8. Changes of more than 10% in the membrane input conductance or series resistance resulted in the recording being discarded. Drugs and solutions were rapidly applied to the cells using a modified Y-tube positioned approximately 300 µm from the recorded cell.

Analysis of whole-cell current data

Peak amplitude GABA-activated currents were determined at –50 mV holding potential. To construct concentration–response relationships for GABA, the current (I) was measured in the presence of each concentration of GABA applied at 2 min intervals to allow recovery from desensitization. The currents were normalized to the maximum GABA response at pH 7.4 (I_max) and the concentration response relationship fitted with the Hill equation:

(1)

where the EC₅₀ represents the concentration of GABA ([A]) inducing 50% of the maximal current evoked by a saturating concentration of GABA and n_H is the Hill coefficient. The GABA concentration–response curve data were subjected to an analysis of variance with Bonferroni's post hoc test. Significance for all data was determined at the P < 0.05 level.

The pH titration data were curve fitted as previously described providing estimates of pK_a values assuming the receptor protein can behave as a weak diprotic acid possessing two sites for proton binding that will influence the GABA-activated conductance (Krishek et al. 1996). The proportions of charged and uncharged species of amino acids that coexisted in solution at particular pH values, were calculated using:

The level of spontaneous receptor activation was assessed by using picrotoxin to block active GABA_A receptors. Spontaneity was manifest by the generation of an outward current superimposed on the holding current. This current was not observed for the wild-type 1ß2 or 1ß22 receptors. The extent of spontaneity was established by examining the maximum outward current induced by picrotoxin (I_PTX) which was summed with the maximum current activated by a saturating concentration of GABA (I_GABA,max). The level of spontaneous receptor activation was quantified according to:

Molecular modelling

The transmembrane domains of GABA_A receptor 1, ß2 and 2 subunits were aligned with those of nACh receptors using Clustal W (Thompson et al. 1994) and a homology model based on the structure of Torpedo nACh receptor transmembrane domains (Miyazawa et al. 2003; PDB accession code, 1oed) was generated using Deep View (Guex & Peitsch, 1997). Deep View was used to dock the model transmembrane domains with Ernst and colleagues' (Ernst et al. 2003) model of the GABA_A receptor ligand binding domain, which is based upon the crystal structure of the acetylcholine binding protein (AChBP; PDB accession code, 1i9b; Brejc et al. 2001). All images were generated with POV-Ray.

	Results

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External alkaline pH and recombinant 1ß2 and 1ß22 GABA_A receptors

The effect of alkaline pH on GABA_A receptors was assessed by comparing GABA concentration–response relationships for both 1ß2 and 1ß22 GABA_A receptors at pH 7.4 and 8.4 (Fig. 1A and B). For either receptor construct, considering the entire curves, there appeared to be no significant shift in the concentration-dependence or in the maximum response to GABA when the pH was increased. Accordingly, the GABA EC₅₀s for 1ß2 (means ±S.E.M.; pH 7.4, 2.9 ± 0.3 µM; pH 8.4, 2.3 ± 0.2 µM; P > 0.05; n= 24) and 1ß22 (pH 7.4, 3.4 ± 0.2 µM; pH 8.4, 2.6 ± 0.1 µM; P > 0.05; n= 22) receptors remained unaffected. However, close inspection of the lower segment of the concentration response curves at pH 8.4 revealed a small, leftward shift tendency that was apparent only at the lowest concentrations of GABA (EC_10-40) for both receptor constructs (Fig. 1C and D).

View larger version (31K): [in this window] [in a new window]   Figure 1.  Alkaline pH and the GABA concentration response relationships for 1ß2 and 1ß22 GABAA receptors A and B, GABA concentration–response curves for 1ß2 (A) and 1ß22 (B) GABAA receptors (n= 8–32 cells). Peak GABA currents were measured at pH 7.4 () and 8.4 () and then normalized to the maximum GABA response at pH 7.4 (= 1). In this and other figures, all points represent the mean ±S.E.M. The insets illustrate sample GABA-activated currents induced by applying 10 µM GABA (continuous lines) at the indicated external pH. Calibration bars represent: 500 pA, 2 s (A) and 1000 pA, 2 s (B). C and D, expansions of the GABA curves shown by the boxed regions in A and B for both 1ß2 (C) and 1ß22 (D) at pH 7.4 () and 8.4 ().

To further examine the dependence of pH modulation on low GABA concentrations, pH titration profiles were constructed for responses to 1 and 5 µM GABA using the 1ß2 receptor (Fig. 2A). For 1 µM GABA, which is near the EC₂₀, the titration was complex revealing a potentiation of the GABA-activated current in both acidic and alkaline pH relative to pH 7.4, with apparent pK_a values of 6.1 ± 0.22 and 7.7 ± 0.38, respectively (Fig. 2A; n= 14). By increasing the GABA concentration to 5 µM, alkaline pH had a small inhibitory effect on the GABA response for 1ß2 receptors, as reported for 1ß1 (Krishek et al. 1996), with the titration curve now described by a single pK_a of 6.9 ± 0.04 (Fig. 2A; n= 13). The effect of alkaline pH was examined further using 0.3–100 µM GABA applied to ß and ß receptors at pH 7.4 and 8.4; however, only with 0.3–3 µM GABA were clear potentiations observed (P < 0.05; n= 12; Fig. 2B and C).

View larger version (26K): [in this window] [in a new window]   Figure 2.  Modulation of 1ß2 and 1ß22 receptors by alkaline pH is dependent on low GABA concentrations A, pH titration profiles for 1ß2 receptors determined for 1 µM () and 5 µM (•) GABA-activated responses (n= 4–12 cells). Data were normalized to the responses evoked by 1 or 5 µM GABA at pH 7.4 (= 100%). B, bar graph of GABA-activated current determined at different GABA concentrations for 1ß2 (white bars) and 1ß22 (grey bars) receptors at pH 8.4 (n= 4–12). *P < 0.05 compared with the control response (= 100%) determined at pH 7.4 for the corresponding GABA concentration. C, GABA-activated currents induced by 1 µM (EC20) GABA at the indicated external pH for 1ß2 and 1ß22 GABAA receptors. Calibration bars represent: 400pA, 2 s.

Alkaline pH-induced modulation does not involve histidine or tyrosine residues

The involvement of histidine and tyrosine residues with the modulation of the GABA_A receptor at pH 8.4 was investigated using the covalent modifying reagent, diethylpyrocarbonate (DEPC) (Miles, 1977) and site-directed mutagenesis. The mutation of the previously identified proton-sensitive H267 on the ß subunit, forming 1ß2^H267A and 1ß2^H267A2 receptors, did not prevent alkaline pH from potentiating the 1 µM (EC₂₀) GABA-activated currents by 55 ± 11% and 36 ± 4% from their control current amplitudes (n= 4–6). This level of potentiation was similar to that observed with the respective wild-type receptors, 1ß2 (38 ± 8%) and 1ß22 (43 ± 8%; n= 10; P > 0.05), suggesting that H267 was unimportant for the modulation at alkaline pH. On a similar basis, previous mutation experiments suggested that other external histidines were not mediating the alkaline pH modulation (data not shown; Dunne et al. 2002; Wilkins et al. 2002).

As the pH effect was GABA concentration dependent we considered if external tyrosines were crucial for the alkaline pH regulation, particularly Y157 and Y205 in the ß subunit, which are thought to reside at the GABA binding site (Cromer et al. 2002; Korpi et al. 2002). These residues were mutated to phenylalanines and separately expressed with wild-type 1 subunits. As previously reported, the sensitivity to GABA for the mutant receptors was reduced (Amin & Weiss, 1993; data not shown) which required a new GABA EC₂₀ (3 µM) to be determined. Exposure of 1ß2^Y157F and 1ß2^Y205F to pH 8.4 potentiated the GABA-activated currents by 33 ± 5% and 39 ± 7%, respectively, which is comparable to that observed with the wild-type receptor (n= 5; P > 0.05), suggesting that these GABA binding site tyrosine residues are not responsible for the potentiation at pH 8.4. It appeared unlikely that other external, accessible tyrosines and histidines were involved in the alkaline pH regulation, since applying 1 mM DEPC for 2 min prior to and during intermittent GABA (10 µM) application did not affect the potentiation of 1 µM GABA-activated responses on 1ß2 receptors at pH 8.4 (24 ± 6%, n= 5; P > 0.05).

Role of ß subunit lysines in alkaline pH modulation of GABA_A receptors

The prospect of lysine residues mediating the effects of alkaline pH was investigated following the reported distortion of their pK_a by charged environments in channels (Schulte et al. 1999). Such a charge distortion in the GABA_A receptor, by nearby arginines in TM2 or in the Cys loop, might bring lysine pK_a values within range of the alkaline pH shift (from 10 to 7.4). The two lysines, K274 and K279, selected for mutation, form part of the ß2 subunit TM2/TM2–TM3 linker (Fig. 3), a region associated with ion channel gating (Cromer et al. 2002; Kash et al. 2003). Furthermore, they also lie in close proximity to the proton sensitive H267 (Wilkins et al. 2002). Sequence homology comparisons reveal that K274 is conserved between the isoforms of the , ß and subunit families, with the sole exception of 3, whils, K279 is only present in the ß and subunit families.

View larger version (10K): [in this window] [in a new window]   Figure 3.  Alignment of the TM2–TM3 linkers for 1, ß2 and 2 subunits The lysine at position 274 (in the ß2 subunit) is conserved between all three subunits (shaded grey). However, the lysine at position 279 and H267 (black boxes in the ß2 subunit) and also E270 (open box) are only conserved between the ß subunit family. The grey shaded region denotes the traditional end of TM2 at 20' and the grey hatched box to 26' reflects the increased length of TM2 as reported previously (Horenstein et al. 2001). The numbering refers to the mature ß2 subunit and the prime numbers are referenced from the cytoplasmic ends of TM2.

View larger version (23K): [in this window] [in a new window]   Figure 4.  ß subunit lysines and the potentiation of the GABA-activated current for 1ß2 receptors at pH 8.4 A, normalized GABA concentration–response curves for 1ß2K274A () and 1ß2K279A () at pH 7.4. Peak currents were normalized to the maximum responses at pH 7.4 (n= 4–12 cells). The curve for the wild type 1ß2 receptor at pH 7.4 is shown as a dashed line for comparison (data from Fig. 1A). B, bar graph of the modulation of EC20 GABA-activated currents at pH 8.4 for 1ß2, 1ß2K274A and 1ß2K279A receptors (n= 4–12) compared with their corresponding control responses at pH 7.4 (= 100%). *P < 0.05 compared with the control response at pH 7.4; **P < 0.05 between the potentiated responses. C, GABA-activated currents induced by ECmax, EC50 and EC20 GABA concentrations applied at the indicated external pH for 1ß2K274A and 1ß2K279A receptors. D, 1 mM GABA- and 100 µM PTX-activated currents for the 1ß2K279A receptor at pH 7.4. All calibration bars represent: 200 pA, 2 s.

In a similar manner to the 1ß2^K279A GABA_A receptor, the GABA concentration–response curve for 1ß2^K279A2 at pH 7.4 also reflected an increased GABA potency (Fig. 5A) with the EC₅₀ (0.79 ± 0.22µM; P < 0.05) and Hill slope (0.5 ± 0.06; P < 0.05; n= 8) both significantly reduced in comparison to the 1ß22 receptor (EC₅₀ 3.4 ± 0.3 µM; Hill slope 1.3 ± 0.2; n= 10). As for the 1ß2^K279A receptor, the EC₂₀ GABA-activated current for the 1ß2^K279A2 receptor was now inhibited at pH 8.4 (Fig. 5B and C) and these receptors were also spontaneously active to a level of 26 ± 7% (n= 6; Fig. 5D).

View larger version (26K): [in this window] [in a new window]   Figure 5.  ß subunit K279 and the potentiation of the GABA-activated current for 1ß22 receptors at pH 8.4 A, normalized GABA concentration–response curves for the 1ß2K279A2, 1ß2K279Q2, 1ß2K279F2 and 1ß2K279R2 GABAA receptors at pH 7.4 (n= 5–12 cells). The curve for the wild-type 1ß22 receptor at pH 7.4 (dashed line) is taken from Fig 1B. B, bar graph of the modulation of the EC20 GABA-activated current at pH 8.4 for the 1ß22, 1ß2K279A2, 1ß2K279Q2, 1ß2K279F2 and 1ß2K279R2 GABAA receptors compared to their controls at pH 7.4 (= 100%; n= 5–13 cells). *P < 0.05 compared with the control response at pH 7.4. C, GABA-activated currents induced by ECmax, EC50 and EC20 GABA concentrations applied at the indicated external pH for the 1ß2K279A2 GABAA receptor. Calibration bars represent: 200pA, 2 s. D, 1 mM GABA- and 100 µM PTX-activated currents at pH 7.4 for the 1ß2K279A2 GABAA receptor. Calibration bar represents: 500pA, 2 s.

Modulation of 1ß22 GABA_A receptors by acidic pH at low GABA concentrations

Previously, 1ß22 GABA_A receptors were considered to be either insensitive to, or inhibited by, acidic pH (Krishek et al. 1996; Huang & Dillon, 1999), contrasting with 1ß2 and 1ß3 receptors, whose function was potentiated (Krishek et al. 1996; Wilkins et al. 2002; Feng & MacDonald, 2004). This differentiation was attributed to the disruption, by the subunit, of either the protonation site, or a transduction mechanism that operates following protonation (Krishek et al. 1996). Given that the pH sensitivity of the 1ß2 receptor clearly changed at low GABA concentrations (Fig. 2A), it was conceivable that the 1ß22 receptor might behave similarly. Indeed, for 1ß22 receptors, 1 µM GABA currents were potentiated at pH 6.4, but at pH 5.4, inhibition was observed when compared to control currents at pH 7.4 (Fig. 6A). The potentiation, at pH 6.4, was comparable to that observed with the 1ß2 receptor; moreover, the ß2^H267A mutation was also just as effective at abolishing the potentiation (Fig. 6B). However, in contrast to the 1ß2 receptor, the enhanced GABA response at pH 6.4 for the 1ß22 receptor was only observed at low GABA concentrations (1 µM; Fig. 6A). These data demonstrate the importance of ßH267 in the modulation of ß GABA_A receptors by protons, but this is only operative over a defined GABA concentration range.

View larger version (26K): [in this window] [in a new window]   Figure 6.  Role of ßH267 in the acidic pH modulation of the GABAA receptor A, bar graph representing the pH titration profile of the 1ß22 GABAA receptor over the pH range 5.4–6.9, determined for 1 µM (open bars) and 5 µM (grey bars) GABA-activated responses compared to their control responses at pH 7.4 (n= 13). B, bar graph for the modulation of 1 µM GABA-activated responses determined for 1ß2, 1ß2H267A, 1ß22 and 1ß2H267A2 GABAA receptors at pH 6.4 (n= 5–10). *P < 0.05 compared with the control responses determined at pH 7.4 (= 100%).

At pH 5.4, the pH sensitivities for 1ß2 and 1ß22 receptors diverged, with 1 µM GABA currents mediated via the latter now clearly inhibited (Figs 6A and 7A). The ß2^H267A mutation increased the inhibition of the GABA-activated current at pH 5.4 for the 1ß2^H267A2 receptor to a level similar to that observed with the 1ß2^H267A. receptor (Fig. 7A). This complex pH profile of the 1ß22 receptor may result from the replacement of a ß subunit by a subunit in the receptor complex, decreasing the number of H267 protonation sites in the receptor or affecting the transduction mechanism involved at pH 5.4. To examine the first possibility, a histidine residue (I282H) was introduced into the 2 subunit at the homologous position to H267 in the ß2 subunit restoring (as for 1ß2) three histidines around the entry to the ion channel, presuming a receptor stoichiometry of 2: 2ß: 1 (Farrar et al. 1999). A further discrepancy in the charged residue complement within the TM2-TM3 linker was also corrected, by replacing 2K285 with the homologous ß2E270 With these mutations, it was conceivable that the 1ß22^I282H,K285E receptor might now support a potentiation of the GABA-activated current at pH 5.4. The GABA concentration–response curve for the 1ß22^I282H,K285E receptor was not different to the wild-type receptor (GABA EC₅₀: 1ß22, 3.4 ± 0.3 µM; 1ß22^I282H,K285E, 3.3 ± 0.3 µM; n= 7; Fig. 7B). However, the double mutation did not allow any potentiation of the EC₂₀ GABA-activated current at pH 5.4, revealing just inhibition to a similar extent to that observed with the wild-type 1ß22 receptor (Fig. 7A and C). These data suggested that the provision of three histidine residues around the ion channel in the 1ß22 receptor is insufficient for proton-induced potentiation of the GABA-activated current at pH 5.4.

View larger version (29K): [in this window] [in a new window]   Figure 7.  Modulation of the GABAA receptor at pH 5.4 is dependent on K279 in the ß2 subunit A, bar graph for the modulation of EC20 GABA-activated currents at pH 5.4 for wild-type 1ß2 and 1ß22 (open bars) and mutant ß GABAA receptors, incorporating: ß2H267A (black bars), 2I282H,K285E (HE), 2I282H,K285E,T294K (HEK), 2T294K (K) and ß2K279A2T294K (AK), all grey bars. *P < 0.05 compared with the respective control responses determined at pH 7.4 (= 100%; n= 5–12). B, normalized GABA concentration–response curves for 1ß22I282H,K285E, 1ß22I282H,K285E,T294K, 1ß22T294K and 1ß2K279A2T294K GABAA receptors (n= 4–10). The dashed line represents the curve for the wild-type 1ß22 receptor at pH 7.4 (from Fig. 1B). C, GABA-activated currents induced by EC20 GABA applied at the indicated external pH for the 1ß22 and mutant ß GABAA receptors. See A, for key. Calibration bar represents: 200pA, 1 s.

Since the double mutant, 1ß22^I282H,K285E receptor failed to support any potentiation at acidic pH, we considered that T294K alone might be sufficient to enable potentiation at pH 5.4. The GABA concentration–response curve revealed that the mutant 1ß22^T294K receptor was similarly sensitive to GABA (GABA EC₅₀ 4.2 ± 0.4 µM; n= 7; P > 0.05) when compared to the wild-type receptor (Fig. 7B). Moreover, on exposure to pH 5.4, the 1 µM GABA-activated response was significantly potentiated (53 ± 8%) compared with the control at pH 7.4, approaching the level of potentiation expected for a wild-type 1ß2 receptor (103 ± 15%; Fig. 7A and C).

To evaluate if the lysine incorporated into the 2 subunit was solely responsible for the modulation of the EC₂₀ GABA-activated current in acidic pH, a double mutant 1ß2^K279A2^T294K was examined. As for the 1ß2^K279A receptor, GABA potency was increased for the 1ß2^K279A2^T294K receptor compared to the wild-type receptor at pH 7.4 (EC₅₀ 0.9 ± 0.1 µM; n= 5; P < 0.05; Fig. 7B). In addition, by using 100 µM PTX, this receptor was spontaneous active at a level of 11 ± 1.2% (n= 4); however, at pH 5.4, the EC₂₀ (0.1 µM) GABA-activated response was still inhibited, thus appearing no different from the wild-type and 1ß22^I282H,K285E receptors (Fig. 7A and C). These data suggest that three copies of the lysine, each one at position 279 in the ß subunits or the homologous position in the subunit, are required for modulation of the GABA_A receptor at pH 5.4, and that inclusion of a lysine only in the subunit is in sufficient.

	Discussion

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This study reveals that the regulation of GABA_A receptor function can change over quite small excursions in external pH. It establishes that the concentration of GABA activating the receptor is critical for pH regulation and also identifies a vital lysine residue, located in the TM2–TM3 linker in the ß subunit, which is critical for the potentiation of GABA-activated currents at alkaline pH. Furthermore, for the potentiation observed at acidic pH, lysine residues must be present, not only in the GABA binding ß subunits but also in the fifth subunit of the pentamer, be it a ß or subunit, which is the only subunit not directly involved in GABA binding. These results are entirely in accord with K279 playing a vital role in the pH modulation of GABA_A receptors as well as being an important residue in GABA_A receptor activation.

GABA concentration dependence of pH regulation

The regulation by external pH of GABA-activated currents is dependent on the receptor subunit composition, but even so, it is clear that the pH sensitivities of neuronal GABA_A receptors do not always correlate with the behaviour expected from recombinant receptor studies, particularly at alkaline pH (Robello et al. 1994; Krishek et al. 1996; Pasternack et al. 1996; Zhai et al. 1998; Huang & Dillon, 1999; Wilkins et al. 2002). Another factor that may affect alkaline pH regulation is the GABA concentration. In acutely isolated hippocampal neurones, using high GABA concentrations (500 µM), alkaline pH reduced the GABA current amplitude, whereas for low GABA concentrations (5 µM) pH 8.4 increased the current amplitude (Pasternack et al. 1996). In contrast, for spinal neurones, pH dependent modulation was abolished at high GABA concentrations (300 µM), whilst at lower concentrations (10 µM), alkaline pH increased the GABA current amplitude (Li et al. 2003). Using fast perfusion techniques, the potentiation of responses to low GABA concentrations in alkaline pH was thought to reflect increases in the agonist binding rate thus promoting channel opening (Mozrzymas et al. 2003), whilst at higher GABA concentrations, the reduced current amplitude was thought to be due to rapid desensitization. These findings are broadly in agreement with the single channel analyses of cerebellar granule cell GABA_A receptors (< 7 DIV) (Krishek & Smart, 2001) and recombinant 3ß22 subunit receptors (Huang & Dillon, 1999). In these studies, alkaline pH excursions increased channel open probabilities, without any change in the open time distributions, but reduced the long shut times causing the channel opening frequencies to increase. In the present study, alkaline pH induced a very small leftward shift in the lower part (< EC₅₀) of the GABA concentration–response curves for 1ß2 and 1ß22 GABA_A receptors. This shift was more clearly resolved by pH titration using low GABA concentrations. Under these conditions, both 1ß2 and the 1ß22 GABA_A receptors were affected by pH 8.4 in a similar manner possibly via the same specific molecular mechanism.

Molecular mechanism underlying alkaline pH modulation

The nature of the residues that are involved in the alkaline pH regulation and where they are located has remained elusive. Following a pH titration of the 1ß2 wild-type receptor, several residues were identified as prospective candidates. However, in the ß subunit, H267, initially considered due to its regulatory role at acidic pH (Wilkins et al. 2002), and Y157 and Y205, which are located in the GABA binding site (Amin & Weiss, 1993; Cromer et al. 2002), played no role in the modulation of the GABA_A receptor at pH 8.4. However, the modulation of GABA_A receptors in alkaline pH has been reported to involve the GABA binding site with Y205 and F64 in the ß2 subunit being involved (Huang et al. 2004). Whilst in our study, Y205 appears to play no role in the alkaline pH modulation, and the excessive decrease in GABA potency for the F64 mutant makes it very difficult to assess its impact in this context. In addition, the observation that ß GABA_A receptors are inhibited in acidic pH (Huang et al. 2004) is also at variance with other groups that have reported potentiation based on the protonation of H267 (Wilkins et al. 2002; Feng & MacDonald, 2004). Furthermore, the use of DEPC, on active or quiescent GABA_A receptors, suggested that all other accessible histidines and tyrosines were most likely not involved in pH regulation, leaving only lysine residues as possible candidates.

The ß subunit lysines selected for mutation, K274 and K279, form part of the TM2–TM3 linker and lie close to H267 in the ion channel. Lysine 274 is conserved between all of the GABA_A receptor subunits and may play an important role in GABA_A receptor gating (Sigel et al. 1999; Kash et al. 2003; Kash et al. 2004). Notably, the GABA concentration–response curve for 1ß2^K274A was laterally displaced to higher GABA concentrations, as reported for the 1ß2^K274A2 receptor (Sigel et al. 1999); however, this same mutation accentuated the potentiation of the GABA current by alkaline pH. The alternative lysine, K279, is only present in ß subunits and its substitution in either 1ß2^K279A or 1ß2^K279A2 receptors ablated the alkaline pH potentiation of the GABA-activated current, revealing inhibition.

The GABA concentration dependence of the alkaline pH modulation could conceivably be induced by the deprotonation of K279, with this reaction being less likely at high GABA concentrations, perhaps because of restricted access of protons to K279. However, the pK_a of the lysine side chain is 10 and would therefore be unlikely to act as a proton acceptor at physiological pH or indeed at pH 8.4, as we would expect little change in its ionization state (99.7% or 97.5% positively charged, respectively). For lysine to act as a proton acceptor, the pK_a would have to shift into the neutral pH range. Such a perturbation (from 10 to 7) can occur but depends upon the close proximity of other basic residues (Schulte et al. 1999). If such a pK_a shift occurred in the GABA_A receptor, then, increasing the pH to 8.4 would cause a much greater loss of H⁺ from the amine group of the lysine, leaving 3.8% of the side-chain positively charged compared with 28.5% at pH 7.4. Interestingly, by altering the charge and size of the side chain at position 279, alkaline pH modulation was prevented, suggesting that the structure of the lysine side chain was critical for potentiation at alkaline pH.

Location of K279 using molecular modelling

An emerging consensus of Cys-loop receptor gating suggests that TM1, 3 and 4 form a rigid structure from which the channel lining the TM2 domain is suspended. The TM1–TM2 and TM2–TM3 linkers act as hinges to allow rotation of TM2 during channel opening, with the TM2–TM3 linker also contacting the ligand-binding extracellular domain (Absalom et al. 2004). The importance of the TM2–TM3 linker for receptor activation is demonstrated by mutating the conserved lysines, K279 ( subunit) and K274 (ß subunit). Both residues are thought to interact with acidic residues in loops 2 and 7 of the extracellular domains (AChBP nomenclature; Kash et al. 2003; Kash et al. 2004). Homology modelling, using the electron microscopic images of nicotinic acetylcholine receptors (Miyazawa et al. 2003), suggests that K279 in the TM2–TM3 linker is ideally situated to either interact with the N-terminal domains, or influence the conformational changes that follow agonist binding (Fig. 8). Whilst the mutation of ß^K279 to aspartate (Kash et al. 2004) did not affect coupling with the N-terminal domain, this residue may participate in channel gating since both 1ß2^K279A and 1ß2^K279A2 receptors are spontaneously active. Although alkaline pH modulation of GABA_A receptors requires a lysine residue at ß^K279 it is unaffected by the subunit and thus apparently oblivious to the homologous residue in the fifth subunit in the pentamer. By contrast, lysine residues are required in both the ß and subunits (as well as H267 in the ß subunit) for modulation by acid pH (see below). Whether pH changes can alter the protonation of K279 cannot be determined at present, but it is conceivable that the differential structural dependence of these pH effects may be caused by the receptor adopting different open conformations in acid compared to alkaline pH.

View larger version (63K): [in this window] [in a new window]   Figure 8.  Molecular model of the GABAA receptor ß2 and 2 subunits A, molecular model showing the ß2 subunit (orange) and the 2 subunit (green) for the GABAA receptor. Both TM2, which lines the receptor channel, and the TM2–TM3 linker, are shown in yellow, with the residues involved in pH modulation space filled. B, magnified view of the area involved in pH modulation. In the ß2 subunit; K279 (red), H267 (light yellow) are thought to be either directly involved in protonation, or form part of an allosteric pH modulatory mechanism; whilst K274 (pink) and E270 (dark yellow) are probably not involved. In the 2 subunit, residues in the homologous positions to those in the ß subunit are also shown.

Protonation and the 1ß22 receptor

The potentiation of the GABA-activated current, at pH 6.4, for low GABA concentrations on 1ß22 receptors, was similar to that observed with the 1ß2 receptor but at pH 5.4, the potentiation was absent. The introduction of ß subunit residues, H267 and E270, into homologous positions in the 2 subunit for 1ß22 receptors failed to potentiate the GABA-activated current at pH 5.4. These data implied that an allosteric mechanism may be required to augment the response at pH 5.4 and this was seemingly achieved by the mutation 2^T294K. Overall, three lysines were required for potentiation of the GABA-activated current at pH 5.4 on 1ß22 receptors (one in each ß subunit and one in the mutated 2 subunit). Interestingly, GABA currents of GABA_A receptors containing the subunit are also potentiated by acidic pH (Feng & MacDonald, 2004; Krishek et al. 1996). These subunits also have a lysine residue (K292) (Shivers et al. 1989) in the homologous position to K279 in the ß subunit. The pivotal role that this lysine residue plays, not only in the alkaline pH modulation but also at acidic pH, emphasizes that it is unlikely to be a target for protonation per se, but most probably participates as a key component in allosteric modulation. Clearly, the TM2–TM3 linker, is not just involved in the gating of the GABA_A receptor, but is a vital component in acidic and alkaline pH-induced modulation.

Physiological relevance

The importance of the pH regulation of GABA_A receptor function, as described in this study, may become physiologically relevant under conditions where low GABA concentrations occur, for example around extrasynaptic receptors where basal GABA concentrations are estimated in the low micromolar range (hippocampus, 0.3–1 µM, M. Mortensen & T. G. Smart, unpublished observations; 0.8–3 µM, Lerma et al. 1986; 0.1 µM, Petrini et al. 2004). Under these conditions, minor external pH excursions of only 0.2 pH units would be sufficient to affect GABA_A receptor function causing subtle effects to the level of tonic inhibition and neuronal excitability which could become more apparent during pathological events. Furthermore, studies investigating the changes in extracellular pH during ischaemia, hypothermia and brain trauma have shown that the extracellular pH of the brain can alter by approximately 1 pH unit which will be expected to impact on the function of GABA_A receptors (Kraig et al. 1987; Hoffman et al. 1996; Hoffman et al. 1999; Anderson & Meyer, 2002).

	References

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