Proton modulation of recombinant GABAA receptors: influence of GABA concentration and the subunit TM2–TM3 domain
1 Department of Pharmacology, University College London, Medical Sciences Building, Gower Street, London WC1E 6BT, UK
Abstract
Regulation of GABAA receptors by extracellular pH exhibits a dependence on the receptor subunit composition. To date, the molecular mechanism responsible for the modulation of GABAA receptors at alkaline pH has remained elusive. We report here that the GABA-activated current can be potentiated at pH 8.4 for both and subunit-containing receptors, but only at GABA concentrations below the EC40. Site-specific mutagenesis revealed that a single lysine residue, K279 in the subunit TM2–TM3 linker, was critically important for alkaline pH to modulate the function of both 12 and 122 receptors. The ability of low concentrations of GABA to reveal different pH titration profiles for GABAA receptors was also examined at acidic pH. At pH 6.4, GABA activation of receptors was enhanced at low GABA concentrations. This effect was ablated by the mutation H267A in the subunit. Decreasing the pH further to 5.4 inhibited GABA responses via receptors, whereas those responses recorded from receptors were potentiated. Inserting homologous subunit residues into the 2 subunit to recreate, in receptors, the proton modulatory profile of receptors, established that in the presence of 2H267, the mutation 2T294K was necessary to potentiate the GABA response at pH 5.4. This residue, T294, is homologous to K279 in the 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 GABAA receptor.
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Introduction
The regulation of GABAA 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 GABAA receptors predominantly composed of subunits (McKernan & Whiting, 1996). In comparison, extrasynaptic GABAA 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 isoforms, but also receptors comprising 4, 5 or 6 and subunits, as well as the potential for isoforms (Brickley et al. 2001; Mody, 2001).
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Protons can exert various effects on GABAA 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 , and GABAA receptors (Krishek et al. 1996). For the isoform, this modulation has been largely attributed to the protonation of a single histidine residue, located in the ion channel lining contributed by the subunit (H267) (Wilkins et al. 2002). However, the alkaline pH sensitivity profiles of recombinant GABAA 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 GABAA 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 GABAA or glutamate receptors (Chen & Chesler, 1991; Chen & Chesler, 1992; Kaila et al. 1992). Physiologically, during synaptic transmission, bicarbonate ions are thought to flow through GABAA 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).
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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 GABAA receptor in alkaline pH. In addition, by examining the differential modulation of and GABAA receptors in acidic pH we have identified key residues that are involved in the modulation of GABAA receptors at both alkaline and acidic pH.
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Methods
cDNA constructs and site-specific mutagenesis
Murine GABAA receptor 1, 2 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).
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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.
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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.
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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.
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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 (Imax) and the concentration response relationship fitted with the Hill equation:
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where the EC50 represents the concentration of GABA ([A]) inducing 50% of the maximal current evoked by a saturating concentration of GABA and nH 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 pKa 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:
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The level of spontaneous receptor activation was assessed by using picrotoxin to block active GABAA 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 12 or 122 receptors. The extent of spontaneity was established by examining the maximum outward current induced by picrotoxin (IPTX) which was summed with the maximum current activated by a saturating concentration of GABA (IGABA,max). The level of spontaneous receptor activation was quantified according to:
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Molecular modelling
The transmembrane domains of GABAA receptor 1, 2 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 GABAA 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.
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Results
External alkaline pH and recombinant 12 and 122 GABAA receptors
The effect of alkaline pH on GABAA receptors was assessed by comparing GABA concentration–response relationships for both 12 and 122 GABAA 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 12 (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 122 (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 (EC10-40) for both receptor constructs (Fig. 1C and D).
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A and B, GABA concentration–response curves for 12 (A) and 122 (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 12 (C) and 122 (D) at pH 7.4 () and 8.4 ().
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Modulation at alkaline pH is dependent on GABA concentration
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 12 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 pKa 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 12 receptors, as reported for 11 (Krishek et al. 1996), with the titration curve now described by a single pKa 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 and 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).
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A, pH titration profiles for 12 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 12 (white bars) and 122 (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 12 and 122 GABAA receptors. Calibration bars represent: 400pA, 2 s.
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As both types of GABAA receptor were regulated in a similar manner by alkaline pH, it was conceivable that the underlying molecular mechanism(s) involved identical residues. To facilitate their identification using site-directed mutagenesis, we targeted residues whose pKa values were similar to those determined from the pH titrations. For the 12 receptor, the pKa of 7.7 implicated cysteine (pKa8.3) and/or histidine (pKa6) residues. As only external pH excursions affected GABAA receptor function (Krishek et al. 1996), only extracellular residues were considered. On this basis, the two extracellular cysteine residues are probably precluded since they form a disulphide bridge (Amin et al. 1994; Lu, 1997). Although histidines are alternatives, the involvement of H267 in receptor modulation at acidic pH suggested that they may not be involved (Wilkins et al. 2002). Further alternatives included lysines or tyrosines and although their side-chain pKa values are 10, these can be considerably lower and within range of the alkaline pH change, depending upon their microenvironment in the receptor protein (Schulte et al. 1999).
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Alkaline pH-induced modulation does not involve histidine or tyrosine residues
The involvement of histidine and tyrosine residues with the modulation of the GABAA 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 subunit, forming 12H267A and 12H267A2 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, 12 (38 ± 8%) and 122 (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).
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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 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 12Y157F and 12Y205F 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 12 receptors at pH 8.4 (24 ± 6%, n= 5; P > 0.05).
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Role of subunit lysines in alkaline pH modulation of GABAA receptors
The prospect of lysine residues mediating the effects of alkaline pH was investigated following the reported distortion of their pKa by charged environments in channels (Schulte et al. 1999). Such a charge distortion in the GABAA receptor, by nearby arginines in TM2 or in the Cys loop, might bring lysine pKa 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 2 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 , and subunit families, with the sole exception of 3, whils, K279 is only present in the and subunit families.
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The lysine at position 274 (in the 2 subunit) is conserved between all three subunits (shaded grey). However, the lysine at position 279 and H267 (black boxes in the 2 subunit) and also E270 (open box) are only conserved between the 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 2 subunit and the prime numbers are referenced from the cytoplasmic ends of TM2.
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The expression of either 12K274A or 12K279A resulted in the formation of functional receptors, but the GABA concentration–response curves were displaced to either the right (K274A) or the left (K279A) when compared with the curve for the wild-type 12 receptor at pH 7.4 (Fig. 4A). These displacements were reflected in GABA EC50 values (12K274A, 8.6 ± 3.2 μM; 12K279A, 0.7 ± 0.2 μM; n= 12) and Hill slopes (12K274A, 0.7 ± 0.18; 12K279A, 0.5 ± 0.06; n= 12), compared to their wild-type equivalents (12, EC50, 2.9 ± 0.3 μM; Hill slope, 1.0 ± 0.08; P < 0.05).
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A, normalized GABA concentration–response curves for 12K274A () and 12K279A () 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 12 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 12, 12K274A and 12K279A 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 12K274A and 12K279A receptors. D, 1 mM GABA- and 100 μM PTX-activated currents for the 12K279A receptor at pH 7.4. All calibration bars represent: 200 pA, 2 s.
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Using the appropriate GABA EC20 (1 μM for 12 and 12K274A and 0.1 μM for 12K279A) at pH 8.4, the potentiation of GABA-activated current for 12K274A receptors was increased (79 ± 20%; Fig. 4B and C; P < 0.05) compared to that at pH 7.4. In contrast, exposing the 12K279A receptor to pH 8.4 abolished the potentiation, revealing a significant inhibition of the GABA response to 78 ± 5% compared to that at pH 7.4 (Fig. 4B and C). Another notable feature of the 12K279A receptor was that the deactivation kinetics of the GABA-activated currents were quite slow, a feature not apparent for the 12K274A mutant (Fig. 4C). Given the position of the lysines in the TM2–TM3 linker, we investigated whether the receptor was capable of spontaneous activation. Both 12K274A and 12K279A receptors were exposed to 100 μM picrotoxin (PTX) in the absence of GABA, which would inhibit any spontaneous channel openings (Sigel et al. 1989; Wooltorton et al. 1997). The K274A mutation was unaffected by PTX (n= 5), in contrast to the outward current observed for the 12K279A receptor (Fig. 4D), which was in accord with a level of spontaneous gating estimated at 22 ± 1.4% (n= 7: see Methods).
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In a similar manner to the 12K279A GABAA receptor, the GABA concentration–response curve for 12K279A2 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 122 receptor (EC50 3.4 ± 0.3 μM; Hill slope 1.3 ± 0.2; n= 10). As for the 12K279A receptor, the EC20 GABA-activated current for the 12K279A2 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).
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A, normalized GABA concentration–response curves for the 12K279A2, 12K279Q2, 12K279F2 and 12K279R2 GABAA receptors at pH 7.4 (n= 5–12 cells). The curve for the wild-type 122 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 122, 12K279A2, 12K279Q2, 12K279F2 and 12K279R2 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 12K279A2 GABAA receptor. Calibration bars represent: 200pA, 2 s. D, 1 mM GABA- and 100 μM PTX-activated currents at pH 7.4 for the 12K279A2 GABAA receptor. Calibration bar represents: 500pA, 2 s.
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To ensure that the mutation of lysine to alanine, with its smaller side chain, was not distorting receptor structure and thus indirectly affecting the response to alkaline pH, we mutated K279 to uncharged glutamine (Q), non-polar phenylalanine (F) and basic arginine (R), with their comparable side chain volumes. All three mutant receptors, 12K279Q2, 12K279F2 and 12K279R2, were functional with reduced EC50 values (1.08 ± 0.04 μM, 0.42 ± 0.06 μM and 0.88 ± 0.21 μM, respectively; n= 5–16) and Hill slopes (0.77 ± 0.02, 0.85 ± 0.1 and 0.49 ± 0.21, respectively; Fig. 5A) compared to 122 (EC50 3.4 ± 0.3 μM; Hill slope 1.3 ± 0.2). Potentiation of the EC20 GABA responses at pH 8.4 was abolished by all three lysine mutations and only for the 12K279R2 receptor was the GABA response inhibited in a similar manner to that observed with the K279A mutant (Fig. 5B). In addition, as for the 12K279A2 receptor, all the other mutants were spontaneously active with levels of 14 ± 2% (K279Q), 13 ± 4% (K279F) and 34 ± 4% (K279R). Taken overall, these findings suggest that the subunit K279 was critically important for the potentiation of the current activated by low GABA concentrations at both and subunit-containing receptors in alkaline pH, as well as a potentially important residue in ion channel gating.
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Modulation of 122 GABAA receptors by acidic pH at low GABA concentrations
Previously, 122 GABAA receptors were considered to be either insensitive to, or inhibited by, acidic pH (Krishek et al. 1996; Huang & Dillon, 1999), contrasting with 12 and 13 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 12 receptor clearly changed at low GABA concentrations (Fig. 2A), it was conceivable that the 122 receptor might behave similarly. Indeed, for 122 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 12 receptor; moreover, the 2H267A mutation was also just as effective at abolishing the potentiation (Fig. 6B). However, in contrast to the 12 receptor, the enhanced GABA response at pH 6.4 for the 122 receptor was only observed at low GABA concentrations (1 μM; Fig. 6A). These data demonstrate the importance of H267 in the modulation of GABAA receptors by protons, but this is only operative over a defined GABA concentration range.
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A, bar graph representing the pH titration profile of the 122 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 12, 12H267A, 122 and 12H267A2 GABAA receptors at pH 6.4 (n= 5–10). *P < 0.05 compared with the control responses determined at pH 7.4 (= 100%).
Role of lysine 279 in the regulation by acidic pH
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At pH 5.4, the pH sensitivities for 12 and 122 receptors diverged, with 1 μM GABA currents mediated via the latter now clearly inhibited (Figs 6A and 7A). The 2H267A mutation increased the inhibition of the GABA-activated current at pH 5.4 for the 12H267A2 receptor to a level similar to that observed with the 12H267A. receptor (Fig. 7A). This complex pH profile of the 122 receptor may result from the replacement of a 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 2 subunit restoring (as for 12) three histidines around the entry to the ion channel, presuming a receptor stoichiometry of 2: 2: 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 2E270 With these mutations, it was conceivable that the 122I282H,K285E receptor might now support a potentiation of the GABA-activated current at pH 5.4. The GABA concentration–response curve for the 122I282H,K285E receptor was not different to the wild-type receptor (GABA EC50: 122, 3.4 ± 0.3 μM; 122I282H,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 122 receptor (Fig. 7A and C). These data suggested that the provision of three histidine residues around the ion channel in the 122 receptor is insufficient for proton-induced potentiation of the GABA-activated current at pH 5.4.
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A, bar graph for the modulation of EC20 GABA-activated currents at pH 5.4 for wild-type 12 and 122 (open bars) and mutant GABAA receptors, incorporating: 2H267A (black bars), 2I282H,K285E (HE), 2I282H,K285E,T294K (HEK), 2T294K (K) and 2K279A2T294K (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 122I282H,K285E, 122I282H,K285E,T294K, 122T294K and 12K279A2T294K GABAA receptors (n= 4–10). The dashed line represents the curve for the wild-type 122 receptor at pH 7.4 (from Fig. 1B). C, GABA-activated currents induced by EC20 GABA applied at the indicated external pH for the 122 and mutant GABAA receptors. See A, for key. Calibration bar represents: 200pA, 1 s.
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The second possibility, regarding disruption to the transduction pathway by the 2 subunit, was addressed by identifying residues that may mediate the potentiation of the GABA current at acid pH. Lysine 279 in the 2 subunit was selected because of its importance for alkaline pH modulation. Indeed, its mutation in 12K279A receptors, resulted in much smaller potentiations of 1 or 10 μM GABA-activated currents at pH 5.4 compared to the wild-type receptor (12, potentiation of 94 ± 17% (1 μM GABA) and 91 ± 10% (10 μM), n= 25; 12K279A, 15 ± 7% (1 μM) and 41 ± 11% (10 μM), n= 7). These data are in accord with K279 playing a critical role in the potentiation of the GABA-activated current for the 12 receptor at pH 5.4 as well as at pH 8.4. As the K279 is normally only present in the subunit, we introduced a lysine into the 2 subunit at the homologous position (T294K), together with the previously introduced residues, I282H and K285E, as found in the 12 receptor. The GABA concentration–response relationship for the 122I282H,K285E,T294K receptor at pH 7.4 was marginally displaced (2-fold) with a reduced EC50 (1.7 ± 0.2 μM; n= 10) compared with the wild-type receptor (3.4 ± 0.3 μM; P < 0.05; Fig. 7B). Of importance, the 1 μM (EC20) GABA-activated current at pH 5.4 was now potentiated, contrasting with the inhibition normally observed with the wild-type 122 receptor (Fig. 7A and C).
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Since the double mutant, 122I282H,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 122T294K 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 12 receptor (103 ± 15%; Fig. 7A and C).
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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 12K279A2T294K was examined. As for the 12K279A receptor, GABA potency was increased for the 12K279A2T294K 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 122I282H,K285E receptors (Fig. 7A and C). These data suggest that three copies of the lysine, each one at position 279 in the subunits or the homologous position in the subunit, are required for modulation of the GABAA receptor at pH 5.4, and that inclusion of a lysine only in the subunit is in sufficient.
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Discussion
This study reveals that the regulation of GABAA 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 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 subunits but also in the fifth subunit of the pentamer, be it a 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 GABAA receptors as well as being an important residue in GABAA receptor activation.
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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 GABAA 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 GABAA receptors (< 7 DIV) (Krishek & Smart, 2001) and recombinant 322 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 12 and 122 GABAA receptors. This shift was more clearly resolved by pH titration using low GABA concentrations. Under these conditions, both 12 and the 122 GABAA receptors were affected by pH 8.4 in a similar manner possibly via the same specific molecular mechanism.
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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 12 wild-type receptor, several residues were identified as prospective candidates. However, in the 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 GABAA receptor at pH 8.4. However, the modulation of GABAA receptors in alkaline pH has been reported to involve the GABA binding site with Y205 and F64 in the 2 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 GABAA 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 GABAA receptors, suggested that all other accessible histidines and tyrosines were most likely not involved in pH regulation, leaving only lysine residues as possible candidates.
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The 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 GABAA receptor subunits and may play an important role in GABAA receptor gating (Sigel et al. 1999; Kash et al. 2003; Kash et al. 2004). Notably, the GABA concentration–response curve for 12K274A was laterally displaced to higher GABA concentrations, as reported for the 12K274A2 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 subunits and its substitution in either 12K279A or 12K279A2 receptors ablated the alkaline pH potentiation of the GABA-activated current, revealing inhibition.
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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 pKa 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 pKa 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 pKa shift occurred in the GABAA 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.
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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 (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 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 12K279A and 12K279A2 receptors are spontaneously active. Although alkaline pH modulation of GABAA receptors requires a lysine residue at 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 and subunits (as well as H267 in the 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.
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A, molecular model showing the 2 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 2 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 subunit are also shown.
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Protonation and the 122 receptor
The potentiation of the GABA-activated current, at pH 6.4, for low GABA concentrations on 122 receptors, was similar to that observed with the 12 receptor but at pH 5.4, the potentiation was absent. The introduction of subunit residues, H267 and E270, into homologous positions in the 2 subunit for 122 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 2T294K. Overall, three lysines were required for potentiation of the GABA-activated current at pH 5.4 on 122 receptors (one in each subunit and one in the mutated 2 subunit). Interestingly, GABA currents of GABAA 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 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 GABAA receptor, but is a vital component in acidic and alkaline pH-induced modulation.
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Physiological relevance
The importance of the pH regulation of GABAA 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 GABAA 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 GABAA receptors (Kraig et al. 1987; Hoffman et al. 1996; Hoffman et al. 1999; Anderson & Meyer, 2002).
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Abstract
Regulation of GABAA receptors by extracellular pH exhibits a dependence on the receptor subunit composition. To date, the molecular mechanism responsible for the modulation of GABAA receptors at alkaline pH has remained elusive. We report here that the GABA-activated current can be potentiated at pH 8.4 for both and subunit-containing receptors, but only at GABA concentrations below the EC40. Site-specific mutagenesis revealed that a single lysine residue, K279 in the subunit TM2–TM3 linker, was critically important for alkaline pH to modulate the function of both 12 and 122 receptors. The ability of low concentrations of GABA to reveal different pH titration profiles for GABAA receptors was also examined at acidic pH. At pH 6.4, GABA activation of receptors was enhanced at low GABA concentrations. This effect was ablated by the mutation H267A in the subunit. Decreasing the pH further to 5.4 inhibited GABA responses via receptors, whereas those responses recorded from receptors were potentiated. Inserting homologous subunit residues into the 2 subunit to recreate, in receptors, the proton modulatory profile of receptors, established that in the presence of 2H267, the mutation 2T294K was necessary to potentiate the GABA response at pH 5.4. This residue, T294, is homologous to K279 in the 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 GABAA receptor.
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Introduction
The regulation of GABAA 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 GABAA receptors predominantly composed of subunits (McKernan & Whiting, 1996). In comparison, extrasynaptic GABAA 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 isoforms, but also receptors comprising 4, 5 or 6 and subunits, as well as the potential for isoforms (Brickley et al. 2001; Mody, 2001).
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Protons can exert various effects on GABAA 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 , and GABAA receptors (Krishek et al. 1996). For the isoform, this modulation has been largely attributed to the protonation of a single histidine residue, located in the ion channel lining contributed by the subunit (H267) (Wilkins et al. 2002). However, the alkaline pH sensitivity profiles of recombinant GABAA 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 GABAA 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 GABAA or glutamate receptors (Chen & Chesler, 1991; Chen & Chesler, 1992; Kaila et al. 1992). Physiologically, during synaptic transmission, bicarbonate ions are thought to flow through GABAA 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).
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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 GABAA receptor in alkaline pH. In addition, by examining the differential modulation of and GABAA receptors in acidic pH we have identified key residues that are involved in the modulation of GABAA receptors at both alkaline and acidic pH.
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Methods
cDNA constructs and site-specific mutagenesis
Murine GABAA receptor 1, 2 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).
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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.
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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.
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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.
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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 (Imax) and the concentration response relationship fitted with the Hill equation:
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where the EC50 represents the concentration of GABA ([A]) inducing 50% of the maximal current evoked by a saturating concentration of GABA and nH 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 pKa 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:
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The level of spontaneous receptor activation was assessed by using picrotoxin to block active GABAA 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 12 or 122 receptors. The extent of spontaneity was established by examining the maximum outward current induced by picrotoxin (IPTX) which was summed with the maximum current activated by a saturating concentration of GABA (IGABA,max). The level of spontaneous receptor activation was quantified according to:
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Molecular modelling
The transmembrane domains of GABAA receptor 1, 2 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 GABAA 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.
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Results
External alkaline pH and recombinant 12 and 122 GABAA receptors
The effect of alkaline pH on GABAA receptors was assessed by comparing GABA concentration–response relationships for both 12 and 122 GABAA 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 12 (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 122 (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 (EC10-40) for both receptor constructs (Fig. 1C and D).
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A and B, GABA concentration–response curves for 12 (A) and 122 (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 12 (C) and 122 (D) at pH 7.4 () and 8.4 ().
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Modulation at alkaline pH is dependent on GABA concentration
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 12 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 pKa 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 12 receptors, as reported for 11 (Krishek et al. 1996), with the titration curve now described by a single pKa 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 and 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).
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A, pH titration profiles for 12 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 12 (white bars) and 122 (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 12 and 122 GABAA receptors. Calibration bars represent: 400pA, 2 s.
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As both types of GABAA receptor were regulated in a similar manner by alkaline pH, it was conceivable that the underlying molecular mechanism(s) involved identical residues. To facilitate their identification using site-directed mutagenesis, we targeted residues whose pKa values were similar to those determined from the pH titrations. For the 12 receptor, the pKa of 7.7 implicated cysteine (pKa8.3) and/or histidine (pKa6) residues. As only external pH excursions affected GABAA receptor function (Krishek et al. 1996), only extracellular residues were considered. On this basis, the two extracellular cysteine residues are probably precluded since they form a disulphide bridge (Amin et al. 1994; Lu, 1997). Although histidines are alternatives, the involvement of H267 in receptor modulation at acidic pH suggested that they may not be involved (Wilkins et al. 2002). Further alternatives included lysines or tyrosines and although their side-chain pKa values are 10, these can be considerably lower and within range of the alkaline pH change, depending upon their microenvironment in the receptor protein (Schulte et al. 1999).
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Alkaline pH-induced modulation does not involve histidine or tyrosine residues
The involvement of histidine and tyrosine residues with the modulation of the GABAA 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 subunit, forming 12H267A and 12H267A2 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, 12 (38 ± 8%) and 122 (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).
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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 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 12Y157F and 12Y205F 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 12 receptors at pH 8.4 (24 ± 6%, n= 5; P > 0.05).
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Role of subunit lysines in alkaline pH modulation of GABAA receptors
The prospect of lysine residues mediating the effects of alkaline pH was investigated following the reported distortion of their pKa by charged environments in channels (Schulte et al. 1999). Such a charge distortion in the GABAA receptor, by nearby arginines in TM2 or in the Cys loop, might bring lysine pKa 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 2 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 , and subunit families, with the sole exception of 3, whils, K279 is only present in the and subunit families.
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The lysine at position 274 (in the 2 subunit) is conserved between all three subunits (shaded grey). However, the lysine at position 279 and H267 (black boxes in the 2 subunit) and also E270 (open box) are only conserved between the 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 2 subunit and the prime numbers are referenced from the cytoplasmic ends of TM2.
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The expression of either 12K274A or 12K279A resulted in the formation of functional receptors, but the GABA concentration–response curves were displaced to either the right (K274A) or the left (K279A) when compared with the curve for the wild-type 12 receptor at pH 7.4 (Fig. 4A). These displacements were reflected in GABA EC50 values (12K274A, 8.6 ± 3.2 μM; 12K279A, 0.7 ± 0.2 μM; n= 12) and Hill slopes (12K274A, 0.7 ± 0.18; 12K279A, 0.5 ± 0.06; n= 12), compared to their wild-type equivalents (12, EC50, 2.9 ± 0.3 μM; Hill slope, 1.0 ± 0.08; P < 0.05).
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A, normalized GABA concentration–response curves for 12K274A () and 12K279A () 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 12 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 12, 12K274A and 12K279A 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 12K274A and 12K279A receptors. D, 1 mM GABA- and 100 μM PTX-activated currents for the 12K279A receptor at pH 7.4. All calibration bars represent: 200 pA, 2 s.
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Using the appropriate GABA EC20 (1 μM for 12 and 12K274A and 0.1 μM for 12K279A) at pH 8.4, the potentiation of GABA-activated current for 12K274A receptors was increased (79 ± 20%; Fig. 4B and C; P < 0.05) compared to that at pH 7.4. In contrast, exposing the 12K279A receptor to pH 8.4 abolished the potentiation, revealing a significant inhibition of the GABA response to 78 ± 5% compared to that at pH 7.4 (Fig. 4B and C). Another notable feature of the 12K279A receptor was that the deactivation kinetics of the GABA-activated currents were quite slow, a feature not apparent for the 12K274A mutant (Fig. 4C). Given the position of the lysines in the TM2–TM3 linker, we investigated whether the receptor was capable of spontaneous activation. Both 12K274A and 12K279A receptors were exposed to 100 μM picrotoxin (PTX) in the absence of GABA, which would inhibit any spontaneous channel openings (Sigel et al. 1989; Wooltorton et al. 1997). The K274A mutation was unaffected by PTX (n= 5), in contrast to the outward current observed for the 12K279A receptor (Fig. 4D), which was in accord with a level of spontaneous gating estimated at 22 ± 1.4% (n= 7: see Methods).
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In a similar manner to the 12K279A GABAA receptor, the GABA concentration–response curve for 12K279A2 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 122 receptor (EC50 3.4 ± 0.3 μM; Hill slope 1.3 ± 0.2; n= 10). As for the 12K279A receptor, the EC20 GABA-activated current for the 12K279A2 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).
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A, normalized GABA concentration–response curves for the 12K279A2, 12K279Q2, 12K279F2 and 12K279R2 GABAA receptors at pH 7.4 (n= 5–12 cells). The curve for the wild-type 122 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 122, 12K279A2, 12K279Q2, 12K279F2 and 12K279R2 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 12K279A2 GABAA receptor. Calibration bars represent: 200pA, 2 s. D, 1 mM GABA- and 100 μM PTX-activated currents at pH 7.4 for the 12K279A2 GABAA receptor. Calibration bar represents: 500pA, 2 s.
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To ensure that the mutation of lysine to alanine, with its smaller side chain, was not distorting receptor structure and thus indirectly affecting the response to alkaline pH, we mutated K279 to uncharged glutamine (Q), non-polar phenylalanine (F) and basic arginine (R), with their comparable side chain volumes. All three mutant receptors, 12K279Q2, 12K279F2 and 12K279R2, were functional with reduced EC50 values (1.08 ± 0.04 μM, 0.42 ± 0.06 μM and 0.88 ± 0.21 μM, respectively; n= 5–16) and Hill slopes (0.77 ± 0.02, 0.85 ± 0.1 and 0.49 ± 0.21, respectively; Fig. 5A) compared to 122 (EC50 3.4 ± 0.3 μM; Hill slope 1.3 ± 0.2). Potentiation of the EC20 GABA responses at pH 8.4 was abolished by all three lysine mutations and only for the 12K279R2 receptor was the GABA response inhibited in a similar manner to that observed with the K279A mutant (Fig. 5B). In addition, as for the 12K279A2 receptor, all the other mutants were spontaneously active with levels of 14 ± 2% (K279Q), 13 ± 4% (K279F) and 34 ± 4% (K279R). Taken overall, these findings suggest that the subunit K279 was critically important for the potentiation of the current activated by low GABA concentrations at both and subunit-containing receptors in alkaline pH, as well as a potentially important residue in ion channel gating.
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Modulation of 122 GABAA receptors by acidic pH at low GABA concentrations
Previously, 122 GABAA receptors were considered to be either insensitive to, or inhibited by, acidic pH (Krishek et al. 1996; Huang & Dillon, 1999), contrasting with 12 and 13 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 12 receptor clearly changed at low GABA concentrations (Fig. 2A), it was conceivable that the 122 receptor might behave similarly. Indeed, for 122 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 12 receptor; moreover, the 2H267A mutation was also just as effective at abolishing the potentiation (Fig. 6B). However, in contrast to the 12 receptor, the enhanced GABA response at pH 6.4 for the 122 receptor was only observed at low GABA concentrations (1 μM; Fig. 6A). These data demonstrate the importance of H267 in the modulation of GABAA receptors by protons, but this is only operative over a defined GABA concentration range.
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A, bar graph representing the pH titration profile of the 122 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 12, 12H267A, 122 and 12H267A2 GABAA receptors at pH 6.4 (n= 5–10). *P < 0.05 compared with the control responses determined at pH 7.4 (= 100%).
Role of lysine 279 in the regulation by acidic pH
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At pH 5.4, the pH sensitivities for 12 and 122 receptors diverged, with 1 μM GABA currents mediated via the latter now clearly inhibited (Figs 6A and 7A). The 2H267A mutation increased the inhibition of the GABA-activated current at pH 5.4 for the 12H267A2 receptor to a level similar to that observed with the 12H267A. receptor (Fig. 7A). This complex pH profile of the 122 receptor may result from the replacement of a 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 2 subunit restoring (as for 12) three histidines around the entry to the ion channel, presuming a receptor stoichiometry of 2: 2: 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 2E270 With these mutations, it was conceivable that the 122I282H,K285E receptor might now support a potentiation of the GABA-activated current at pH 5.4. The GABA concentration–response curve for the 122I282H,K285E receptor was not different to the wild-type receptor (GABA EC50: 122, 3.4 ± 0.3 μM; 122I282H,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 122 receptor (Fig. 7A and C). These data suggested that the provision of three histidine residues around the ion channel in the 122 receptor is insufficient for proton-induced potentiation of the GABA-activated current at pH 5.4.
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A, bar graph for the modulation of EC20 GABA-activated currents at pH 5.4 for wild-type 12 and 122 (open bars) and mutant GABAA receptors, incorporating: 2H267A (black bars), 2I282H,K285E (HE), 2I282H,K285E,T294K (HEK), 2T294K (K) and 2K279A2T294K (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 122I282H,K285E, 122I282H,K285E,T294K, 122T294K and 12K279A2T294K GABAA receptors (n= 4–10). The dashed line represents the curve for the wild-type 122 receptor at pH 7.4 (from Fig. 1B). C, GABA-activated currents induced by EC20 GABA applied at the indicated external pH for the 122 and mutant GABAA receptors. See A, for key. Calibration bar represents: 200pA, 1 s.
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The second possibility, regarding disruption to the transduction pathway by the 2 subunit, was addressed by identifying residues that may mediate the potentiation of the GABA current at acid pH. Lysine 279 in the 2 subunit was selected because of its importance for alkaline pH modulation. Indeed, its mutation in 12K279A receptors, resulted in much smaller potentiations of 1 or 10 μM GABA-activated currents at pH 5.4 compared to the wild-type receptor (12, potentiation of 94 ± 17% (1 μM GABA) and 91 ± 10% (10 μM), n= 25; 12K279A, 15 ± 7% (1 μM) and 41 ± 11% (10 μM), n= 7). These data are in accord with K279 playing a critical role in the potentiation of the GABA-activated current for the 12 receptor at pH 5.4 as well as at pH 8.4. As the K279 is normally only present in the subunit, we introduced a lysine into the 2 subunit at the homologous position (T294K), together with the previously introduced residues, I282H and K285E, as found in the 12 receptor. The GABA concentration–response relationship for the 122I282H,K285E,T294K receptor at pH 7.4 was marginally displaced (2-fold) with a reduced EC50 (1.7 ± 0.2 μM; n= 10) compared with the wild-type receptor (3.4 ± 0.3 μM; P < 0.05; Fig. 7B). Of importance, the 1 μM (EC20) GABA-activated current at pH 5.4 was now potentiated, contrasting with the inhibition normally observed with the wild-type 122 receptor (Fig. 7A and C).
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Since the double mutant, 122I282H,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 122T294K 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 12 receptor (103 ± 15%; Fig. 7A and C).
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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 12K279A2T294K was examined. As for the 12K279A receptor, GABA potency was increased for the 12K279A2T294K 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 122I282H,K285E receptors (Fig. 7A and C). These data suggest that three copies of the lysine, each one at position 279 in the subunits or the homologous position in the subunit, are required for modulation of the GABAA receptor at pH 5.4, and that inclusion of a lysine only in the subunit is in sufficient.
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Discussion
This study reveals that the regulation of GABAA 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 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 subunits but also in the fifth subunit of the pentamer, be it a 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 GABAA receptors as well as being an important residue in GABAA receptor activation.
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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 GABAA 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 GABAA receptors (< 7 DIV) (Krishek & Smart, 2001) and recombinant 322 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 12 and 122 GABAA receptors. This shift was more clearly resolved by pH titration using low GABA concentrations. Under these conditions, both 12 and the 122 GABAA receptors were affected by pH 8.4 in a similar manner possibly via the same specific molecular mechanism.
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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 12 wild-type receptor, several residues were identified as prospective candidates. However, in the 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 GABAA receptor at pH 8.4. However, the modulation of GABAA receptors in alkaline pH has been reported to involve the GABA binding site with Y205 and F64 in the 2 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 GABAA 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 GABAA receptors, suggested that all other accessible histidines and tyrosines were most likely not involved in pH regulation, leaving only lysine residues as possible candidates.
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The 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 GABAA receptor subunits and may play an important role in GABAA receptor gating (Sigel et al. 1999; Kash et al. 2003; Kash et al. 2004). Notably, the GABA concentration–response curve for 12K274A was laterally displaced to higher GABA concentrations, as reported for the 12K274A2 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 subunits and its substitution in either 12K279A or 12K279A2 receptors ablated the alkaline pH potentiation of the GABA-activated current, revealing inhibition.
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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 pKa 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 pKa 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 pKa shift occurred in the GABAA 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.
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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 (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 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 12K279A and 12K279A2 receptors are spontaneously active. Although alkaline pH modulation of GABAA receptors requires a lysine residue at 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 and subunits (as well as H267 in the 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.
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A, molecular model showing the 2 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 2 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 subunit are also shown.
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Protonation and the 122 receptor
The potentiation of the GABA-activated current, at pH 6.4, for low GABA concentrations on 122 receptors, was similar to that observed with the 12 receptor but at pH 5.4, the potentiation was absent. The introduction of subunit residues, H267 and E270, into homologous positions in the 2 subunit for 122 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 2T294K. Overall, three lysines were required for potentiation of the GABA-activated current at pH 5.4 on 122 receptors (one in each subunit and one in the mutated 2 subunit). Interestingly, GABA currents of GABAA 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 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 GABAA receptor, but is a vital component in acidic and alkaline pH-induced modulation.
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Physiological relevance
The importance of the pH regulation of GABAA 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 GABAA 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 GABAA receptors (Kraig et al. 1987; Hoffman et al. 1996; Hoffman et al. 1999; Anderson & Meyer, 2002).
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