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Journal of Physiology (2001), 537.1, pp. 101-113
© Copyright 2001 The Physiological Society
GABAA receptors |
ABSTRACT |
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subunit cDNA into a human embryonic kidney (HEK) cell line stably expressing 132 receptors (WSS-1 cells) to establish whether the subunit competes with the 2 subunit for assembly into receptors. GABA-evoked currents were recorded using the patch-clamp technique from cells transfected with cDNA encoding green fluorescent protein (GFP) alone or in combination with the subunit cDNA.
subunit did not change the potency of GABA: the GABA EC50 was 34 ± 6 µM in control WSS-1 cells and 37 ± 6 µM in cells expressing the subunit. The introduction of the subunit reduced the peak current amplitude activated by GABA (1 mM) from 1.8 ± 0.2 nA in control cells to 0.9 ± 0.2 nA in cells expressing the subunit (P < 0.05).
subunit caused the appearance of leak currents recorded in the absence of GABA. Outside-out patches excised from subunit-containing WSS-1 cells exhibited spontaneously opening GABAA channels not seen in patches excised from control GFP-expressing WSS-1 cells. Introduction of the subunit did not alter the GABA-evoked single-channel cord conductance.
subunit reduced potentiation by both agents 48-96 h after transfection.
subunit had no effect on the ability of propofol (3-30 µM) relative to GABA (1 mM) to activate GABAA receptors in WSS-1 cells. High concentrations of propofol (
100 µM) produced a more marked desensitization of GABAA receptor activity in WSS-1 cells transfected with cDNA for the subunit than in control cells.
subunit (IC50 = 179 ± 11 µM).
subunit reversed in sign at the Cl- equilibrium potential and exhibited outward rectification.
subunit changes the functional properties of GABAA receptors in WSS-1 cells. The resulting receptors have a unique combination of properties indicative of the co-assembly of , , and subunits.
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INTRODUCTION |
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GABAA receptors are heterogeneous by virtue of the diversity of genes that encode their subunits, 1-6, 1-3, 1-3, 
, , 
and 
(Bonnert et al. 1999; Mehta & Ticku, 1999). With five subunits making up each receptor-Cl- channel complex (Nayeem et al. 1994) the theoretical number of unique combinations is large. The actual number is limited by the failure of many subunits to form functional receptors. An additional limitation on receptor diversity in the brain is imposed by regional segregation of subunit expression (McKernan & Whiting, 1996).
Recombinant and subunits form functional receptors when expressed in cell lines, but most mature neurons predominantly express receptors. The 2 subunit confers increased single channel conductance, benzodiazepine sensitivity and reduced sensitivity to blockade by Zn2+ (Pritchett et al. 1989; Draguhn et al. 1990; Smart et al. 1991; Angelotti & Macdonald, 1993). In some neurons subunits other than 2 (i.e. or ) may combine with and subunits producing receptors with unique properties (Saxena & Macdonald, 1994; Davies et al. 1997a). The 2, and subunits have overlapping distributions and in some cases all three transcripts are expressed in the same cell (Brooks-Kayal et al. 1998, 1999). This could result in the existence of multiple receptor subtypes in a single cell as in the case of and receptors in cerebellar granule cells (Nusser et al. 1998). Alternatively, 2, and/or subunits may co-assemble within the same receptor complex. If so, it is important to know if any of the co-assembled subunits has a dominant effect on receptor function.
Both kindling- and pilocarpine-induced seizures change the properties of GABAA receptors expressed by rodent dentate granule cells (Buhl et al. 1996; Brooks-Kayal et al. 1998). Following seizures GABAA receptors exhibit an increased sensitivity to being blocked by Zn2+ and a reduced sensitivity to the potentiating effects of anxiolytic benzodiazepines. These functional changes are associated with increases in the levels of and subunit mRNAs without a significant change in the level of the 2 transcript (Brooks-Kayal et al. 1998). The loss of -subunit properties could be explained in three ways. (1) Receptors appear that lack , and subunits. (2) The 2 subunit and or subunits compete for occupation of the receptor. After seizures this competition is won by and/or subunits. (3) Receptors form that contain 2 subunits and either or subunits, their properties being dominated by the or subunit.
In this study we mimicked the induction of subunit expression in neurons by transiently transfecting subunit cDNA into WSS-1 cells (HEK cells already stably expressing 132 receptors) (Wong et al. 1992; Davies et al. 2000). We used the patch-clamp technique to examine whether the 2 and subunits compete for control of GABAA receptor function. Receptors containing , and subunits can be distinguished by their distinctive functional properties. The well-described anaesthetic potentiation of and GABAA receptor function is markedly reduced in receptors (Davies et al. 1997a). Furthermore, receptors, but not or receptors, are sensitive to benzodiazepines. The , and receptors can also be distinguished by their differential sensitivities to Zn2+, an inhibitor of GABAA receptor function, and by their current-voltage relationships (Draguhn et al. 1990; Davies et al. 1997a; Whiting et al. 1997). Using these properties we provide evidence for the existence of receptors.
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METHODS |
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Cell culture and transfection
HEK cells (ATCC CRL-1573) were grown in medium comprising Dulbecco's modified Eagle's medium supplemented with 10 % calf serum, 100 i.u. ml-1 penicillin, and 100 µg ml-1 streptomycin. WSS-1 cells (ATCC, CRL-2029) were grown in HEK medium supplemented with 400 µg ml-1 geneticin (G-418), used to positively select cells that express GABAA receptors. Resistance to the antibiotic is conferred by the vector containing cDNA encoding the rat 1 subunit (Wong et al. 1992). Cells were maintained for 1 week in a humid environment of 5 % CO2, 95 % air at 37 oC before subculturing. Once the cells approached confluence they were suspended and seeded into 35 mm diameter dishes for transfection as described previously (Davies et al. 1997b). Cells were transfected in HEK medium using the calcium phosphate precipitation method. WSS-1 cells were transiently transfected with either green fluorescent protein (GFP) cDNA or the human subunit and GFP cDNAs (both in pCDM8). HEK cells were transiently transfected with human 1, 3, 2 and (all in pCDM8) cDNAs to produce recombinant 13, 132, 13, GABAA receptors. After transfection cells were incubated (5 % CO2, 95 % air at 37 oC) for 24 h, washed and incubated for a further 24-144 h before experimentation.
Electrophysiology
The whole-cell configuration of the patch-clamp technique was used to record GABA-activated currents from voltage-clamped HEK or WSS-1 cells. GABA (100 µM) was applied by pressure (70 kPa, 0.04 Hz) ejection from modified patch pipettes; other compounds were applied by perfusion into the recording chamber. In experiments investigating the modulation of GABA-evoked currents by anaesthetics and flunitrazepam the duration of GABA application was sufficient to activate ~10 % of the maximum GABA (100 µM)-activated current. GABA or propofol were applied by prolonged (1 s) pressure ejection from low resistance micropipettes in order to determine their concentration dependence as GABAA receptor agonists as described previously (Adodra & Hales, 1995). The recording chamber was continuously perfused (5 ml min-1) with an extracellular solution comprising (mM): NaCl, 140; KCl, 4.7; MgCl2, 1.2; CaCl2, 2.5; glucose, 11; Hepes-NaOH, 10 (pH 7.4). The electrode solution contained (mM): KCl, 140; MgCl2, 2.0; EGTA, 11; ATP (Mg2+ salt) 0.1; Hepes-KOH, 10 (pH 7.4). Junction potentials were nulled with an open electrode in the recording chamber prior to each experiment. The liquid junction potential was trivial (1.7 mV) and its inappropriate compensation was ignored. Cells were voltage clamped at -60 mV except in those experiments in which the relationship between holding potential and current amplitude was examined. Experiments were performed at room temperature (20-24 oC).
Acquisition and analysis of data
GABA-evoked currents were monitored by an Axopatch-200A amplifier, then low-pass filtered with a cut-off frequency of 2 kHz, recorded on chart paper (Gould, Brush 2200) and simultaneously digitized, using a DigiData 1200 interface (Axon Instruments, Burlingame, CA, USA), for acquisition onto the hard drive of a personal computer. Currents were averaged, superimposed and measured using pCLAMP software (Axon Instruments). Graphs of concentration-response relationships were fitted using the logistic function as described previously (Adodra & Hales, 1995). To test the possibility that 132 and 13 exist in WSS-1 cells expressing subunits we plotted curves generated by adding two logistic equations using the parameters determined when these receptor subtypes were expressed alone. All data are expressed as the arithmetic mean ± S.E.M.
Drugs and reagents used
-Aminobutyric acid (GABA) was from Sigma (St Louis, MO, USA). 2,6-Diisopropylphenol (propofol) was from Aldrich (Milwaukee, WI, USA). Stock solutions of flunitrazepam and propofol, in ethanol, were diluted to achieve a final ethanol concentration < 0.1 %. This concentration of ethanol had no effect on GABA (100 µM)-activated currents. Tissue culture reagents were purchased from Gibco-BRL (Gaithersburg, MD, USA) and all other reagents were from Sigma.
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RESULTS |
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Introduction of the subunit alters GABAA receptor function
We examined whether the human subunit alters GABAA receptor function upon transient introduction of its cDNA into WSS-1 cells (HEK cells that stably express recombinant 1 and 2 subunits and an endogenous 3 subunit) (Wong et al. 1992; Davies et al. 2000). Cells were transfected with cDNAs encoding the subunit and green fluorescent protein (GFP), then located using fluorescence microscopy. Control WSS-1 cells were transfected with GFP cDNA alone. We focused on pharmacological properties conferred specifically by or subunits as a means to verify the incorporation of subunits into functional GABAA receptors. These properties are benzodiazepine sensitivity and resistance to anaesthetic modulation of GABA-evoked currents, respectively (Pritchett et al. 1989; Davies et al. 1997a). GABA-activated currents recorded from control WSS-1 cells 24-144 h post-transfection showed no significant change in sensitivity to the anaesthetic agent propofol (3 µM) after transfection with GFP cDNA (Fig. 1A and B). The inclusion of subunit cDNA, in contrast, caused a decline in the modulatory effect of propofol. The potentiation of GABA-activated currents evoked by propofol 48 h after transfection was greatly reduced (Fig. 1A), indicating that the subunit was incorporated into functional GABAA receptors of WSS-1 cells. Consistent with the eventual dilution and degradation of subunit cDNA, the modulation of GABA-activated currents by propofol showed signs of recovery 120 h after transfection and was indistinguishable from control after 144 h. While the potentiation of GABA-evoked currents by propofol (3 µM) was markedly reduced after subunit expression, there was a significant increase in current elicited by the anaesthetic in the absence of GABA (Fig. 1A, P < 0.005, n = 11). From Fig. 3 it is clear that this effect cannot be entirely accounted for by direct GABAA receptor activation by propofol (3 µM) and may represent the ability of this agent to potentiate the spontaneous activity of subunit-containing channels. The data for propofol (3 µM) modulation of GABA-evoked currents recorded from WSS-1 cells transfected with GFP cDNA alone or in combination with the subunit cDNA 48-96 h after transfection are shown in Table 1. The levels of propofol-evoked potentiation of GABA-evoked currents mediated by recombinant 13, 13 and 132 receptors transiently expressed in HEK cells are included for comparison (Table 1).
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Figure 1. Transient expression of the A, left panel: sub-maximal GABA-evoked currents (equivalent in amplitude to currents activated by GABA EC10) recorded from GFP-expressing WSS-1 cells were potentiated by propofol (3 µM). Dotted line represents zero current. A, right panel: WSS-1 cells expressing | ||
The benzodiazepine receptor agonist flunitrazepam (1 µM) potentiated GABA-activated currents recorded from control cells 24-144 h after transfection (Fig. 1C). By contrast, cells transfected with subunit cDNA exhibited a significant decline in flunitrazepam sensitivity 48 h after transfection. By 120 h after transfection the flunitrazepam-evoked potentiation was beginning to recover and, as for propofol-potentiation, was indistinguishable from control at 144 h. Together, these data demonstrate that expression of the subunit can displace subunit-dependent pharmacology in cells stably expressing receptors.
The mean flunitrazepam-elicited modulation of GABA-evoked currents recorded from WSS-1 cells expressing the subunit 48-96 h after transfection (131 ± 11 % of control, n = 11) was significantly reduced (P < 0.00005) compared to that seen in control WSS-1 cells (228 ± 13 % of control, n = 14, Fig. 1D). Recombinant 13 receptors lack flunitrazepam sensitivity (101 ± 8 % of control, n = 4). The low-level residual flunitrazepam potentiation seen in WSS-1 cells expressing the subunit could be caused by the formation of mixed 13/132 receptors, alternatively 132 receptors with a modest sensitivity to flunitrazepam may form. If mixed 13/132 receptors do exist in WSS-1 cells expressing subunits then on average 24 % of receptors would have to be 132 to enable flunitrazepam to potentiate GABA-activated currents to 131 % of control.
The concentration dependence of GABAA receptor activation was examined by pressure applying GABA (1 µM to 1 mM) for 1 s. Currents were recorded from fluorescent cells between 48 and 96 h after transfection. The introduction of the subunit had no effect on the potency of GABA (Fig. 2C). GABA activated currents recorded from control WSS-1 cells and cells transfected with the subunit cDNA with EC50 values of 34 ± 6 µM and 36 ± 6 µM, respectively (Table 1). The introduction of the subunit significantly reduced the peak amplitude of the current activated by GABA (1 mM) from 1.8 ± 0.2 nA (n = 11) in control cells to 0.9 ± 0.2 nA (n = 13) in cells expressing the subunit (P < 0.05). WSS-1 cells expressing subunits may contain a mixture of 13 and 132 receptors. Alternatively they may contain 132 receptors. For comparison HEK cells lacking GABAA receptors were transiently transfected with cDNAs encoding 1, 3 and subunits. These cells exhibited a GABA EC50 that was markedly different from GABA EC50 values determined for control WSS-1 cells and WSS-1 cells expressing the subunit (Table 1). We used parameters derived from fits of GABA concentration-response relationships in control WSS-1 cells and HEK cells expressing 13 receptors to determine whether a mixed 13/132 receptor population could cause the relationship seen in WSS-1 cells expressing the subunit. As discussed above, on the basis of the low level of flunitrazepam sensitivity of WSS-1 cells after transfection with subunit cDNA we generated a theoretical GABA concentration-response curve assuming 24 % of the receptors were 132 and 76 % were 13. The fact that the theoretical curve is markedly different from the experimental data suggests that WSS-1 cells transfected with subunit cDNA do not express mixed 13/132 receptors. Regardless of the ratio of 13 to 132 receptors used it was not possible to accurately represent the observed GABA concentration-response relationship.
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Figure 2. Activation of GABAA receptors expressed by WSS-1 cells transfected with GFP cDNA alone or in combination with A, currents activated by increasing concentrations of GABA, applied for 1 s, recorded from WSS-1 cells 48 to 96 h after transfection with GFP cDNA alone. Continuous line represents the 1 s application of GABA. Dotted line represents zero current. B, GABA-evoked currents recorded from WSS-1 cells expressing the | ||
Leak currents routinely appeared upon achieving the whole-cell configuration in WSS-1 cells 48-96 h after transfection with subunit cDNA (Fig. 1-3 and Fig. 6). The mean amplitude of the leak currents was -198 ± 46 pA (n = 13). Such spontaneous currents were not seen in control WSS-1 cells at similar times after transfection with GFP cDNA alone (Fig. 2C). The mean amplitude of leak currents recorded from control cells was -5 ± 13 pA (n = 8). This observation is consistent with the subunit causing the appearance of spontaneously active GABAA receptors (Neelands et al. 1999). In support of this hypothesis, picrotoxin (50 µM, data not shown) and Zn2+ (100 µM, Fig. 6A) inhibited the spontaneous current recorded from subunit-expressing WSS-1 cells.
Propofol and other general anaesthetic modulators of GABA responses are able to directly activate GABAA receptors in the absence of GABA (Hales & Lambert, 1991) regardless of whether the receptors contain either or subunits (Jones et al. 1995; Davies et al. 1997a,b). The introduction of the subunit had no discernible effect on the ability of propofol (3-30 µM) relative to GABA (1 mM) to activate GABAA receptors in WSS-1 cells (Fig. 3). High concentrations of propofol (100 µM and 300 µM) produced a more marked reduction of GABAA receptor activity in subunit-expressing WSS-1 cells than in control cells. This may be due to an increase in receptor desensitization (Whiting et al. 1997) and/or increased GABAA receptor blockade by propofol (Adodra & Hales, 1995).
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Figure 3. Propofol directly activates GABAA receptors in WSS-1 cells transfected with GFP cDNA alone or in combination with the A, propofol causes a concentration-dependent activation of GABAA receptors recorded from GFP-expressing WSS-1 cells. Surge currents occur on cessation of the 1 s application of propofol at 100 µM. Bar represents a 1 s application of propofol. Dotted line indicates zero current. B, WSS-1 cells transfected with cDNA encoding the | ||
Spontaneous and GABA-activated channels
Spontaneous single channel events were apparent in recordings from outside-out patches excised from WSS-1 cells expressing subunits (n = 3, Fig. 4). These events varied substantially in their durations and had two different cord conductances, 13 ± 0.3 and 36 ± 3 pS, measured at -80 mV (Fig. 4B). No spontaneous events were observed in recordings from outside-out patches excised from control cells (n = 5, Fig. 4A). GABA (1 µM) activated channels in patches excised from WSS-1 cells expressing GFP with or without the subunit (Fig. 5). At least two conductance levels were apparent in these recordings. The most frequent conductance activated by GABA in all patches tested was the larger of the two conductance levels, 28 ± 1 pS (n = 5) recorded from control patches and 30 ± 4 pS (n = 3) recorded from patches excised from WSS-1 cells expressing the subunit (Fig. 5). In all cases the application of GABA (1 µM) revealed the presence of multiple channels in outside-out patches preventing us from undertaking kinetic analyses of these events. In future studies ultra-rapid GABA application will be employed to examine differences in gating kinetics between channels of control cells and cells expressing the subunit.
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Figure 4. Spontaneous channel events recorded from outside-out patches excised from cells expressing the A, recording from an outside-out patch, voltage clamped at -80 mV, excised from a control WSS-1 cell transfected with GFP cDNA shows very little channel activity in the absence of GABA. B, spontaneous channel opening of | ||
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Figure 5. GABA-activated channels recorded from outside-out patches excised from control cells and cells expressing the A, GABA (1 µM) was bath-applied to an outside-out patch excised from a GFP-positive WSS-1 cell. At least two GABA-activated channels are present in this patch, recorded at -80 mV. B, GABA-activated single channels recorded from an outside-out patch excised from a WSS-1 cell expressing the | ||
receptors have unique functional properties
The inhibition of GABAA receptor function by Zn2+ is strongly influenced by incorporation of or subunits (Draguhn et al. 1990; Whiting et al. 1997; Fisher & Macdonald, 1998; Neelands et al. 1999). Zn2+ caused a concentration-dependent inhibition of GABA-evoked currents recorded from HEK cells expressing 13 receptors (Table 1). Zn2+ was less potent as an inhibitor of 13 receptors and had the lowest potency as an inhibitor of 132 receptors (Table 1).
We examined whether the modulation by Zn2+ of GABA-activated currents recorded from WSS-1 cells 48-96 h after transfection was influenced by the incorporation of the subunit (Fig. 6A and B). Zn2+ (1 µM to 3 mM) caused a concentration-dependent inhibition of GABA (100 µM)-activated currents recorded from GFP-expressing WSS-1 cells, with an IC50 of 165 ± 34 µM (Fig. 6B, Table 1). Surprisingly, GABA-activated currents recorded from WSS-1 cells expressing subunits had similar sensitivity to Zn2+ (IC50 of 179 ± 11 µM). This observation was surprising in view of the fact that Zn2+ is more effective as an inhibitor of receptors compared to receptors (Table 1). It demonstrates that receptors can form with -like anaesthetic pharmacology and a much-reduced Zn2+ sensitivity. The small flunitrazepam modulation of GABAA receptors in WSS-1 cells after transfection with subunit cDNA (Fig. 2B) indicates that if 132 receptors are present they would on average make up only 24 % of the total receptor population. We generated a theoretical Zn2+ concentration-response relationship assuming 24 % 132 receptors and 76 % 13 receptors, using the parameters listed in Table 1. The theoretical curve does not fit the data points obtained from WSS-1 cells expressing subunits. In fact, regardless of the ratio of 13 to 132 receptors used, the theoretical curves always failed to adequately describe the experimental observations. The low Zn2+ sensitivity of GABA-evoked currents argues against the presence of both and receptors in WSS-1 cells transfected with subunit cDNA. Taken together the data suggest the occurrence of receptors containing , , and subunits.
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Figure 6. The potency of inhibition by Zn2+ and the GABA-evoked current-voltage relationship are similar in control cells and cells expressing the A, examples of superimposed GABA-evoked currents recorded before and after the application of Zn2+ (100 µM). The dotted line represents zero current. In the recording from cells expressing the | ||
A lack of receptors in WSS-1 cells transfected with subunit cDNA is also suggested by the relationship between current amplitude and holding potential (Fig. 6C). Currents mediated by receptors exhibit either a linear relationship to voltage (Davies et al. 1997a) or inward rectification (Neelands et al. 1999), properties that distinguish them from the outward rectification seen in recordings from cells expressing either or receptors. This can be appreciated by examining the ratio of GABA-evoked current amplitudes recorded at +60 and -60 mV (Table 1). Recombinant or receptors exhibit +60/-60 mV current ratios of > 1, while currents recorded from cells expressing receptors have +60/-60 mV current ratios < 1 (Table 1). GABA-activated currents recorded from control WSS-1 cells and cells expressing the subunit were indistinguishable, both exhibiting outward rectification. The absence of a linear current-voltage relationship in subunit-expressing cells demonstrates a lack of receptors and, taken together with the Zn2+ data, suggests that receptors are formed with a distinctive combination of functional properties.
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DISCUSSION |
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The subunit has greater amino acid sequence similarity to the subunits than to any of the other classes of GABAA receptor polypeptides. Furthermore, the and subunits both require the presence of and subunits to incorporate within functional receptors (Davies et al. 1997a). Therefore we initially hypothesized that and subunits compete for a common site in the GABAA receptor complex. In this study we have demonstrated that expression of the subunit does indeed modify the functional properties of 132 receptors stably expressed in WSS-1 cells. However, rather than the subunit simply displacing the 2 subunit from the receptor, the distinctive functional properties of the resulting receptors suggest that an receptor combination is formed.
WSS-1 cells stably express GABAA receptors that are modulated by benzodiazepines, loreclezole and intravenous general anaesthetics (Davies et al. 2000). The transient introduction of cDNA encoding GFP had no effect on the modulation of GABA-activated currents by the intravenous anaesthetic propofol, or the benzodiazepine flunitrazepam. By contrast, GABA-activated currents recorded from WSS-1 cells transiently transfected with GFP and subunit cDNAs became resistant to benzodiazepine modulation 48-96 h after transfection, and had a greatly diminished modulation by propofol. These properties suggest that neither nor receptors are present at detectable levels. Instead, the resistance to benzodiazepine and anaesthetic potentiation suggests that either or receptors are expressed. We were able to distinguish between these two possibilities by examining the GABA concentration-response relationship and by using Zn2+, a potent non-competitive inhibitor of GABAA receptors that lack subunits (Draguhn et al. 1990; Krishek et al. 1998). The GABA EC50 was not altered upon incorporation of the subunit into WSS-1 cell GABAA receptors despite the fact that 13 receptors are more potently activated by GABA than are 132 receptors in WSS-1 cells (Table 1). This observation demonstrates that there are a lack of 13 receptors in WSS-1 cells expressing subunits and supports the existence of 132 receptors with similar GABA sensitivity to that of 132 receptors.
The potencies of Zn2+ as an inhibitor of GABA-activated currents recorded from control WSS-1 cells and cells transfected with subunit cDNA were similar despite the fact that receptors are more potently blocked by Zn2+ than are receptors (Table 1). This indicates the formation of receptors with some -like properties and a high resistance to Zn2+. It is not yet known whether the inhibition of subunit-containing receptors by Zn2+ is competitive as in the case of receptors or non-competitive as in the case of and receptors (Draguhn et al. 1990; Krishek et al. 1998).
Studying the voltage dependence of GABA-activated currents recorded from WSS-1 cells expressing subunits provided further evidence against the existence of mixed / receptor populations and, therefore, in favour of the formation of receptors. Currents mediated by receptors exhibit either a linear relationship to voltage (Davies et al. 1997a) or inward rectification (Neelands et al. 1999), properties that clearly distinguish them from both and receptors (Table 1). Currents recorded from control WSS-1 cells and cells expressing the subunit displayed similar relationships to voltage, exhibiting outward rectification indistinguishable from that seen in recordings from cells expressing recombinant receptors.
It is not known whether GABAA receptors containing both and subunits exist in vivo. Immunoprecipitation studies, in which subunit-specific antibodies were used to identify combined subunits, support the coexistence of more than one type of subunit in some receptors (McKernan & Whiting, 1996). In view of the amino acid sequence similarities between and subunits it would not be surprising if similar studies in the future identify receptors in specific brain regions into which both subunits assemble. Immunobiochemical studies provide evidence for (Mertens et al. 1993) and against (Quirk et al. 1995; Araujo et al. 1998) the existence of neuronal receptors containing both and subunits. The distinctive properties of recombinant receptors expressed by cells transfected with , , 2 and subunits suggest that receptors containing all four subunits can form (Saxena & Macdonald, 1994; Krishek et al. 1996). Such receptors may be confined to a small subset of neurons making their functional or biochemical detection problematic. The occurrence of receptors containing and or subunits may become more widespread following seizures as a result of increased levels of and subunit expression (Brooks-Kayal et al. 1998). GABAA receptors in dentate granule cells become more Zn2+ sensitive and less benzodiazepine sensitive following pilocarpine-induced seizures. This cannot be entirely explained on the basis of the formation of receptors alone, since these have a similar Zn2+ sensitivity to receptors. However, it is possible that mixed and subunit-containing receptor populations may exist in neurons following seizures.
Functional and biochemical evidence supports the existence of GABAA receptors with stoichiometries of 2:2:1 and 2:1:2 subunits (Backus et al. 1993; Im et al. 1995; Chang et al. 1996; Farrar et al. 1999). Studies involving site-directed mutagenesis of these subunits support the hypothesis that GABA binds to residues within adjacent and subunits while benzodiazepines bind to amino acids on adjacent and subunits (Sigel & Buhr, 1997; Horenstein et al. 2001). It is not clear how the additional inclusion of the subunit would affect subunit stoichiometry. The modest benzodiazepine sensitivity of 132 receptors may indicate the existence of an interface between and subunits. The subunit differs in amino acid sequence from both and subunits within the putative GABA binding domains. The lack of effect of the subunit on the GABA EC50 may indicate that there is the same number of / interfaces in 132 and 132 receptors. These data could be explained in the light of the current dogma regarding GABA and benzodiazepine binding by WSS-1 receptors having the stoichiometry of 2:1:2, changing to 2:1:1:1 in cells expressing subunits. Alternatively if the WSS-1 cell receptor has a stoichiometry of 2:2:1 then the subunit must displace and/or subunits to create receptors of unknown stoichiometry. It is not clear how this could occur without altering the GABA EC50. We are currently using cDNA constructs expressing concatenated subunits to further examine this issue.
Although receptors in cells expressing , , and subunits exhibit greatly reduced potentiation of GABA-activated currents by propofol, the anaesthetic was able to directly activate the receptor in the absence of GABA. Anaesthetics are also able to directly activate receptors (Davies et al. 1997a). There was no significant difference in the ability of propofol (3-30 µM) relative to GABA to directly activate GABAA receptors in subunit- containing and control WSS-1 cells. In both cases currents activated by high concentrations of propofol (100 and 300 µM) were associated with rebound or 'surge' currents upon cessation of propofol application. The surge current appears to be caused by propofol unbinding from a low affinity inhibitory site on the subunit (Adodra & Hales, 1995; Davies et al. 1997b). Compared to control WSS-1 cells, subunit-expressing cells had smaller propofol-evoked current amplitudes when higher concentrations of the anaesthetic were applied (P < 0.05 for 100 µM propofol). The reduced peak current amplitudes could be caused by increased receptor blockade or desensitization induced by propofol. Increased GABAA receptor desensitization by GABA has previously been reported for compared to receptors (Whiting et al. 1997).
Both and receptors exhibit spontaneous channel openings in the absence of GABA. These events were evident from the picrotoxin- and Zn2+-sensitive leak currents that routinely developed upon achieving the whole-cell configuration in recordings from WSS-1 cells expressing subunits. Spontaneous single channel activity was also directly observed in recordings from outside-out patches excised from WSS-1 cells expressing the subunit. Neither leak currents nor spontaneous channels were seen in whole-cell or outside-out patch recordings from control WSS-1 cells. GABA activated single channels in patches excised from either control WSS-1 cells or cells expressing the subunit. The main state conductance of GABA-activated channels was similar to the conductance of single channels recorded from outside-out patches excised from cells expressing either or receptors, but larger than that observed in recordings of single channels (Angelotti & Macdonald, 1993; Neelands et al. 1999). This similarity between - and 2-containing receptors suggests that the amino acids responsible for controlling channel conductance are homologous in the two subunits.
The prolonged (> 20 s) bath application of a low concentration of propofol (3 µM) to WSS-1 cells containing subunits caused the appearance of a current that was significantly larger than that seen in similar recordings from control WSS-1 cells. By contrast currents activated by brief (1 s) local application of propofol (3 µM) to WSS-1 cells with or without the subunit were not significantly different in amplitude. One explanation for these data is that propofol is able to potentiate spontaneous channel openings. This effect may be delayed by a requirement for the channels to enter the open state. In order to examine the possibility that propofol modulates the kinetics of spontaneous channels it will be necessary to directly compare spontaneous single channel openings in the absence and presence of propofol at a concentration below that required for direct activation.
Interestingly, recent reports suggest that specific single amino acid substitutions in GABAA and/or subunits result in receptors with spontaneous channel activity, together with a decrease in sensitivity of the channel to several GABA receptor modulators including benzodiazepines and anaesthetics (Thompson et al. 1999; Findlay et al. 2000). Findlay and colleagues suggest that their tryptophan mutations at serine 270 in the 2 subunit or 265 in the 1 subunit decrease the free energy of the open state thus increasing the tendency of the channel to open in the absence of agonist. As would be expected from this hypothesis the mutant receptors show an increased sensitivity to activation by GABA. The mutation may mimic the effect of positive allosteric modulators such as benzodiazepines and anaesthetics making the actions of these agents redundant. Spontaneous openings induced by the subunit may decrease anaesthetic and benzodiazepine modulation through a similar mechanism. However, it is unlikely that such a simple scheme accounts for the resistance to modulation induced by the subunit, because there is little increase in the sensitivity of (Davies et al. 1997a) and receptors to activation by agonists.
The peak GABA-evoked current amplitude seen in recordings from subunit-expressing WSS-1 cells was approximately 60 % of that seen in control WSS-1 cell recordings. This can be explained by the fact that spontaneous channel activity accounts for approximately 40 % of the GABA-evoked current amplitude in subunit-expressing cells. Therefore the total number of and receptors in transfected and untransfected cells remains unchanged.
By transiently introducing subunit cDNA into cells stably expressing receptors we have mimicked the induction of subunit expression in dentate granule neurons seen after pilocarpine-induced seizures in rats (Brooks-Kayal et al. 1998). Although the level of subunit mRNA increases relative to other GABAA mRNAs in the pilocarpine model of epilepsy the level of 2 subunit mRNA is unchanged. The incorporation of the subunit into functional receptors may provide a mechanism for changing receptor properties without requiring either a reduction of 2 transcript or the omission of 2 subunits from functional receptors.
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REFERENCES |
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Acknowledgements
We are grateful to Megan Dankovich for her expert technical assistance. Research support was provided by the National Institute of Health grants GM58037 (T.G.H.) and NS34702 (E.F.K.).
Corresponding author
T. G. Hales: Department of Pharmacology, Ross Hall, 2300 Eye Street NW, Washington DC 20037, USA.
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). After 48 h post-transfection, GABA-evoked currents recorded from cells expressing 
) exhibited a significantly reduced modulation by propofol. The modulation of GABA-evoked currents by propofol was indistinguishable from control levels by 144 h post-transfection of WSS-1 cells with