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The Journal of Neuroscience, December 3, 2003, 23(35):11158-11166
 Previous Article | Next Article 
Cellular/Molecular
Individual Properties of the Two Functional Agonist Sites in GABAA Receptors
Sabine W. Baumann,
Roland Baur, and
Erwin Sigel
Department of Pharmacology, University of Bern, CH-3010 Bern, Switzerland
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Abstract
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The members of the pentameric ligand-gated receptor channel family are involved in information transfer in synapses and the neuromuscular junction. They often contain several copies of the same subunit isoform. Here, we present a method to functionally dissect the role of individual subunits that occur in multiple copies in these receptors.
Opening of the inherent chloride channel in the GABAA receptor is achieved through the binding of two agonist molecules; however, it has been difficult to obtain information on the contribution of the two individual binding sites. The sites are both located at  (+)/ (-) subunit interfaces, suggesting similar properties. One pair of subunits is flanked by  and (site 1) and the other by and (site 2), the different environment possibly affecting the binding sites. Here, we used concatenated subunits and two point mutations of amino acid residues, each in and subunits, both located in the agonist binding pocket, to investigate the properties of these two sites. The sites were individually mutated, and consequences of these mutations on GABA and muscimol-induced channel opening and its competitive inhibition by bicuculline were studied. A model predicts that opening also occurs for receptors occupied with a single agonist molecule but is promoted approximately 60-fold in those occupied by two agonists and that site 2 has an approximately threefold higher affinity for GABA than site 1, whereas muscimol and bicuculline show some preference for site 1.
Key words: GABA; GABAA receptor; ion channel; linked subunits; agonist; bicuculline
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Introduction
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GABAA receptors are the major inhibitory neuronal ion channels in the mammalian brain. They belong to the family of ligand-gated ion channels that includes nicotinic acetylcholine, glycine, and serotonin type 3 receptors. Biochemical purification of a bovine GABAA receptor (Sigel et al., 1983 ) was followed by cloning, which led to the identification of 18 different subunit isoforms (Macdonald and Olsen, 1994; Rabow et al., 1995; Davies et al., 1997; Hedblom and Kirkness, 1997; Whiting et al., 1997, 1999; Barnard et al., 1998). Most GABAA receptors are thought to be pentameric assemblies containing , , and or , , and  subunits (McKernan and Whiting, 1996). The five subunits are arranged pseudosymmetrically around a central Cl--selective channel (Macdonald and Olsen, 1994). The most likely stoichiometry is two subunits, two subunits, and one or subunit (Backus et al., 1993; Chang et al., 1996; Tretter et al., 1997; Farrar et al., 1999; Baumann et al., 2001). The major receptor isoform of the GABAA receptor in the brain probably consists of 1, 2, and 2 subunits (Laurie et al., 1992; Benke et al., 1994; Macdonald and Olsen, 1994; Rabow et al., 1995; McKernan and Whiting, 1996). The inferred arrangement of subunits around the channel pore is counterclockwise when viewed from the synaptic cleft (Baumann et al., 2002).
The functional GABA binding site in GABAA receptors is located at intersubunit contacts between and subunits (Sigel et al., 1992; Amin and Weiss, 1993; Smith and Olsen, 1994; Westh-Hansen et al., 1997; Boileau et al., 1999), and homologous amino acid residues of and subunits form the benzodiazepine binding pocket (Wieland et al., 1992; Amin et al., 1997; Buhr and Sigel, 1997; Buhr et al., 1997a, b; Sigel and Buhr, 1997; Teissere and Czajkowski, 2001).
Here, we address the question of whether or not the two agonist sites on the receptor have identical properties. To our knowledge, this question has never been addressed for GABAA receptors. In the homologous nicotinic acetylcholine receptor, there is indirect evidence both for (Edelstein et al., 1997) and against (Neubig and Cohen, 1979) different properties of the agonist at the two sites. Some competitive antagonists (e.g., D-tubocurarine) differ widely in their affinity for the two sites (Martinez et al., 2000).
In the present study, we used two mutations, 1F65L located on the minus side and 2Y205S located on the plus side, to dissect the contribution of each of the two individual functional GABA binding sites. Concatenated subunits of the GABAA receptor were used for a forced assembly. The point mutations were introduced in the 1 and 2 subunits flanked by the subunit and a subunit (site 1), by an subunit and the subunit (site 2), or both. Mutation of the two sites leads to subtly different consequences.
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Materials and Methods
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Construction of tandem and triple subunit cDNAs. The tandem constructs, 2-1 and 2-2, and triple constructs, 2-2-1 and 1-2-1, were made as described previously (Baumann et al., 2001, 2002). Site-directed mutagenesis of 1F65 to L, 2Y62 to L, or 2Y205 to S were done in the tandem constructs 2-1 or 2-2 and 1-2 using the QuickChange mutagenesis kit (Stratagene, Amsterdam, The Netherlands). Subsequently, the triple constructs 2-2-1F65L, 2-2Y62L-1, and 1-2Y205S-1 were made similar as described by Baumann et al. (2001, 2002).
Expression of linked constructs in Xenopus oocytes. Capped cRNAs were synthesized (Ambion, Lugano, Switzerland) from linearized pCMV vectors containing different tandem or triple constructs, respectively, and from the vector pVA2580 (Kuhn and Greeff, 1999) encoding a neuronal voltage-gated sodium channel. A poly-A tail of  400 residues was added to each transcript using yeast poly-A polymerase (United States Biochemicals, D bendorf, Switzerland). The concentration of the cRNA was quantified on a formaldehyde gel using Radiant Red stain (Bio-Rad, Reinach, Switzerland) for visualization of the RNA and known concentrations of RNA ladder (Invitrogen, Basel, Switzerland) as standard on the same gel. cRNA combinations were precipitated in ethanol/isoamylalcohol, 19:1, and stored at -20°C. For injection, the alcohol was removed, and the cRNAs were dissolved in water. Isolation of oocytes from the frogs, culturing of the oocytes, injection of cRNA, and defolliculation were done as described previously (Sigel, 1987). Oocytes were injected with 50 nl of the cRNA solution. For cRNA combinations of the triple constructs with the tandem construct, ratios of 10:10 nM were investigated. The combination of wild-type 1, 2, and 2 subunits was expressed at a ratio of 10:10:50 nM (Boileau et al., 2002). To allow standardization of expressed GABA currents, cRNA coding for the voltage-gated sodium channel was always added to a concentration of 40 nM. The injected oocytes were incubated in modified Barth's solution [10 mM HEPES, pH 7.5, 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.34 mM Ca(NO3)2, 0.41 mM CaCl2, 100 U/ml of penicillin, 100 µg/ml of streptomycin] at 18°C for 2 d before the measurements.
Two-electrode voltage-clamp measurements. All measurements were done in medium containing (in mM): 90 NaCl, 1 MgCl2, 1 KCl, 1 CaCl2, and 5 HEPES, pH 7.4, at a holding potential of -80 mV. For the determination of maximal current amplitudes, 10 mM GABA (Fluka, Buchs, Switzerland) was applied for 20 sec. The perfusion solution (6 ml/min) was applied through a glass capillary with an inner diameter of 1.35 mm, the mouth of which was placed 0.4 mm from the surface of the oocyte. The rate of solution change under our conditions has been estimated 70% within <0.5 sec (Sigel et al., 1990). Voltage-dependent sodium currents were determined by a potential jump from a holding potential of -100 to -15 mV. Functional GABAA receptor expression was determined by application of 10 mM GABA. The elicited current amplitude was normalized to the Na current measured. GABA-evoked currents (at 8-12% of the maximal current amplitude) were inhibited by varying concentrations of bicuculline methiodide (Sigma/RBI, Buchs, Switzerland). Concentration-response curves for GABA were fitted with the equation I(c) = Imax/[1 + (EC50/c)n], where c is the concentration of GABA, EC50 is the concentration of GABA eliciting half-maximal current amplitude, Imax is the maximal current amplitude, I is the current amplitude, and n is the Hill coefficient. Inhibition curves for bicuculline were fitted with the equation I(c) = I(0)/[1 + (IC50/c)n], where I(0) is the control current in the absence of bicuculline standardized to 100%, I(c) is the relative current amplitude, c is the concentration of bicuculline, IC50 is the concentration of bicuculline causing 50% inhibition of the current, and n is the Hill coefficient. Data are given as mean ± SEM (number of experiments, number of batches of oocytes). Relative current stimulation by diazepam (DZ) was determined at a GABA concentration evoking 2-5% of the maximal current amplitude in combination with varying concentrations of DZ (Roche Pharma, Reinach, Switzerland) and expressed as [(I(GABA + DZ)/I(GABA)) - 1] · 100.
Western blotting. Oocytes were homogenized in lysis buffer (10 mM HEPES, pH 8.0, 100 mM NaCl, 10 mM EDTA, 1% Triton X-100, Pepstatin, Leustatin, Antipain, and PMSF, each at 5 µg/ml) using a Teflon glass homogenizer. The homogenate was incubated on ice for 15 min and centrifuged at 15,000 x g for 15 min at 4°C. The supernatant was extracted with chloroform-methanol and subjected to SDS-PAGE (Laemmli, 1970). Proteins were transferred to nitrocellulose membranes (HybondECL; Amersham Pharmacia, Dbendorf, Switzerland) according to Towbin et al. (1979) and decorated with the monoclonal antibodies bd24 and bd17 (Häring et al., 1985; Ewert et al., 1990), which recognize the N-terminal of the 1 and 2 subunits of the GABAA receptor, respectively. Bands were detected using the ECL system (Amersham Pharmacia).
Model. Because some of the parameters were in common for linked and mutated receptors, the averaged standardized data including SDs were fitted simultaneously with using a Levenberg-Marquardt algorithm (ProFit 5.1 software; ProFit, Zurich, Switzerland) in combination with the following equations: loose subunits, I(c) = [100 · (1 + L)]/(1/[1 + (L1 · K1)/(L2 · K2) + (L1 · c)/(L · K2)]) · [1 + (K1 · L1)/c + L1 + (L1 · K1)/K2 + (c · L1)/(K2) + (L1 · K1)/(L2 · K2) + (c · L1)/(K2 · L)]; receptors linked /, I(c) = [100 · (1 + fA · L)]/(1/[1 + (L1 · K1)/(L2 · K2) + (fa · L1 · c)/(fA · L · K2)]) · [1 + (K1 · fa · L1)/c + fa · L1 + (fa · L1 · K1)/K2 + (c · fa · L1)/(K2) + (L1 · K1)/(L2 · K2) + (c · fa · L1)/(K2 · fA · L)]; receptors linked /, I(c) = [100 · (1 + fG · L)]/(1/[1 + (L1 · K1)/(L2 · K2) + (fg · L1 · c)/(fG · L · K2)]) · [1 + (K1 · fg · L1)/c + fg · L1 + (fg · L1 · K1)/K2 + (c · fg · L1)/(K2) + (L1 · K1)/(L2 · K2) + (c · fg · L1)/(K2 · fG · L)]; / receptors mutated in site 1 and an analogous equation for the mutation in residue 165 in site 1, I(c) = [100 · (1 + fA · L)]/(1/[1 + (L1 · K1205)/(L2 · ta205 · K2) + (fa · L1 · c)/(fA · L · ta205 · K2)]) · [1 + (K1205 · fa · L1)/c + fa · L1 + (fa · L1 · K1205)/(ta205 · K2) + (c · fa · L1)/(ta205 · K2) + (L1 · K1205)/(L2 · ta205 · K2) + (fa · L1 · c)/(fA · L · ta205 · K2)]; / receptors mutated in site 2 and an analogous equation for the mutation in residue 165 in site 2, I(c) = [100 · (1 + fA · L)]/(1/[1 + (L1 · ta205 · K1)/(L2 · K2205) + (fa · L1 · c)/(fA · L · K2205)]) · [1 + (ta205 · K1 · fa · L1)/c + fa · L1 + (fa · L1 · ta205 · K1)/K2205 + (c · fa · L1)/(K2205) + (L1 · ta205 · K1)/(L2 · K2205) + (fa · L1 · c)/(fA · L · K2205)]; and / receptors mutated both sites, I(c) = [100 · (1 + fG · L)]/(1/[1 + (L1 · K165)/(L2 · K265) + (fg · L1 · c)/(fG · L · ta65 · K265)]) · [1 + (ta65 · K165 · fg · L1)/c + fg · L1 + (fg · L1 · K165)/K265 + (c · fg · L1)/(t65 · K265) + (L1 · K165)/(L2 · K265) + (c · fg · L1)/(ta65 · K265 · fG · L)]); I is the current, K1 and K2 are dissociation constants for agonists of the unmuted and K165, K265, K1205, and K2205 of the mutated receptors illustrated in Figure 5, L describes the isomerization between the closed (ARA) and open (ARA*) channel [L = (ARA)/(ARA*)], L1 and L2 describe the isomerization between closed receptors occupied by a single agonist molecule (AR) or (RA) and the corresponding open channels (AR*) and (RA*), respectively, c is the concentration of GABA or muscimol, b is the concentration of bicuculline or SR95531, fG and fA, fg, and fa are coefficients modulating L, L1, and L2, respectively (gating of the channel), as a consequence of subunit linkage, and ta65 and ta205 coefficients describe the allosteric effect of the mutation in one site on the dissociation constant for GABA of the second site.
Inhibition was fitted with the equation I(c) = Imax/(1/[1 + (L1 · K1)/(L2 · K2) + (L1 · c)/(L · K2)]) · [1 + (K1 · L1)/c + L1 + (L1 · K1)/K2 + (c · L1)/(K2) + (L1 · K1)/(L2 · K2) + (c · L1)/(K2 · L)] + [(b · K1 · L1)/c] · [1/K1i + 1/K2i + b/(K1i · K2i)] for loose subunits, I(c) = Imax/[(1/[1 + (L1 · K1205)/(L2 · ta205 · K2) + (fa · L1 · c)/(fA · L · ta205 ·K2)]) · [1 + (K1205 · fa · L1)/c + fa · L1 + (fa · L1 · K1205)/(ta205 · K2) + (c · fa · L1)/(ta205 · K2) + (L1 · K1205)/(L2 · ta205 · K2) + (fa · L1 · c)/(fA · L · ta205 · K2)] + [(b · K1 · fa · L1)/c] · [1/(m · K1I) + 1/(tb205 · K2I) + b/(m · K1i · tb205 · K2i)])] for / receptors mutated in site 1, and analogously for the other receptors. K1i and K2i are dissociation constants for antagonists, m is the ratio between dissociation constants of mutated divided by that of unmutated receptors, and tb65 and tb205 coefficients describe the allosteric effect of the mutation in one site on the dissociation constant for bicuculline of the second site.
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Results
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Mutations in the agonist binding site
We made use of the two point mutations, 1F65L and 2Y205S, to alter agonist binding sites in recombinant 122 GABAA receptors. The first mutation has been shown previously to decrease the apparent affinity of GABA for channel gating and the apparent affinity for the competitive antagonist bicuculline (Sigel et al., 1992). The mutation 2Y205S has been reported to compromise GABA binding without affecting channel opening induced by pentobarbital (Amin and Weiss, 1993). It is now known that a receptor pentamer contains two and two subunits. Therefore, in each case, both subunits and thereby both GABA sites were affected by the point mutations in the previous studies (Fig. 1, top). We have shown previously that it is feasible to covalently link , , and subunits of the GABAA receptor while retaining full receptor function (Baumann et al., 2001, 2002). This allows the introduction of a point mutation in one individual 1 or 2 subunit exclusively. Thus, the concatenated subunits 2-2-1 (), 2-1 (), 2-2-1F65L (65), and 2-1F65L (65) were prepared and functionally expressed in Xenopus oocytes as a channel containing no mutation /, one mutation 65/ and /65, or two mutations 65/65 (Fig. 1). 2-2Y62L-1 (62) and 2Y62L-1 (62) were also prepared for control purposes. To study a second contact point of agonists with the receptor and a differently concatenated channel, we similarly prepared 2-2 (), 2-2Y205S (205), 1-2-1 (), and 1-2Y205S-1 (205). /, 205/205, 205/, and /205 (Fig. 1) were expressed in Xenopus oocytes and functionally characterized.
Nonlinked receptors and linked receptors
Linked subunits might be proteolysed during expression. If the linker region(s) specifically would be affected, nonlinked subunits could be produced from dual constructs and nonlinked subunits and dual subunit constructs could be produced from triple constructs, all products carrying part of the corresponding linkers. Such an event would complicate interpretation of the results in the event that the newly liberated subunits retain the ability to reassemble to form functional channels. Because the assembly of multi-subunit proteins is taking place in an early phase after translation in the endoplasmic reticulum, this hypothetical process would have to take place there. Figure 2A shows a Western blot analysis of noninjected oocytes (lane 1), oocytes expressing loose subunits // (lane 3), or the combination of concatenated subunits / (lane 2). All lanes were decorated with the 1-specific antibody bd24. As expected for the case of absent proteolysis, we detected no band in lane 1, a single specific band at 50 kDa in lane 3, and a single specific band at 170 kDa in lane 2. Different exposures allowed an estimate of the detection limit, which was 2% for a single proteolyzed fragment. Thus, our results obtained with / and its mutational variants (see below) cannot be explained by proteolysis and subsequent reassembly. Figure 2B shows a similar analysis for oocytes injected with / and decorated with bd17, which recognizes free N-termini of subunits. In this case, a stronger nonspecific reaction occurred. However, the band corresponding to is clearly seen at 110 kDa. A similar analysis using antibodies directed against loops of and and the N-terminal of subunits failed because of small amounts of antigens formed in the Xenopus oocyte that were not detected with the corresponding antibodies (data not shown).
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Figure 2. Western blot analysis. A, Resistance to proteolysis of the fusion protein . Lanes 1-3 were decorated with bd24, reacting with free termini of subunits. Lane 1, Noninjected oocytes (n.i.). No signal was detected in noninjected oocytes. Lane 2, Oocytes injected with /. The triple construct migrates at 170 kDa. No specific signal was detected at 50 or 110 kDa, the size of a monomeric 1 subunit or dimeric subunit carrying 1 at the N-terminus, as expected as a consequence of proteolysis in one of the linker regions. The absence of specific signals in other areas indicates that no N-terminal breakdown product of this triple subunit construct larger than 21 kDa is formed. Lane 3, Oocytes injected with //. The two loose 1 subunits from the wild-type 122 receptor migrate at 50 kDa. B, Lanes 1-3 were decorated with bd17 and reacted with free termini of subunits. Lane 1, Noninjected oocytes. Lane 2, Oocytes injected with /. The dual construct migrates at 110 kDa. No specific signal was detected at 53 kDa, the size of a monomeric 2 subunit, as expected during proteolysis in the linker region. Lane 3, Oocytes injected with //. The two loose 2 subunits from the wild-type 122 receptor migrate at 53 kDa.
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Apart from these observations, we believe it for several reasons unlikely that proteolysis of subunits is followed by assembly of the fragments to form functional channels. First, we did not obtain functional channels when we chose the linker too short. Second, we tried to functionally express a huge variety of combinations of dual and triple constructs without succeeding unless a channel could be formed directly (Baumann et al., 2001, 2002). If proteolysis would lead to functional channels, many of the non- channels should have resulted in functional expression. Specifically, we expressed alone (Baumann et al., 2001) and in combination with or subunits (Baumann et al., 2002) and observed very little, if any, current. Third, the hypothetical proteolysis leading to different functional channels than envisaged would be predicted to lead to a channel mixture. We never observed evidence for a two or more phasic dose-response curve for nonmutated channels.
At a functional level, the effect of subunit concatenation was first studied. Thus, the functional properties of nonlinked receptors, //, and linked receptors, / and /, were compared. Concentration-response curves for GABA and muscimol, as agonists, and inhibition of currents elicited by GABA by bicuculline were measured for all three types of receptors. Results are summarized in Table 1. Subunit concatenation resulted in a 2- to 3.5-fold loss in agonist sensitivity. In contrast, subunit concatenation did not significantly affect the IC50 for bicuculline in / receptors and only to a very small extent in / receptors.
Effect of the mutation 1F65L on agonist concentration-response properties for GABA and muscimol
Figure 3A summarizes average concentration-reponse curves for wild-type / and mutant 65/65 channels and also shows curves derived from channels harboring a single mutation 65/ and /65. The curves obtained from the channels containing one mutation displayed properties more similar to channels with two mutated sites. Data are summarized in Table 1.
GABA concentration-response curves were also obtained for mutant 62/62 channels (data are given in Table 1). 62 is the homologous residue in the 2 subunit to 65 in the 1 subunit and has been proposed to contribute to high-affinity binding of agonists (Newell et al., 2000). The mutation introduced in both 2 subunits had only a very small effect on EC50. Each of the channels harboring a single mutated subunit was studied only twice. As expected, an intermediate EC50 resulted (data not shown).
The concentration-response curves for the agonist muscimol was also performed with wild-type / and mutant 65/65 channels (Fig. 3B). Quantitative data are given in Table 1. Although double mutation caused a 12-fold shift in the EC50 for GABA, it amounted to only fourfold for muscimol.
Effect of the mutation 2Y205S on agonist concentration-response properties for GABA and muscimol
Figure 4A illustrates concentration-response curves obtained from wild-type / channels with GABA and muscimol, respectively (values are given in Table 1). The channel containing two mutations did not result in any detectable current for GABA concentrations up to 10 mM. At 100 mM, the elicited current amounted to 0.1% of the maximal current amplitude of wild-type / channels. Expression of all channels was verified by determining the amplitude of the coexpessed voltage-gated Na channel and using direct activation of the GABA channel by 1 mM pentobarbital. The observed current amplitudes elicited by pentobarbital were in the range of 200-1200 nA for the wild-type channel (10 oocytes), 300-2800 nA for the /205 channel (13 oocytes), 70-400 nA for 205/ channels (14 oocytes), and 2-60 nA for 205/205 channels (15 oocytes). The reasons for the small currents in the latter case is not known; however, because the corresponding oocytes expressed microampere-sized Na currents, it cannot be explained by either failure of injection or presence of RNase activity in the injection solution.
The mutation 2Y205S destroys the agonist binding site and theoretically should uncover the properties of the second site. Thus, in 205/ mutant receptors, only site 1 should be visible and in /205 mutant receptors, only site 2 should be visible. The maximal current amplitudes of 205/ and /205 mutant receptors could not be determined because the affinity of the second site is in the molar range. For evaluation of data, it was assumed that this parameter was the same as for / channels. Figure 4B summarizes the dose-response curves in response to the agonists GABA and muscimol on 205/ and /205 mutant receptors after normalization to the maximal current amplitude of unmutated / receptors. All average curves were fitted with a two component logistic equation (Fig. 4B, solid lines). Data are summarized in Table 1. The absolute amplitudes should be judged with care because they depend on the expression rate. Our observations are in line with a preference of site 2 for GABA and a preference of site 1 for muscimol. It was interesting to see whether the assumption that 205/ and /205 mutant receptors display the same maximal current amplitudes as / receptors affected our basic findings. Therefore, three different alternative possibilities were tested. First, it was assumed that the maximal currents by both mutant receptors were only one-fifth of the standardized currents. Second, it was assumed that they were both five times the standardized currents and, third, that /205 and 205/ mutant receptors had a twofold, respectively one-third, maximal current amplitude of wild-type receptors, as suggested by the currents elicited by 1 mM pentobarbital. Our basic conclusions were not affected in any of the three cases.
Inhibition by bicuculline
The concentration-dependent inhibition of GABA-induced currents by the competitive antagonist bicuculline was also studied. Opening of the GABA receptor channel was standardized by performing experiments approximately at the respective EC10 (EC8.9-EC11.2), except for the receptors containing a 2Y205S mutation. For the mutant 65/65 channels, as compared with wild-type / channels, IC50 for the competitive antagonist bicuculline is shifted to 56-fold higher concentrations. Figure 5A summarizes these experiments and, in addition, shows average curves obtained with channels harboring a single mutation, 65/ and /65, which were shifted eightfold and fourfold, respectively. Quantitative data are summarized in Table 1.
Bicuculline concentration-inhibition curves were also obtained from mutant 62/62 channels (Table 1). The mutation introduced in both 2 subunits had no significant effect. Therefore, channels harboring a single mutated subunit were not studied.
Figure 5B summarizes bicuculline inhibition curves for wild-type / and single mutant 205/ and /205 channels, respectively. Both curves of mutant receptors would be expected to superimpose with those of concatenated wild-type receptors, unless the effects of mutations were allosterically communicated to the other site. Because the shift amounts to approximately sevenfold in both cases, this seems to be the case. Quantitative data are summarized in Table 1. Inhibition for /205 and 205/ receptors was measured at a GABA concentration of 100 and 300 µM, respectively.
Inhibition by SR95531
Inhibition by the competitive inhibitor SR95531 was determined for /, 65/, /65, 65/65, /, 205/, and /205 under conditions described for bicuculline. In all cases, a qualitatively similar behavior was found. The respective IC50 were 10-fold lower than with bicuculline.
Diazepam responsiveness
All receptors, /, 65/, /65, 65/65, 62/, /62, and 62/62, were tested for stimulation of the current amplitude by 1 µM diazepam at an EC5 for GABA. In all cases, the current was stimulated to 240-330% of the control amplitude (data not shown).
Model
Different models were used to analyze the experimental data. Figure 6 shows the model that was in best agreement with our findings and then used for the fit of the data. The model incorporates two binding sites for agonists and competitive antagonists, site 1 and site 2. At least a single site has to be occupied by an agonist to produce an open state AR*, RA*, or ARA*. Concentration-response curves, including those with nonlinked subunits, were averaged and standardized. We initially tried to fit individual curves with the equations given in Materials and Methods containing variables describing binding (K1, K2) as well as gating (L, L1, L2) phenomena. However, many acceptable fits were obtained. Because different curves share at least some variables, a combined Levenberg-Marquardt fit was applied to the activation curve data collected with GABA as an agonist in receptors composed of nonlinked subunits, containing linked subunits (two receptor types), mutated in site 1 (two receptor types), mutated in site 2 (two receptor types), and containing the 1F65L mutation in both sites. Combined fitting led to a single solution. Another combined fit was applied to the data on bicuculline inhibition. Here, the fitted parameters of the activation curves were used. Results of the fits are given in the legend for Figure 6. Fitting suggested that concatenation of subunits modified gating of the channel, favoring closed channels by a factor of 4-31 (fa, fA, fg, fG). For unmutated channels, transition into the open state is 60 times less efficient when occupied by a single agonist molecule as compared with those occupied by two agonist molecules. Fitting also predicted that site 2 has an approximately threefold higher affinity for GABA than site 1. Data presented in Figure 4B indicate a preference of site 1 for muscimol. Occupation of a single site by bicuculline keeps the channel in a closed state. Site 1 has a slight preference for bicuculline. Simulated data were obtained with the above fit results for the gating of the channel by GABA and its inhibition by bicuculline for the mutation in the 1 subunit 1F65L (Fig. 7A,B) and the mutation in the 2 subunit 2Y205S (Fig. 7C,D). The model predicts the behavior of the channels quite precisely in both cases. The coefficients ta65 and tb65, and ta205 and tb205, describing an allosteric effect on agonist and antagonist binding elicited by the mutations, was indicative of little effect in both cases of the 1F65L mutation and an approximately twofold to fivefold effect in the case of the 2Y205S mutation.
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Figure 6. Model of the receptor with two agonist binding sites 1 and 2. The receptor (R) can first bind GABA (A) to either site 1 (AR) or site 2 (RA). Analogously, the receptor can first bind bicuculline (I) to either site 1 (IR) or site 2 (RI). The receptor occupied by two agonist molecules (ARA) can isomerize to the open-state ARA*, and the receptors occupied by a single agonist molecule can isomerize to the open-states AR* and RA*. The model at the top left describes a receptor composed of loose subunits. Concentration modifies L and L1 with fG and fg, respectively. Effects of mutation in site 1 are allosterically transferred to site 2 and vice versa (ta, agonists; tb, antagonists). Constants are taken as dissociation constants and gating constants as closed state divided by open state. Combined Levenberg-Marquardt fit gave the following estimates for the parameters: L, 0.23; L1, 15.3; L2, 13.6; K1, 128 µM; K2, 42 µM; fA, 4.5; fa, 8.6; fG, 20.4; fg, 31.2; K165, 1440 µM; K265, 1320 µM; K1205, 1.3 M; K2205, 14.9 M; ta65, 0.84; tb65, 1.00; ta205, 4.6; tb205, 2.2; K1I, 2.2 µM; K2I, 3.0 µM; m, 97.
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Figure 7. A-D, Simulation of the GABA dose-response curves (A, C) and bicuculline inhibition curves (B, D). A, C, /, 65/65 (sites 1 and 2 mutated), 65/ (site 1 mutated), and /65 (site 2 mutated) receptors. B, D, /, /205 (site 1 mutated), and 205/ (site 2 mutated) receptors. The parameters obtained in a fit are given in Figure 6.
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Discussion
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Top
Abstract
Introduction
),
),
), and
) receptors. Bicuculline was applied in increasing concentrations together with a GABA concentration eliciting 9-11% of the maximal current amplitude. B, Bicuculline inhibition curves of
), and
) receptors. Mean values with SEM from four to five oocytes from two batches for each subunit combination are shown. Individual curves were normalized to the current found in the absence of bicuculline and were subsequently averaged.
