Amino acid substitutions in the human homomeric β₃ GABA_A receptor that enable activation by GABA

Abstract

GABA_A receptors (GABA_ARs) are pentameric ligand-gated ion channels that mediate synaptic inhibition throughout the central nervous system. The α₁β₂γ₂ receptor is the major subtype in the brain; GABA binds at the β₂(+)α₁(−) interface. The structure of the homomeric β₃ GABA_AR, which is not activated by GABA, has been solved. Recently, four additional heteromeric structures were reported, highlighting key residues required for agonist binding. Here, we used a protein engineering method, taking advantage of knowledge of the key binding residues, to create a β₃(+)α₁(−) heteromeric interface in the homomeric human β₃ GABA_AR that enables GABA-mediated activation. Substitutions were made in the complementary side of the orthosteric binding site in loop D (Y87F and Q89R), loop E (G152T), and loop G (N66D and A70T). The Q89R and G152T combination enabled low-potency activation by GABA and potentiation by propofol but impaired direct activation by higher propofol concentrations. At higher concentrations, GABA inhibited gating of β₃ GABA_AR variants containing Y87F, Q89R, and G152T. Reversion of Phe⁸⁷ to tyrosine abolished GABA's inhibitory effect and partially recovered direct activation by propofol. This tyrosine is conserved in homomeric GABA_ARs and in the Erwinia chrysanthemi ligand-gated ion channel and may be essential for the absence of an inhibitory effect of GABA on homomeric channels. This work demonstrated that only two substitutions, Q89R and G152T, in β₃ GABA_AR are sufficient to reconstitute GABA-mediated activation and suggests that Tyr⁸⁷ prevents inhibitory effects of GABA.

Introduction

GABA_ARs² are members of the pentameric ligand-gated ion channel family and mediate fast synaptic inhibition (1). Consequently, they are important pharmacological targets (2, 3).

GABA_AR subunits are composed of three domains (4): 1) the extracellular domain (ECD), with 10 β-strands (β1–10), one α-helix, and the orthosteric binding site; 2) the transmembrane domain (TMD) comprising four helices (TM1–4), with the TM2 of each subunit forming the ion pore; and 3) the intracellular domain (ICD), between TM3 and TM4, which is a site for posttranslational modification that interacts with trafficking proteins (4–6).

The orthosteric binding site is located between the α and β subunits that comprise the complementary (−) and principal (+) components, respectively. The site contains seven noncontiguous binding loops (A–G): A–C belong to the principal side, whereas loops D–G belong to the complementary side (7–9).

There are 19 different GABA_AR subunits that form at least 14 distinct combinations in vivo (10, 11), accounting for the physiological versatility and pharmacological selectivity of these channels (2). The major subtype in the central nervous system is the α₁β₂γ₂ GABA_AR. The β₁, β₃, and ρ subunits can form homomers when recombinantly expressed in vitro. Although the homomeric β₃ has not been identified in vivo, it is of considerable interest as the first GABA_AR to yield to high-resolution structural analysis (12) and for functional studies because histaminergic ligands and propofol activate the receptor (13–16). Recently, four heteromeric GABA_AR structures were published, including the major subtype (17–19). These studies determined the important residues for GABA binding and suggest that variability on the complementary subunit influences ligand selectivity (19). The homomeric β₃ cannot be activated by GABA (16, 20). This raises questions about which residues in the complementary side are required to reconstitute activation. The availability of the β₃ structure provides an opportunity to locate candidate residues.

In the present study, we investigated whether substituting amino acids in the complementary side of the β₃ GABA_AR to corresponding residues in the α₁ subunit would reconstitute activation by GABA. Four β₃ mutants were designed and used for patch-clamp electrophysiology. We analyzed the activation by GABA and propofol, potentiation of GABA-evoked currents by propofol, and the kinetics of GABA-evoked currents. Comparative modeling and molecular docking calculations were used to predict the orientation of GABA at the orthosteric site of the mutant β₃ GABA_AR. Using these approaches, we demonstrated that Q89R and G152T substitutions reconstituted GABA activation of GABA_AR β₃ and potentiation by propofol. In addition, we found that the Y87F substitution caused GABA to inhibit receptor function.

Results

Designing the constructs

We modified the β₃ GABA_AR by replacing the ICD (residues 346–396) with the SQPARAA sequence to mimic the construct used to crystallize the β₃ GABA_AR (12), referred to from this point as β₃-cryst (Table 1).

Table 1.

Protein constructs

Construct name	ECD interface	Substitutions
GABAAR β3-cryst	β3(+)β3(−)	Substitute ICD (346–396) for SQPARAA
GABAAR β3 C1	β3(+)α1(−)	β3-cryst + Y87F,Q89R,G152T
GABAAR β3 C1 N66D	β3(+)α1(−)	C1 + N66D
GABAAR β3 C1 A70T	β3(+)α1(−)	C1 + A70T
GABAAR β3 C1 F87Y	β3(+)α1(−)	β3-cryst + Q89R,G152T

Docking GABA into the β₃ orthosteric site

Docking calculations were performed between GABA and the β₃-cryst model (Fig. 1A). As far as we are aware, there are no prior reports of docking GABA into the homomeric β₃ receptor. The best GABA pose presented an energy of −38 kcal/mol, suggesting binding. Examination of residues within the orthosteric binding domain on the α₁ subunit revealed amino acids that are not shared by β₃ at key locations known to affect activation by GABA (Fig. 1B). Substitution of these residues into the β₃-cryst model (GABA_AR β₃ C1) improved the binding energy of GABA as evidenced by docking calculations (−46 kcal/mol). The model suggests that the GABA amino group forms a salt bridge with Glu¹⁸⁰ on the (+) interface of the β₃ subunit (Fig. 1C) and that the GABA carboxyl makes a bidentate interaction with Arg⁸⁹ and a hydrogen bond with the Thr¹⁵² hydroxyl group, substituted in the (−) interface. These interactions are in agreement with the cryo-EM structures of the human GABA_AR α₁β₂γ₂ and rat GABA_AR α₁β₁γ₂ (18, 19). In addition, they were described by other studies using docking calculations with human GABA_AR α₁β₂γ₂ (21) and insect GABA_AR models (21, 22).

Figure 1.

Docking results for GABA_AR constructs. The predicted GABA orientations (pink and yellow) in GABA_AR β₃-cryst (A) and GABA_AR β₃ C1 (C) show the carboxyl group facing the complementary side (green cartoon) and the amino group facing the principal side (cyan cartoon). Residues interacting with GABA are depicted as gray sticks, and polar interactions are depicted as black dashes. B, sequence alignment of the orthosteric site (loops A–G) based on structural comparisons of GABA_AR β₃ (PDB code 4COF) and the GABA_AR α₁ model (21), showing the substitutions (gray box). Secondary structure, loops (black line) and β-strands (yellow arrows), is depicted below the sequence. Residues are numbered according to the initiating Met¹.

Three substitutions reconstituted GABA activation of homomeric β₃ receptors

The Y87F, Q89R, and G152T substitutions were introduced into the β₃-cryst construct using site-directed mutagenesis. This β₃ C1 cDNA was transiently transfected into human embryonic kidney 293 (HEK293) cells for whole-cell electrophysiology recordings. Concentrations of GABA that are maximally efficacious at heteromeric GABA_ARs (1 mm) fail to activate homomeric β₃ GABA_ARs (16, 20). We therefore applied higher concentrations of GABA (10 mm) to HEK293 cells expressing either β₃-cryst or β₃ C1. GABA (10 mm) evoked negligible currents mediated by β₃-cryst with current densities of 2.3 ± 0.8 pA/pF (Fig. 2A). By contrast, GABA (10 mm)–evoked currents mediated by β₃ C1 were larger with current densities of 15.7 ± 12.5 pA/pF (Fig. 2B). This was significantly different from β₃-cryst (n = 7, p = 0.003, t test; Fig. 2C). These results indicate that the amino acid substitutions (Y87F, Q89R, and G152T) were sufficient to reconstitute activation by GABA.

Figure 2.

GABA_AR β₃ C1 is activated and inhibited by GABA. A and B, examples of currents recorded when GABA (10 mm) was applied to cells expressing GABA_AR β₃-cryst (A) and GABA_AR β₃ C1 (B) indicate that the latter is functional and activated by the neurotransmitter. C, mean ± S.D. current densities evoked by GABA (10 mm), with an asterisk indicating significant differences between the proteins (n = 7, p = 0.003, t test). D, examples of currents mediated by GABA_AR β₃ C1, evoked by increasing concentrations of GABA. Currents in gray are declining due to inhibition by GABA (>10 mm). The bar indicates GABA application (5 s). E, concentration-response relationships obtained using the percentage of the maximum amplitude recorded for each cell (n = 5). Logistic equations were fitted to the data points (see “Experimental procedures”). From the double-logistic fit, two distinct potencies were observed for activation (EC₅₀ = 2.9 mm) and inhibition (IC₅₀ = 50.5 mm). A summary of the data is in Table 2. F, graph of mean current 10–90% rise time. Activation rates are slowed somewhat by increasing the GABA concentration in β₃ C1 (n = 6, F(4,25) = 42.2, one-way ANOVA post hoc Dunnett's, p = 0.04 comparing 10 with 1 mm GABA), whereas currents evoked by 100 mm GABA were activated faster (n = 6, p < 0.0001, F(4,25) = 42.2, one-way ANOVA post hoc Dunnett's, comparing 100 with 1 mm GABA). G, values for weighted τ of deactivation exhibited a similar trend with increasing GABA concentration (p = 0.04, one-way ANOVA, n = 6, F(4,19) = 3.2), although there was no significant difference comparing 1 mm with the other GABA concentrations tested. Detailed information about the components is in Table 3. Error bars represent S.D.

We subsequently determined the concentration-response relationship of β₃ C1 to characterize the potency of GABA (Table 2). GABA was applied at increasing concentrations to cells expressing β₃ C1. A representative example of these currents is shown in Fig. 2D. GABA-evoked current amplitudes were expressed as a percentage of the maximum and plotted as a concentration-response relationship (Fig. 2E). The data indicate that GABA exhibits a biphasic concentration-response relationship, which suggests two effects: activation and inhibition. We therefore fitted a two-component logistic function to the data (see “Experimental procedures”). GABA, up to 10 mm, activates β₃ C1 with an EC₅₀ of ∼3 mm (Table 2). Higher concentrations of GABA caused a reduction in current amplitude with an IC₅₀ of ∼50 mm. This inhibitory effect has not been observed previously in GABA_ARs (9, 23–26) or in the bacterial pentameric ligand-gated ion channel ELIC (27), which, like β₃ C1, also requires high concentrations of GABA for its activation (Fig. S1). We also observed a lack of inhibitory effect in heteromeric GABA_ARs formed from β₃-cryst and β₃ C1 subunits (Fig. S2).

Table 2.

Summary of Hill slope, EC₅₀, and current density values obtained for GABA activation of GABA_AR β₃ C1 and F87Y

Mean ± S.D. Hill slope and EC₅₀ values obtained from logistic function fit parameters of individual experiments and mean ± S.D. current densities evoked by peak concentrations of GABA are shown. No significant differences between GABA_AR β₃ C1 and F87Y were observed (t test; EC₅₀ p = 0.2; current densities p = 0.6). n = number of experiments.

Receptor	Hill slope	EC50	Current density	n
		mm	pA/pF
GABAAR β3 C1	1.3 ± 0.4	2.9 ± 2.1	−15.7 ± 12.5	4
GABAAR β3 C1 F87Y	1.2 ± 0.3	1.3 ± 0.6	−17.1 ± 11.9	4

Kinetics of β₃ C1

In addition to the biphasic nature of the GABA concentration-response relationship, the representative currents shown in Fig. 2D also display unusual kinetics. Therefore, we analyzed the current activation and deactivation rates by measuring the 10–90% rise time and by fitting a two-component exponential function, respectively (see “Experimental procedures”). The mean values of rise times and weighted τ were plotted (Fig. 2, F and G). The individual components of the double-exponential fits for deactivation can be found in Table 3. Currents evoked by lower concentrations of GABA (0.1 and 0.3 mm) were excluded from the analysis due to their small amplitudes. Consistent with the concentration dependence of peak current activation (Fig. 2E), the concentration dependence of activation and deactivation also appears to be biphasic (Fig. 2, F and G).

Table 3.

Mean ± S.D. of the deactivation components in GABA_AR β₃ C1 and F87Y

No significant differences were observed between the mutants (n = 4, p = 0.1, F(9,34) = 1.8, one-way ANOVA post hoc Tukey's).

[GABA]	τf	%f	τs	%s	Weighted τ
mm
β3 C1
1	34 ± 3	59 ± 5	253 ± 3	41 ± 5	142 ± 54
3	56 ± 32	48 ± 14	347 ± 139	52 ± 14	181 ± 97
10	58 ± 45	47 ± 2	342 ± 137	72 ± 26	243 ± 130
30	39 ± 10	37 ± 10	282 ± 98	72 ± 20	260 ± 124
100	37 ± 23	63 ± 33	261 ± 125	37 ± 33	84 ± 29
β3 C1 F87Y
1	40 ± 9	64 ± 13	424 ± 142	36 ± 13	176 ± 56
3	51 ± 19	66 ± 11	345 ± 139	34 ± 11	151 ± 76
10	67 ± 30	56 ± 13	337 ± 79	44 ± 14	194 ± 66
30	79 ± 49	52 ± 15	511 ± 173	48 ± 15	287 ± 114
100	63 ± 7	77 ± 17	518 ± 232	23 ± 17	198 ± 171

GABA does not cause a voltage-dependent channel block

The inhibitory effect of GABA at higher concentrations could be due to binding at a lower-affinity site, which blocks the channel pore. We therefore examined whether GABA causes a voltage-dependent block of β₃ C1 by comparing the current–voltage (I–V) relationships of currents evoked by 1 and 100 mm GABA. These concentrations were chosen because the inhibitory effect was observed at 100 mm but not at 1 mm GABA. Representative examples of the currents evoked by GABA at voltages ranging from −60 to 60 mV are shown in Fig. 3A. GABA (1 mm) produced an outwardly rectifying I–V relationship, consistent with previous observations of currents mediated by β₃ and α₁β₃ GABA_ARs (16), as did 100 mm GABA. We quantified outward rectification by expressing the current amplitudes as a ratio of those evoked at −60 mV (Fig. 3B). The rectification indexes calculated (I_{60 mV}/I_{−60 mV}) were 3.5 ± 0.7 and 3.6 ± 0.5 (n = 4) for 1 and 100 mm GABA, respectively (p = 0.8, t test, n = 4). These results suggest that the inhibitory effect of 100 mm GABA was not caused by voltage-dependent channel block.

Figure 3.

GABA does not block the channel by a voltage-dependent process. A, representative examples of the currents evoked by GABA (1 and 100 mm) recorded at voltages ranging from −60 to 60 mV. B, the amplitude of the currents was expressed as a ratio of those evoked at −60 mV (I/I_{−60 mV}) and plotted against the voltage, indicating similar outward rectification for both concentrations. Error bars represent S.D.

Substitutions in loop G do not affect the activation of β₃ C1 by GABA

Mutagenesis studies in GABA_AR α₁β₂γ₂ indicate that the identities of α₁ loop G residues at positions 71 and 75 influence gating and, thereby, the apparent potency of GABA (9, 23, 25, 28). In an attempt to increase the apparent potency of GABA, substitutions in loop G were made, introducing α₁ residues into β₃ C1 N66D and β₃ C1 A70T GABA_ARs (residues equivalent to those at α1 positions 71 and 75, respectively). Neither the potency nor the efficacy of GABA was affected (one-way ANOVA post hoc Dunnett's, p = 0.8, F(2,12) = 0.28). This is perhaps not surprising due to the conservative nature of the N66D and A40T substitutions (Fig. S3 and Table S1).

A loop D Tyr conserved in homomeric receptors prevents block by GABA

An amino acid sequence alignment of the pentameric ligand-gated ion channel subunits that form homomeric GABA-activated receptors, including ELIC, reveals conservation of the Tyr at the position equivalent to β₃ amino acid 87 (Fig. S4A). We investigated whether replacement of ELIC Tyr³⁸ with Phe affects activation by GABA. Interestingly, GABA failed to evoke currents mediated by ELIC Y38F despite the conservative nature of this substitution (Fig. S4C). These data suggest that the Phe is detrimental to ELIC function. Because ELIC, GABA_A ρ, and GABA_A β all contain a Tyr, this residue may be necessary for preventing block of homomeric receptors by GABA. We tested the hypothesis that the Tyr is required in β₃ receptors to prevent inhibitory effects of GABA at high concentrations by creating the β₃ C1 F87Y in which the Phe⁸⁷ was reverted back to the tyrosine found in WT β₃.

Cells expressing β₃ C1 F87Y were voltage-clamped at −60 mV, and GABA-evoked currents were recorded. Representative examples are shown in Fig. 4A. The current amplitudes were expressed as a percentage of maximum and plotted as a concentration-response relationship, which was fitted with a single-component logistic function (Fig. 4B). The potency of activation by GABA was similar when compared with β₃ C1 (p = 0.2, n = 4, t test; Table 2) as was the maximum current density (p = 0.6, n = 4, t test; Table 2). However, the inhibition by 100 mm GABA was absent in β₃ C1 F87Y with a significant difference in the current amplitude evoked by 100 mm GABA compared with that mediated by β₃ C1 (p = 0.003, n = 4, t test). Similarly, higher concentrations of GABA (300 mm) did not reduce GABA-mediated current amplitude (Fig. S5), indicating that the inhibitory component was abolished with the F87Y substitution. Furthermore, the rate of activation of β₃ C1 F87Y increased with GABA concentration and was not biphasic (Fig. 4C). There was also no apparent influence of GABA concentration on deactivation (Fig. 4D), consistent with the previous data for GABA_ARs (29) and ELIC (Fig. S1, C and D).

Figure 4.

The C1 F87Y substitution abolished the biphasic nature of concentration-response relationship. A, examples of currents mediated by GABA_AR β₃ C1 F87Y, evoked by increasing concentrations of GABA. B, concentration-response relationship obtained using the percentage of the maximum amplitude recorded for each cell. The inhibition caused by 100 mm GABA in β₃ C1 was abolished by the F87Y substitution. A summary of the data is in Table 2. C, mean 10–90% rise times showed no significant change with increasing GABA concentrations in β₃ C1 F87Y except comparing 100 with 1 mm (p = 0.008, n = 4, F(4,15) = 5.2, one-way ANOVA post hoc Dunnett's). D, mean deactivation weighted τ was also independent of GABA concentrations (p = 0.5, one-way ANOVA, F(4,15) = 0.96). Detailed information about the components is in Table 3. Error bars represent S.D.

Effects of propofol on β₃ mutants

Consistent with a previous report of β₃ receptor activation (16), propofol (30 μm) evoked inward currents when applied locally to HEK293 cells expressing β₃-cryst recorded under voltage clamp at −60 mV (Fig. 5A); however, no response was observed in cells expressing β₃ C1 (Fig. 5B). A partial recovery of propofol direct activation was observed in β₃ C1 F87Y (Fig. 5C) as evidenced by the significant difference in current densities between β₃-cryst and β₃ C1 (n = 10, t test, p < 0.0001) and between β₃ C1 and β₃ C1 F87Y (n = 10, t test, p = 0.007; Fig. 5D).

Figure 5.

Propofol does not activate β₃ C1. Examples of currents recorded in the presence of propofol from HEK293 cells expressing β₃-cryst (A), β₃ C1 (B), and β₃ C1 F87Y (C) are shown. D, mean ± S.D. current densities evoked by propofol (30 μm) demonstrate that the function of β₃ C1 is impaired, with values indicated by asterisks significantly different from the β₃-cryst (n = 10, t test, β₃ C1 p < 0.0001). However, propofol direct activation was partially restored in β₃ C1 F87Y with values significantly different from β₃ C1 (n = 10, p = 0.007, t test). Error bars represent S.D.

Potentiation, activation, and blockade of GABA_ARs occur at different propofol concentrations, consistent with the possibility of distinct sites with differing affinities (30–32). The substitutions introduced in β₃ C1 and β₃ C1 F87Y may have affected the gating mechanism or induced structural rearrangements that disrupt the binding site of propofol responsible for the direct activation of the receptor.

We investigated whether propofol can potentiate GABA-induced currents mediated by β₃ C1 and β₃ C1 F87Y. Cells were stepped from GABA (1 mm) to a solution of GABA (1 mm) plus propofol (10 or 30 μm) and back to GABA (1 mm). This concentration of GABA corresponds to EC₂₅ according to the concentration-response relationship, allowing ample scope for enhancement (Fig. 2E).

As observed previously (Fig. 2A), GABA failed to evoke currents when applied to cells expressing β₃-cryst (Fig. 6A). The current observed when GABA was applied with propofol (30 μm) to cells expressing β₃-cryst was equivalent to that observed when propofol was applied alone (Fig. 5), indicating a lack of interaction with GABA (Fig. 6A). By contrast, in cells expressing GABA_AR β₃ C1, propofol (10 and 30 μm) enhanced GABA-evoked currents (Fig. 6, B and C) by 404 ± 183 (n = 3) and 405 ± 176% (n = 7), respectively (Fig. 6F). When applied alone, propofol did not evoke a current. Therefore, the enhancement by propofol of GABA-evoked currents mediated by β₃ C1 is caused by potentiation rather than additive activation. Propofol (10 and 30 μm) also enhanced GABA-induced currents mediated by β₃ C1 F87Y (Fig. 6, D and E) by 242 ± 140 (n = 5) and 663 ± 233% (n = 8), respectively (Fig. 6F). The significant increase (one-way ANOVA post hoc Tukey's, p = 0.008, F(3,19) = 5.3) in current enhancement by propofol (30 μm) is due in this case to the additive effect of direct activation rather than increased potentiation. However, the observation that propofol (10 μm) alone failed to activate a current in the absence of GABA indicates that, similar to GABA_AR β₃ C1, β₃ C1 F87Y also supports propofol-evoked potentiation.

Figure 6.

Potentiation of GABA-evoked currents by propofol was unaffected in the β₃ mutants. A, an exemplar current evoked by propofol mediated by β₃-cryst. GABA had no effect, and the current amplitude evoked by propofol is similar to that seen in the absence of GABA. B and C, examples of GABA (1 mm)–evoked currents mediated by β₃ C1 enhanced in the presence of 10 μm propofol (B) and 30 μm propofol (C). D and E, examples of GABA (1 mm)–evoked currents mediated by β₃ C1 F87Y enhanced in the presence of 10 μm propofol (D) and 30 μm propofol (E). F, the percentages of potentiation by propofol at 10 and 30 μm for β₃ C1 and β₃ C1 F87Y were plotted, with an asterisk indicating a significant difference for β₃ C1 F87Y between 10 and 30 μm propofol (n = 4, p = 0.008, F(3,19) = 5.3, one-way ANOVA post hoc Tukey's). This difference can be explained by the additive effect of propofol (30 μm) activation of β₃ C1 F87Y (Fig. 5C). The bars indicate application of GABA (1 mm) or propofol (30 and 10 μm) with GABA (1 mm). Error bars represent S.D.

Discussion

This study demonstrates that the replacement of two key residues in the orthosteric binding site of the β₃ subunit (Gln⁸⁹ and Gly¹⁵²) by the equivalent ECD loci in the α subunit, Arg and Thr, respectively, enables gating of β₃ receptors by GABA. Docking to the β₃ C1 model, which includes these substitutions plus the additional F87Y substitution, confirmed the interaction of GABA with all three of these binding residues. The favored GABA-binding pose was similar to that of heteromeric GABA_AR structures (18, 19) and to observations in previous docking studies using the mammalian heteromeric and the insect homomeric GABA_ARs (21, 22) and in general agreement with the literature (21, 33, 34). The GABA carboxyl makes a bidentate interaction with Arg⁸⁹ and a hydrogen bond with the Thr¹⁵² hydroxyl group in β₃ C1. The same interactions were reported in heteromeric GABA_AR structures solved in the presence of the agonist (18, 19). In addition, site-directed mutagenesis studies demonstrate that substitution of these residues in the α subunit affects GABA potency in GABA_AR α₁β₂γ₂ and GABA_AR α₁β₂ (24, 35, 36). Taken together, the results of docking and functional analysis are consistent with the idea that the introduction of Q89R and G152T substitutions into β₃ generates a heteromeric β₃(+)α₁(−)-like interface capable of activation by GABA albeit at high concentrations (>300 μm).

GABA concentrations above 10 mm caused a blocking effect in β₃ C1. This has not been observed in other physiologically relevant heteromeric GABA_ARs (9, 23–26) or in ELIC (27). The effect was abolished when the phenylalanine in β₃ C1 was reverted back to tyrosine, F87Y. Interestingly, this effect was also abolished in heteromeric GABA_ARs formed from β₃ C1 (where position 87 is a Phe) and β₃-cryst subunits (where position 87 is a Tyr). The apparent potency of GABA-mediated activation is not altered in these heteromeric GABA_ARs. Although the stoichiometries of heteromeric GABA_ARs formed from β₃ C1 and β₃-cryst subunits are not known, our data suggest that the incorporation of one or more Tyr⁸⁷ is sufficient to prevent GABA-mediated blockade while preserving GABA-mediated activation, highlighting the importance of this residue in GABA_AR function.

The kinetics of GABA-evoked currents mediated by β₃ C1 GABA_ARs were also unusual. Activation and deactivation became slower and then faster with increasing concentrations of GABA, whereas the kinetics in β₃ C1 F87Y GABA_ARs were more consistent with those of heteromeric GABA_ARs (29) and ELIC (Fig. S1). Interestingly, activation and deactivation rates of GABA-evoked currents mediated by β₃ C1 GABA_ARs appear similar to those described for GABA_ARs activated in the presence of modulators, such as propofol (37) and benzodiazepines (38). In addition to its role as an agonist and an inhibitor of β₃ C1 GABA_ARs, GABA may also act as a positive allosteric modulator. In the homomeric β₃ C1 GABA_ARs, GABA may bind to all five subunit interfaces, and the Hill slope of 1.3 suggests cooperativity between at least two of these sites. It is possible that binding to additional orthosteric sites may result in potentiation, similar to the effect of benzodiazepines (38). However, our data with β₃ C1 and β₃-cryst heteromeric GABA_ARs suggest that GABA-mediated activation does not require GABA binding to all interfaces.

Moreover, bell-shaped concentration-response curves have been described for allosteric activators and modulators of GABA_ARs, such as propofol (16), valerenic acid (39), and pentobarbital (40–42). Pentobarbital, at low concentrations (low micromolar), can potentiate GABA_AR currents by increasing the mean open duration. Higher concentrations (high micromolar) of pentobarbital can activate GABA_ARs, and millimolar concentrations can inhibit the channel, slowing deactivation (42). Similarly, GABA may act as an agonist, modulator, and inhibitor of β₃ C1. However, the inhibition is not through a voltage-dependent channel block. Instead, there may be a lower-affinity inhibitory site for GABA. A similar mechanism has been proposed for the inhibitory effect observed with high concentrations of propofol (32).

Although the potentiation of GABA-evoked currents was unaffected, propofol's direct activation of β₃ C1 was impaired compared with β₃-cryst. There was partial recovery of propofol-activated current medicated by β₃ C1 F87Y GABA_ARs. It is clear that substitutions in the orthosteric site can influence direct activation by propofol despite its binding site being in the TM region. In keeping with a need for conformational rearrangement in the orthosteric binding site during gating by propofol, the activation is also inhibited by bicuculline (43). Furthermore, we recently demonstrated faster deactivation of propofol-evoked currents with α₁ loop D (F64C) and loop G (T47R) substitutions in GABA_AR α₁β₂γ₂, which adds additional support for a role of residues in or near the orthosteric binding site in the efficacy of gating by an allosteric agonist (26). Several studies suggest that gating by both orthosteric and allosteric agonists involves an interaction of the loops in the ECD with the TMD, particularly the loops between the β1-β2 strands and TM2-TM3 helices (44–47) and between β6-β7 strands and TM2-TM3 helices (12). It is important to note that loop G is located in β1 strand, loop D is in β2, and loop E is in β6. The substitutions in GABA_AR β₃ C1 are located in loops D and E; therefore, they may affect a concerted gating mechanism.

It is not yet clear why the substitution Y87F causes GABA to act as an inhibitor of β₃ C1 GABA_ARs at high concentrations and impair propofol direct activation. The substitution may affect channel gating, consistent with previous mutagenesis studies of homologous residues in GABA_AR ρ₁ that produced spontaneous opening and affected GABA, trans-4-aminocrotonic acid, and imidazole-4-acetic acid potencies (48) and in GABA_AR α₁β_1,2γ₂ that affected GABA potency and kinetics (9, 49).

The tyrosine is found in all GABA_AR β and ρ subunits and in ELIC. The latter two form homomers that can be activated by GABA (20, 27, 50). Tyrosine may prevent an inhibitory effect of GABA in homomeric receptors, and its substitution to phenylalanine may enable GABA to bind at another lower-affinity site and inhibit gating.

In summary, this study demonstrated that only two substitutions (Q89R and G152T) were required to reconstitute activation by GABA in homomeric β₃ constructs. The potency of GABA was 2 orders of magnitude lower compared with heteromeric GABA_ARs. Similar to heteromeric GABA_ARs, propofol potentiated submaximal GABA-evoked currents and caused direct activation of β₃ C1 F87Y receptors. Surprisingly, the conservative replacement of Tyr⁸⁷ by phenylalanine abolished gating by propofol and caused GABA to have inhibitory effects at high concentrations.

These findings identify structural requirements for the reconstitution of a functional GABA-binding site in β₃ homomeric receptors by transplanting key residues of the α subunit at the heteromeric interface. This approach provides a novel method for developing a better understanding of the structural requirements for gating.

Experimental procedures

Constructs

The GABA_AR constructs were designed based on the published GABA_AR β₃ structure, i.e. substituting the ICD for the amino acid sequence SQPARAA (12) and using the human GABA_AR β₃ sequence (UniProt accession number P28472). The ELIC WT construct (UniProt accession number P0C7B7) was modified for expression in HEK293 cells, adding a Kozak sequence before the cDNA and using the human 5-HT3A subunit signal peptide as described previously (51).

Mutagenesis of GABA_AR β₃ subunit

Genes encoding the human GABA_AR β₃ WT, human GABA_AR β₃ C1, and Erwinia chrysanthemi ELIC WT were ordered from GeneWiz and cloned into pRK5 and pcDNA3.1 vectors. Single point mutations were performed by overlap extension PCR (52). The QuikChange® tool (Agilent) was utilized to design the primers. Multiple template-based sequential PCRs were used to obtain the 5-HT3A signal peptide–ELIC WT chimera (53).

PCR products, mutagenesis reactions, and ligations were verified using agarose gel electrophoresis and DNA sequencing (DNA Sequencing and Services, University of Dundee). The PCR and cloning reagents were bought from Agilent and Thermo Fisher, respectively.

The genes cloned into their respective vectors were used to transform Escherichia coli DH5α cells and grow cultures (500 ml of lysogeny broth medium with 50 μg/ml carbenicillin) at 37 °C overnight. The cells were harvested (6000 × g, 4 °C, 20 min) and used for Maxiprep (Qiagen) to obtain a higher yield of the plasmid.

Cell culture and transfection

HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 μg/ml penicillin, and 100 units/ml streptomycin at 37 °C and 5% CO₂. Cells were seeded at low density in 35-mm dishes for electrophysiology. Transfections were performed by calcium phosphate precipitation using 1 μg of total cDNA per dish as described previously (26). The cDNAs encoding GABA_AR β₃ WT and the mutants were cloned into the pRK5 mammalian expression vector. The cDNA encoding ELIC WT was cloned into the pcDNA3.1 vector. The cDNA that encodes enhanced green fluorescent protein (0.1 μg; in pEGFP vector) was included to identify successfully transfected cells using fluorescence microscopy. Cells were washed with medium 16 h after transfection and used for voltage-clamp electrophysiology after 48–72 h. The tissue culture reagents were obtained from Invitrogen.

Electrophysiology

The whole-cell configuration of the patch-clamp technique was used to record propofol- or GABA-evoked currents from HEK293 cells transiently expressing GABA_AR β₃ WT, GABA_AR β₃ mutants, and ELIC WT. Recording electrodes were fabricated from borosilicate glass capillaries with resistances of 1.2–3.5 megaohms when filled with intracellular solution, which contained 140 mm CsCl, 2 mm MgCl₂, 1.1 mm EGTA, 3 mm Mg-ATP, and 10 mm HEPES (pH 7.4 with CsOH). The extracellular solution contained 140 mm NaCl, 4.7 mm KCl, 1.2 mm MgCl₂, 2.5 mm CaCl₂, 10 mm HEPES, and 10 mm glucose (pH 7.4 with NaOH). The solutions for ELIC WT were different. The intracellular solution contained 140 mm NaCl, 0.5 mm CaCl₂, 5 mm EGTA, and 10 mm HEPES (pH 7.4 with NaOH). The extracellular solution contained 140 mm NaCl, 4.7 mm KCl, 1.2 mm MgCl₂, 0.2 mm CaCl₂, 10 mm HEPES, and 10 mm glucose (pH 7.4 with NaOH).

Cells were voltage-clamped at an electrode potential of −60 mV unless otherwise stated. Currents were evoked by rapid application of GABA or propofol using the three-pipe Perfusion Fast Step system (Warner Instruments) as described previously (26).

The data were recorded using an Axopatch 200B amplifier (Axon Instruments), low pass–filtered at 2 kHz, digitized at 10 kHz using a Digidata 1320 A interface (Molecular Devices), and acquired using pCLAMP8 software (Molecular Devices).

Data analyses

The analyses were carried out using Clampfit 10 (Molecular Devices), Excel 2011 (Microsoft), and Prism 5 (GraphPad). Peak amplitudes were measured using averaged traces from at least three currents. GABA-evoked current amplitudes were expressed as a percentage of the maximum and plotted as a concentration-response relationship. The following logistic (Equation 1) and bell-shaped equations (Equations 2 and 3) were fitted to the data points to determine the Hill slopes (n_H) and the EC₅₀.

$$f\left(\left[\text{GABA}\right]\right)=\frac{\text{Minimum}+\left(\text{Maximum}-\text{Minimum}\right)}{1+{10}^{\left(\log {\text{EC}}_{50}-\left[\text{GABA}\right]\right)\times n\text{H}}}$$ (Eq. 1)

$$\text{Activation}=\frac{\text{Minimum}-\left(\text{Maximum}+\text{Minimum}/2\right)}{1+{10}^{\left(\log {\text{EC}}_{50}-\left[\text{GABA}\right]\right)\times n\text{H}1}}$$ (Eq. 2)

$$\text{Inhibition}=\frac{\text{Minimum}-\left(\text{Maximum}+\text{Minimum}/2\right)}{1+{10}^{\left(\left[\text{GABA}\right]-\log {\text{EC}}_{50}\right)\times n\text{H}2}}$$ (Eq. 3)

Peak current densities were calculated by normalizing the peak current amplitude to the cell capacitance. The potentiation effect of propofol was calculated using the following formula,

$$\%\hspace{0.17em}\text{potentiation}=\frac{\left(I_{\text{pot}}-I_{\text{GABA}}\right)}{I_{\text{GABA}}}\times 100$$ (Eq. 4)

where I_pot and I_GABA represent the potentiated and control peak current amplitudes, respectively. Activation rates were measured using 10–90% rise time of the GABA-evoked current. Deactivation rate was calculated by fitting a double-exponential function to the decay phase of the GABA-evoked current as follows,

$$f\left(t\right)=A_1{e}^{-t/{\tau }_1}+A_2{e}^{-t/{\tau }_2}$$ (Eq. 5)

where τ_n are time constants and A_n represent the proportion of the particular τ. The best-fit number of exponential terms was determined using an F-test with confidence at the 95% level. Deactivation rates were provided as weighted τ values using the following equation.

$$\text{Weighted}\hspace{0.17em}\tau =A_1\times {\tau }_1+A_2\times {\tau }_2$$ (Eq. 6)

Statistical analyses

Data are presented as mean ± S.D. Differences of three or more groups were compared using one-way ANOVA. Subsequent multiple pairwise comparisons were performed using the Dunnett's or Tukey's correction. Student's t test was used for other pairwise comparisons. In all cases, p < 0.05 was considered statistically significant. Statistical analyses were performed in Prism 5.

Comparative modeling

The model for GABA_AR β₃ C1 was generated in Modeller v9.13 (54) using the GABA_AR β₃ structure (Protein Data Bank (PDB) code 4COF) (12) as a template. The proteins share 99% sequence identity according to MUSCLE sequence alignment (55) and thus are suitable for comparative modeling. The best model according to energy, spatial restraints, and stereochemistry was chosen using the Discrete Optimized Protein Energy (DOPE) score (56) and Ramachandran plot (57).

Molecular docking

Molsoft ICM v.3.8-3 (58) was used to perform docking calculations of GABA into the GABA_AR β₃ WT structure (PDB code 4COF) and the GABA_AR β₃ C1 model. The preparation of the receptor and ligand models involved adding hydrogens, calculating charges at pH 7.0, deleting waters, and treating the receptor as rigid and the ligand as flexible. The whole receptor or potentially important residues of the binding site were selected (principal side, Asp⁹⁵–Leu⁹⁹, Leu¹⁵²–Thr¹⁶¹, and Asn¹⁹⁷–Arg²⁰⁷; complementary side, Asn⁴¹–Ala⁴⁵, Met⁶¹–Tyr⁶⁶, Asn¹¹³–Leu¹¹⁸, Leu¹²⁵–Ala¹³⁵, and Ala¹⁷⁴–Val¹⁷⁸), and a box was created around the selection with a 3-Å distance between the residues and the edges. The results were ranked according to the ICM score, which takes into consideration the quality of the complex based on van der Waals interactions and the internal force-field energy of the ligand (58).

Author contributions

C. G. C. and D. T. B.-H. data curation; C. G. C. and D. T. B.-H. software; C. G. C. and D. T. B.-H. formal analysis; C. G. C. and D. T. B.-H. validation; C. G. C. and D. T. B.-H. investigation; C. G. C. and D. T. B.-H. visualization; C. G. C. and D. T. B.-H. methodology; C. G. C. and T. G. H. writing-original draft; D. T. B.-H., W. N. H., and T. G. H. supervision; D. T. B.-H., W. N. H., and T. G. H. writing-review and editing; W. N. H. and T. G. H. conceptualization; W. N. H. and T. G. H. resources; W. N. H. funding acquisition; W. N. H. and T. G. H. project administration.