α subunits in GABA_A receptors are dispensable for GABA and diazepam action

Abstract

The major isoform of the GABA_A receptor is α₁β₂γ₂. The binding sites for the agonist GABA are located at the β₂+/α₁− subunit interfaces and the modulatory site for benzodiazepines at α₁+/γ₂−. In the absence of α₁ subunits, a receptor was formed that was gated by GABA and modulated by diazepam similarly. This indicates that alternative subunits can take over the role of the α₁ subunits. Point mutations were introduced in β₂ or γ₂ subunits at positions homologous to α₁− benzodiazepine binding and GABA binding positions, respectively. From this mutation work we conclude that the site for GABA is located at a β₂+/β₂− subunit interface and that the diazepam site is located at the β₂+/γ₂− subunit interface. Computational docking leads to a structural hypothesis attributing this non-canonical interaction to a binding mode nearly identical with the one at the α₁+/γ₂− interface. Thus, the β₂ subunit can take over the role of the α₁ subunit for the formation of both sites, its minus side for the GABA binding site and its plus side for the diazepam binding site.

Introduction

γ-Aminobutyric acid type A (GABA_A) receptors are the major inhibitory neurotransmitter receptors in the mammalian central nervous system. The GABA_A receptor is a pentameric protein complex, whose subunits are drawn from the following different isoforms: α(1–6), β(1–4), γ(1–3), δ, ε, θ, π and ρ(1–3). The five subunits form a chloride selective ion channel^1–3. The most common isoform of this receptor consists of two α₁, two β₂ and one γ₂ subunit(s)^4–6 arranged α₁γ₂β₂α₁β₂ counterclockwise when viewed from the extracellular space^7–9. These receptors have two agonist GABA binding sites and one benzodiazepine binding site¹⁰. By using in vitro mutagenesis the binding sites for the agonist GABA were located to the β₂+/α₁− subunit interfaces^11,12, and the modulatory site for benzodiazepines was at the α₁+/γ₂− subunit interface¹³. Thus, the α₁ subunit is commonly accepted to contribute to the formation of both sites.

The GABA_A receptors can be activated by the agonist GABA and modulated by many drugs¹⁴. Among these drugs are the benzodiazepines, such as diazepam, that have sedative, anxiolytic, anticonvulsant, hypnotic, and muscle relaxant properties¹⁵. Coexpression of different combinations of recombinant subunits has generated GABA_A receptors with distinct pharmacological and electrophysiological properties.

As early as 1990, we observed that β₂γ₂ GABA_A receptors, lacking the α₁ subunit, were activated by GABA and potentiated by diazepam¹⁶. Later this observation was confirmed by several groups for GABA and diazepam^17–19 or other modulators²⁰. Expression was also documented for β₁γ₂ ^21,22 and β₃γ₂ ^22,23 GABA_A receptors. In the present study, we tried to understand this apparent contradiction and decided to investigate whether alternative GABA and benzodiazepine-binding subunit interfaces exist. Site-directed mutagenesis was combined with two-electrode voltage clamp in Xenopus oocytes. Our findings suggest that the β₂ subunit may replace the α₁ subunit for the formation of either site. We have previously utilized experimentally guided computational docking that led to a diazepam bound structure model at the α₁+/γ₂− interface²⁴. Computational docking at the β₂+/γ₂− interface yielded structural models which strongly suggest that diazepam can interact with this site in a binding mode nearly identical with the one observed at the canonical α₁+/γ₂− site, thus explaining the similar apparent potency.

Results

Functional expression of β₂γ₂ GABA_A receptors in Xenopus oocytes

We initially determined whether varying the subunit ratio led to a different extent of expression of β₂γ₂ GABA_A receptors. We injected β₂ and γ₂ cRNAs at the three ratios 1:1 (1 fMol each/oocyte), 2:1 (2 fMol and 1 fMol/oocyte) and 1:3 (1 fMol and 3 fMol/oocyte) into oocytes and measured the maximum current amplitudes elicited by 10 mM GABA. 5–7 days after microinjection of RNA, the β₂γ₂ GABA_A receptors formed by the 1:3 cRNA injection ratio gave the highest maximal current amplitude (131 ± 19 nA, n = 13). In contrast receptors formed from 1:1 and 2:1 cRNA ratios resulted in current amplitudes less than 100 nA. Thus, we used the 1:3 cRNA ratio coding for wild type or mutant β₂ or γ₂ subunits for all following experiments. Possibly the subunit arrangement is affected by the injection ratio as it has been documented in the case of β₃γ₂ GABA_A receptors²⁵. Oocytes injected with 1 fMol coding for the β₂ subunit only, or with 3 fMol coding for the γ₂ subunit only, both did not result in current expression.

β₂γ₂ receptors respond to GABA

5–7 days after injection, Xenopus oocytes expressing β₂γ₂ GABA_A receptors were investigated for the presence of currents elicited by 10 mM GABA. Figure 1a shows original current traces obtained from oocytes clamped at −80 mV. Figure 1b shows an averaged concentration-response curve for β₂γ₂ GABA_A receptors. The curve was characterized by an EC₅₀ of 75 ± 5 µM and a Hill coefficient of 1.0 ± 0.1 (n = 5). This EC₅₀ is similar to that reported for α₁β₂γ₂, which amounts to 51 ± 15 µM⁸.

Figure 1.

Concentration response curve for GABA at β₂γ₂ GABA_A receptors. Receptors were expressed in Xenopus oocytes and exposed to subsequently higher concentrations of GABA and the elicited current amplitude was determined. Individual curves were first normalized to the fitted maximal current amplitude and subsequently averaged. Data are expressed as mean ± S.E.M., n = 5 from two batches of oocytes. (a) Original current traces. GABA applications are indicated by a bar. The numbers indicate the concentration of GABA in μM. (b) Averaged concentration-response curve for β₂γ₂ GABA_A receptors. The dotted line shows for comparison corresponding data on α₁β₂γ₂ GABA_A receptors.

β₂γ₂ receptors respond to diazepam

Diazepam is a positive allosteric modulator of certain GABA_A receptors enhancing the GABA-induced chloride ion influx. We examined the current potentiation by 1 µM diazepam using a GABA concentration that elicited about 5% of the respective maximal current amplitude. Figure 2a shows original current traces from an experiment were oocytes were exposed to either GABA alone or in combination with increasing concentrations of diazepam. Figure 2b shows an averaged concentration-response curve. The curve was characterized by an EC₅₀ of 69 ± 14 nM and a Hill coefficient of 0.6 ± 0.1 (n = 3). The EC₅₀ is similar to that reported earlier for α₁β₂γ₂ with 92 ± 6 nM⁸, while the Hill coefficient is, for reasons we do not understand, significantly lower than 1. No evidence for a possible receptor heterogeneity that could explain this finding was found (see below). Potentiation by 1 µM diazepam amounted to 216 ± 30% (n = 15).

Figure 2.

Concentration response curve for diazepam at β₂γ₂ GABA_A receptors. Receptors were expressed in Xenopus oocytes and exposed to either GABA alone or GABA in the presence of subsequently higher concentrations of diazepam and the elicited current amplitude was determined. At each concentration of diazepam current potentiation was calculated. Individual curves for potentation were first normalized to the fitted maximal current amplitude and subsequently averaged. Data are expressed as mean ± S.E.M., n = 3 from two batches of oocytes. (a) Original current traces. (b) Averaged concentration-response curve for β₂γ₂ GABA_A receptors. The dotted line shows for comparison corresponding data on α₁β₂γ₂ GABA_A receptors.

Selection of point mutations

Obviously the α₁ subunit is dispensable for the formation of a GABA_A receptor responsive to both channel agonist GABA and diazepam as shown above. We aimed to localize both binding sites in β₂γ₂ receptors. For this purpose we selected some point mutations that have been described to affect either the response to GABA or that to diazepam in α₁β₂γ₂ ^{11,12,26–29} (Table 1). A total of nine different point mutations were introduced into either the β₂ subunit or γ₂ subunit. In the β₂ subunit we generated one mutation at the minus side (β₂Y62L) and four mutations at the plus side (β₂T202A, β₂T202S, β₂Y205S, β₂Y205Q) and in the γ₂ subunit one mutation on the minus side (γ₂F77Y) and three mutations on the plus side (γ₂S217A, γ₂Y220S and γ₂Y220Q). A sequence alignment of the corresponding regions in α₁, β₂ and γ₂ is shown in Fig. 3. Table 1 summarizes the consequences of the mutations in α₁β₂γ₂ receptors. In addition the effect of homologous mutations in other subunits is listed.

Table 1.

Point mutations in β₂ and γ₂ used in this study.

mutation	effect in α1β2γ2	homologous residues in α1 or β2	effect of homologous mutations in α1β2γ2	Lit.
β2Y62L	30-fold decrease in EC50 GABA	α1F64L	200-fold decrease in EC50 GABA	11
	no shift in antagonist apparent affinity		200-fold decrease in antagonist apparent affinity
β2T202S	20-fold decrease in EC50 GABA	(α1T206C)	(5-fold increase in EC50 GABA)	12,27
			(no effect on DZ affinity)
		(α1T206V)	(7-fold decrease in DZ affinity)	28
γ2F77Y	230-fold decrease in DZ affinity			29
	no effect on EC50 GABA
γ2S217A	Not available	(β2T202A)	(drastic loss of EC50 GABA)	12
		(α1T206A)	(3-fold decrease in DZ affinity)	26
γ2Y220Q	Not available	α1Y209Q	see above	26

Point mutations were selected on the basis of previous findings in α₁β₂γ₂ GABA_A. The receptors β₂T202Aγ₂, β₂Y205Sγ₂, β₂γ₂Y220S and β₂Y205Qγ₂ were additionally studied, but were not activated by GABA or etomidate.

Figure 3.

Sequence alignment of α₁, β₂ and γ₂ subunits of the rat GABA_A receptor. Mutated residues of β₂ and γ₂ subunits are indicated with numbers. Homologous positions of the α₁ subunit are also highlighted.

All mutated subunits were expressed in Xenopus oocytes in combination with wild-type subunits to result in β₂γ₂ GABA_A receptors. Functional properties were determined by using two-electrode voltage clamp.

Functional expression of receptors was verified using 50 µM etomidate as agonist since mutations affecting the response to GABA are not very likely to influence the response to this agent. Amino acid residues affecting the latter property have been described to be located within the membrane embedded part of the receptor³⁰. The mutation β₂Y62L interfered very strongly with channel activation by etomidate. As the mutation is located far away from the suspected etomidate site³¹, this indicates that this mutation also affects gating by etomidate (see below). Unfortunately, β₂T202Aγ₂, β₂Y205Sγ₂ and β₂Y205Qγ₂ all resulted in no or very little expression (Table 2), so that these receptors could not be further investigated.

Table 2.

Functional properties of expressed wild type and mutated GAB_AA receptor subunits.

Subunit combination	Etomidate (nA; 50 µM)	GABA (nA; 10 mM)	EC50 (µM)	DZ potentiation (%)	Homology to mutated residues
β2	4 ± 1 (4)	−4 ± 1 (4)	—	—	—
γ2	0 ± 0 (5)	−4 ± 1 (5)	—	—	—
β2γ2	946 ± 8 (4)	131 ± 19 (13)	75 ± 5 (5)	216 ± 30 (15)	—
β2Y62Lγ2	13 ± 3 (11)	53 ± 8 (17)	228 ± 50 (7)	121 ± 12 (5)	α1F64, γ2F77
β2T202Aγ2	8 ± 2 (5)	5 ± 0 (5)	—	—	α1T206, γ2S217
β2T202Sγ2	380 ± 77 (5)	203 ± 18 (5)	3130 ± 260 (4)	100 ± 7 (4)	α1T206, γ2S217
β2Y205Sγ2	0 ± 0 (5)	0 ± 0 (16)	—	—	α1Y209, γ2Y220
β2Y205Qγ2	6 ± 2 (5)	1 ± 0 (4)	—	—	α1Y209, γ2Y220
γ2F77Yβ2	193 ± 25 (4)	148 ± 23 (11)	72 ± 15 (5)	5 ± 2 (5)	α1F64, β2Y62
γ2S217Aβ2	141 ± 31 (5)	100 ± 25 (13)	133 ± 20 (3)	185 ± 23 (4)	α1T206, β2T202
γ2Y220Sβ2	76 ± 17 (5)	10 ± 1 (12)	—	—	α1Y209, β2Y205
γ2Y220Qβ2	777 ± 113 (5)	127 ± 40 (6)	367 ± 139 (3)	127 ± 11 (6)	α1Y209, β2Y205

Individual subunits or subunit combinations were expressed in Xenopus oocytes. Assembly of receptors was verified by determining amplitudes of current responses to 50 µM etomidate. Responses to 10 mM GABA and potentiation by 1 µM diazepam were determined.

Effect of a mutation at the plus side of β₂

β₂T202S was the only investigated point mutation at the plus side of β₂ that did not disrupt β₂γ₂ receptor assembly as evidenced by the large response to etomidate (Table 2). In α₁β₂γ₂ receptors this mutation led to a 20-fold decrease in GABA sensitivity¹². In β₂γ₂ receptors the same mutation led to a 42-fold decrease in GABA sensitivity (Fig. 4, Table 2). The EC₅₀ for GABA dependent channel gating was 3130 ± 260 µM and a Hill coeffient of 0.8 ± 0.1 (n = 4). Potentiation by 1 µM diazepam was not significantly affected by the mutation (Fig. 5, Table 2).

Figure 4.

Influence of different point mutations on the concentration dependence of GABA. Wild type or mutant β₂γ₂ GABA_A receptors were expressed in Xenopus oocytes and exposed to subsequently higher concentrations of GABA and the elicited current amplitude was determined. Individual curves for each subunit combination were first normalized to the fitted maximal current amplitude and subsequently averaged. Averaged concentration-response curve are shown. Data are expressed as mean ± S.E.M., n = 3–7 from two batches of oocytes.

Figure 5.

Influence of different point mutations on the potentiation by diazepam. Receptors were expressed in Xenopus oocytes and first exposed to 7 µM GABA alone or the same concentration of GABA in the presence of 1 µM diazepam and the elicited current amplitude was determined. Current potentiation by diazepam was calculated and averaged for each subunit combination. Data are expressed as mean ± S.E.M., n = 4–15 from two batches of oocytes.

Effect of a mutation at the minus side of β₂

In α₁β₂γ₂ receptors the mutation β₂Y62L leads to a 30-fold decrease in GABA sensitivity with no effect on the antagonist affinity as compared to wild-type α₁β₂γ₂ GABA_A receptors¹¹. The homologous mutation in the α₁ subunit, α₁F64L caused a 200-fold drop in both properties¹¹. Here, we found that combination of β₂Y62L with the γ₂ subunit showed 2-fold smaller current amplitude (Table 2) as compared to wild-type β₂γ₂ receptors, indicating that this mutation might somehow disturb efficient assembly of functional channels and/or that gating is affected. In addition the mutation β₂Y62L led to a 3-fold decrease in the sensitivity for GABA with an EC₅₀ of 228 ± 50 µM and a Hill coefficient of 1.0 ± 0.1 (n = 7) (Fig. 4, Table 2). Again, potentiation by 1 µM diazepam was not significantly affected by the mutation (Fig. 5, Table 2).

The notion that gating is affected by the mutation is strongly supported by the fact that 50 µM etomidate elicits about 4-fold smaller currents than saturating concentrations of GABA (Table 2). In wild type β₂γ₂ receptors this current was about 7-fold larger than the one elicited by GABA. Similarly, in mutated α₁β₂Y62Lγ₂ receptors this current was about 30-fold smaller than the one elicited by GABA. As the site for etomidate is located far away from the mutated residue, this is a strong indication that β₂Y62 is not only involved in binding of GABA, but also in gating.

Effect of mutations at the plus side of γ₂

Among the investigated mutations, γ₂S217A and γ₂Y220Q led to sizeable expression in combination with β₂ and were further studied. To our knowledge nothing is known about both mutations. The homologous mutation to γ₂S217A in the β₂ subunit, β₂T202A, led to a drastic loss in GABA sensitivity¹². The homologous mutation to γ₂Y220Q in the α₁ subunit, α₁Y209Q, disrupted the site for diazepam, while leaving GABA sensitivity unaffected²⁶. This residue was also identified with photoaffinity labeling by the benzodiazepine binding site ligand Ro15–4513³². In β₂γ₂S217A and β₂γ₂Y220Q the sensitivity to GABA was decreased 2-fold and 5-fold, respectively with EC_50s of 133 ± 20 µM (n = 3) and 367 ± 139 µM (n = 3), and Hill coefficients of 0.8 ± 0.1 and 0.7 ± 0.1, respectively (Fig. 4, Table 2). Potentiation by 1 µM diazepam was not significantly affected by both mutations (Fig. 5, Table 2).

Effect of a mutation at the minus side of γ₂

We studied the mutation γ₂F77Y. In α₁β₂γ₂ receptors this mutation abolishes the binding site for diazepam, while leaving GABA sensitivity unaffected²⁹. Similar findings were made in β₂γ₂ receptors. The EC₅₀ for GABA was 72 ± 15 µM (n = 5) and the Hill coefficient 0.9 ± 0.1 (Fig. 4, Table 2). Modulation by 1 µM diazepam was nearly lost (Fig. 5, Table 2).

Summary of the findings

No appreciable currents could be elicited upon expression of β₂ or γ₂ subunits alone and the receptors β₂T202Aγ₂, β₂Y205Sγ₂, β₂γ₂Y220S and β₂Y205Qγ₂. Modulation by diazepam was nearly lost in β₂γ₂F77Y receptors, whereas the reponse to GABA remained unaffected. Activation by GABA was strongly affected in β₂T202Sγ₂ receptors and weakly affected in β₂Y62Lγ₂, β₂γ₂S217A and β₂γ₂Y220Q receptors.

Computational Docking

We performed computational docking of diazepam utilizing a homology model of the β₂+/γ₂− interface based on the β₃ crystal structure 4COF³³ as specified in the Methods section. The overal sequence similarity between β₂+ and α₁+ is high, especially in loops B and C where several aromatic and polar amino acids are conserved. We have shown previously that loop C residues are engaged in key interactions with diazepam^24,26. The docking as specified in the Methods section provided for sidechain flexibility (loops D, G, E, B and C,) as well as a limited degree of backbone flexibility in the loop C tip, very similar to the approach used in our previous docking studies at the canonical high affinity α₁+/γ₂− site²⁴. Computational docking usually generates correct binding poses, but they are not always correctly ranked by the different scoring functions³⁴. We therefore analyzed the top 100 poses of the docking run based on multiple different criteria: Only poses that display interactions with γ₂F77 were considered, to limit poses to those that reflect experimental findings. Poses were then filtered by similarity to the binding mode in the high affinity α₁+/γ₂− site²⁴, where similarity was judged on ligand binding mode and major interactions with the pocket. Lastly, two different scoring functions were employed to identify the best candidate poses based on consensus scoring. Overall, six poses were identified that show high similarity with the high affinity binding mode at α₁+/γ₂−. Among these, two were found in rank one and two positions in the ChemScore ranking, and five were among top 30 ChemScored. Similarly, one of the six candidates was found in the rank two position of the GoldScore Fitness ranking, and a total of three were among the GoldScore top 30. Thus, consensus scoring leads to a binding mode model that features a binding mode very similar to the one that is observed at the canonical high affinity site.

Due to the sterically demanding sidechain β₂F200, we find that sidechain rotamers adjust differently to ligand binding compared to the high affinity pocket, but gross binding mode and key interactions are highly similar (Fig. 6) where one of the representative poses is shown in comparison with the pose depicted in our previous study²⁴.

Figure 6.

Structural hypothesis for diazepam binding at the extracellular β₂+/γ₂ interface. Panel (a) shows the reference binding pose from our previous studies¹⁷ at the α₁+ (orange)/ γ₂− (cyan) interface. Panel (b) shows the most closely corresponding binding pose from the computational docking at the β₂+ (red)/γ₂− (cyan) interface. The homologous key amino acids in the binding pockets, as well as diazepam, are rendered in stick representation. While sidechain rotamers show some differences, ligand position, binding mode and key interations are very similar.

In the proposed binding mode the pendant phenyl ring is in close contact to loop A, which bears in the diazepam sensitive α isoforms a histidine, and in α₄ and α₆ an arginine which is known to interfere with diazepam binding³⁵. Several H101X mutations were investigated in the past, and it was demonstrated that F (Phe), Y (Tyr) or Q (Gln) have only a small impact on flunitrazepam modulation³⁶. The homologous position in β₂ is the hydrophobic Leu99, which is sterically similar to Gln, and as hydrophobic as Phe. Thus, the proposed binding mode is compatible with previous mutagenesis studies and with the present observations.

Discussion

Several early studies reported responsiveness of β₂γ₂ GABA_A receptors expressed in heterologous systems to both GABA and diazepam^16–19 or to GABA in β_xγ₂ GABA_A receptors^20–23. Later, the two binding sites for GABA in α₁β₂γ₂ GABA_A receptors were localized to the two β₂+/α₁− subunit interfaces^11,12 and the diazepam binding site to α₁+/γ₂− subunit interface¹³. Thus, the α₁ subunit seemed to be required for the formation of both sites. The major point of this study was to localize the subunit interfaces that harbor the alternative GABA and benzodiazepine binding sites in β₂γ₂ GABA_A receptors lacking the α₁ subunit.

After having shown that β₂γ₂ GABA_A receptors responded similarly to GABA and diazepam as α₁β₂γ₂ GABA_A receptors, we used point mutations abrogating one of the two in α₁β₂γ₂. Unfortunately, some of the chosen mutations interfered with receptor expression or gating, presumably by negatively affecting assembly or by leading to mainly inactive channels.

The γ₂+/β₂− subunit interface may be excluded for both sites

There are four possible subunit interfaces in β₂γ₂ GABA_A receptors: β₂+/β₂−, β₂+/γ₂−, γ₂+/β₂− and γ₂+/γ₂−. Of these, the γ₂+/β₂− subunit interface also occurs in the α₁β₂γ₂ receptors. Mutation β₂T202A dramatically impaired GABA activation in these receptors¹² and mutation α₁Y209Q led to loss of flumazenil sensitivity²⁶. Obviously the γ₂+/β₂− subunit interface can not take over the formation of both sites. In addition three mutations located at this interface in β₂γ₂ receptors, β₂Y62Lγ₂, γ₂S217Aβ₂ and γ₂Y220Qβ₂, had no strong impact on the responses to GABA or diazepam (Table 2). Taken together, we can exclude that the γ₂+/β₂− subunit interface is the location of GABA and benzodiazepine binding sites.

Localization of the diazepam binding subunit interface

Mutations β₂T202S¹² and γ₂F77Y²⁹ have been described to disrupt GABA and diazepam binding sites in α₁β₂γ₂ receptors, respectively. Mutations β₂T202S and γ₂F77Y similarly strongly affect the response of β₂γ₂ receptors to GABA and diazepam. Mutations β₂T202S and γ₂F77Y had little impact on diazepam and GABA sites, respectively. This implies a role of β₂+ and γ₂− in the formation of GABA site and diazepam site, respectively. Thus, the GABA binding site must be located at β₂+/β₂− or β₂+/γ₂− subunit interfaces and that for diazepam at β₂+/γ₂− or γ₂+/γ₂−.

The mutation α₁Y209Q abrogates the diazepam site in α₁β₂γ₂ receptors²⁶. Therefore, it may be expected that the homologous mutation in the γ₂ subunit, γ₂Y220Q, affects the apparent affinity for diazepam. Similarly, the mutation α₁T206A decreases the affinity for diazepam in α₁β₂γ₂ receptors²⁶. Thus, it may be expected that the homologous mutation in the γ₂ subunit, γ₂S217A, affects the apparent affinity for diazepam. Both mutations failed to affect the response to diazepam, arguing strongly against involvement of the γ₂+ subunit interface in the diazepam site. For these reasons, we locate this site to the β₂+/γ₂− subunit interface (Fig. 7). The failure of the mutation β₂T202S to affect the response to diazepam may be explained by the fact that a homologous, similar mutation α₁T206C did not affect the response to diazepam in α₁β₂γ₂ receptors²⁷. The high affinity interaction of diazepam with the β₂+/γ₂− subunit interface is also supported by the docking experiments (Fig. 6), which strongly suggest that diazepam binds in this non-canonical site in a fashion very similar to the one that is observed in the high affinity site.

Figure 7.

Schematic representation of β₂γ₂ GABA_A receptors. The location of amino acid residues of interest is indicated. Point mutations resulting in disrupting assembly are not shown. The binding site for GABA is concluded to locate at the β₂+/ β₂− interface, that for diazepam at the β₂+/γ₂− interface. Please note that the subunit arrangement was not addressed in this study.

Putative localization of the GABA binding subunit interface

Thus, we are left with the β₂+/β₂− and the γ₂+/γ₂− subunit interfaces. The mutation β₂T202S at the β₂+ has very strong effect on the EC₅₀ for GABA shifting the concentration response curve 42-fold, while effects of mutations at the γ₂+ side are much smaller. Therefore we localize the GABA binding site at the β₂+/β₂− subunit interface (Fig. 7). However, we can not fully exclude an additional site at a γ₂+ side. As β₂+/γ₂− has been excluded, this additional GABA site would have to be at the γ₂+/γ₂− interface. It should be noted that a binding site for GABA has also been described at the β₃+/δ− subunit interface³⁷.

Functional expression

We observed that individual β₂ or γ₂ subunits did not form functional receptors on the surface of oocytes. Within 1 day after injection, the α₁β₂γ₂ receptors expressed currents in µA range³⁸. In contrast, we observed in this work that the β₂γ₂ receptors need longer period for channel expession (5–7 days) and the maximal current amplitudes elicited by 10 mM GABA amount to only 100–200 nA. If silent receptors and different single channel open frequency between receptors are ignored, β₂γ₂ receptors form less efficiently than α₁β₂γ₂ receptors. A previous study described a important role of α₁ subunits for receptor trafficking and assembly in α₁β₂γ₂ receptors³⁹. Thus, in the presence of large amounts of α₁, β₂γ₂ may not be formed. Under special circumstances where α subunit expression is low, the formation of a limited amount of β₂γ₂ receptors may occur. Recent single cell RT-PCR data indicate that cells devoid of mRNA coding for α subunits are not present in the hypothalamus, where diversity of neurons is huge. However, the endocrine system may have receptors without α subunits. Chromaffine cells (at least in certain developmental stages) have only mRNA coding for β₃ and ε subunits (personal communication, I. Adameyko).

While so far it is not considered a candidate receptor to exist in the adult mammalian nervous system, the possible existence of such receptors has also not been specifically excluded. Given that fact that in the developing mammalian brain expression of all three γ isoforms is higher than in the postnatal brain, non-canonical receptor arrangements should be considered. In this vein, it is very important to realize that a high affinity benzodiazepine binding site at the β₂+/γ₂− interface implies that such receptors cannot be distinguished from α+/γ₂− “canonical” receptors in radioligand and PET studies where benzodiazepine ligands are used as presumably selective probes for α+/γ₂− canonical benzodiazepine-sites.

What may be the biological relevance of our observations? Ralvenius et al.⁴⁰ studied mice carrying a point mutation in all those four alpha subunits that can form diazepam sensitive GABA_A receptors. At 10 mg/kg diazepam these mice were completely protected from diazepam-induced muscle relaxation and motor impairment. However, they showed a trend towards reduced locomotor activity that was quite prominent at higher doses. At least part of this response could be due to β₂γ₂ GABA_A receptors. We have not tested whether β₁ or β₃ (that may form β₁γ₂ and β₃γ₂) behave as β₂. As their loop C differs (see Supplementary Fig. S1), it is conceivable that the diazepam site described here does not exist or has different properties in these receptors.

Summary

While in α₁β₂γ₂ receptors diazepam binds to the α₁+/γ₂− subunit interface and GABA to β₂+/α₁−, in β₂γ₂ receptors diazepam binds to the β₂+/γ₂− subunit interface and GABA to β₂+/β₂− (Fig. 7). Thus, the β₂ subunit can take over the role of the α₁ subunit for the formation of both sites, its minus side for the GABA binding site and its plus side for the diazepam binding site.

Methods

Construction of mutated receptor subunits

The point mutations β₂Y62Lγ₂, β₂T202Aγ₂, β₂T202Sγ₂, β₂Y205Sγ₂, β₂Y205Qγ₂, β₂γ₂F77Y, β₂γ₂S217A, β₂γ₂Y220S and β₂γ₂Y220Q were prepared using the QuickChange^TM mutagenesis kit (Stratagene, Agilent Technologies, Basel, Switzerland).

Expression in Xenopus oocytes

Animal experiments were carried out in strict accordance to the Swiss ethical guidelines, and have been approved by the local committee of the Canton Bern Kantonstierarzt, Kantonaler Veterinärdienst Bern (BE85/15). Surgery of Xenopus laevis to obtain the oocytes was done under anesthesia, and all efforts were made to diminish animal suffering. Oocytes were prepared, injected and defolliculated as described previously^41,42. Polyadenylated cRNA coding for the subunits of GABA_A receptors were prepared in vitro with the mMESSAGE mMACHINE kit (Ambion, Austin, TX, USA). Oocytes were injected with 50 nl of solution containing cRNA coding for wild type or mutants β₂ (1 fMol) or γ₂ (3 fMol) subunits and then 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 units/ml penicillin, 100 µg/ml streptomycin) at 18 °C for 5–7 days before measurements.

Functional characterization in Xenopus oocytes

Electrophysiological experiments were performed using an Oocyte Clamp OC-725 (Warner Instrument Corp., Hamden, USA) two-electrode voltage clamp amplifier. Currents were digitized at 5 kHz with MacLab/200 (AD Instruments, Spechbach, Germany).

The holding potential was −80 mV. The perfusion medium contained 90 mM NaCl, 1 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂ and 5 mM Na-HEPES (pH 7.4). 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 about 0.5 mm from the surface of the oocyte. Individual concentration response curves for GABA were fitted with the equation I(c) = I_max/[1 + (EC₅₀/c)ⁿ], where c is the concentration of GABA, EC₅₀ the concentration of GABA eliciting half-maximal current amplitude, I_max is the maximal current amplitude, I is the current amplitude, and n is the Hill coefficient. Maximal current amplitudes (I_max) were obtained from the fits of the concentration-response curves. The individual curves were fitted and standardized to I_max and subsequently averaged. For all receptors studied, potentiation was measured at a GABA concentration eliciting 1–5% of the maximal GABA current amplitude. GABA was applied twice alone for 20 s, and then in combination with diazepam for 20 s. The duration of washout periods was 4 min in between agonist or agonist/drug applications to prevent receptor desensitization. At the beginning of the experiments, GABA applications were repeated when the elicited current amplitude altered by >5%. Potentiation was calculated by the following equation: (I_{Modulator + GABA}/I_GABA−1) * 100%. Concentration dependent potentiation was fitted with the equation I(c) = I_max/[1 + (EC₅₀/c)ⁿ], where c is the concentration of diazepam, EC₅₀ the concentration of diazepam eliciting half-maximal current amplitude, I_max is the maximal current amplitude, I is the current amplitude, and n is the Hill coefficient. Maximal current amplitudes (I_max) were obtained from the fits of the concentration-response curves. The individual curves were fitted and standardized to I_max and subsequently averaged.

All data are from at least two different batches of oocytes. Data represent mean ± S.E.M as indicated in each case. An unpaired t-test was used to compare two means. ***p < 0.001.

Computational Modelling and Docking

Homology models of the β₂+/γ₂− interface were generated based on the 4COF GABA_A receptors human β₃ homopentamer structure²⁴. Due to the high homology of the β₂ and β₃ subunits, no insertions or deletions requiring gaps occur in the extracellular domain, while an alignment of the γ₂ subunit as described previously⁴³ was used to account for the lower homology in the loop F region. Computational docking was subsequently performed using the GOLD software v1.6.2⁴⁴, where the binding site was defined to be at the interface between loops A – G of the subunits. Sidechains β₂Y157, β₂T160, β₂Y159, β₂T161, β₂F200, β₂T202, and β₂Y205 as well as γ₂Y58, γ₂F77 and γ₂T142 were kept flexible, soft potential were applied to the tip of loop C (β₂V198 - β₂G203) to allow some degree of backbone flexibility, default settings for the docking run were used and the top 100 ranked poses were retained for subsequent analysis. Ligand interactions of poses were computed using the MOE program (MOE (The Molecular Operating Environment), Version 2011.10, Chemical Computing Group Inc., Montreal), and poses featuring interactions with γ₂F77 were inspected using two scoring functions (ChemScore Fitness as implemented in GOLD, GoldScore Fitness). “Hits” were defined as poses among top 30 ranked in both scoring functions (consensus score hits) and featuring interactions with γ₂F77 and the resulting hit poses were subsequently compared to our poses from previous work at the canonical binding site²⁴.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Author Contributions

N.W., M.C.M., X.S. and R.B. performed molecular biology and electrophysiological experiments, X.S. and M.E. performed computational studies, N.W. and E.S. designed the experiments, analysed the data and wrote the manuscript.

Competing Interests

The authors declare that they have no competing interests.