Unanticipated Structural and Functional Properties of δ-Subunit-containing GABA_A Receptors

Abstract

GABA_A receptors mediate inhibitory neurotransmission in the mammalian brain via synaptic and extrasynaptic receptors. The delta (δ)-subunit-containing receptors are expressed exclusively extra-synaptically and mediate tonic inhibition. In the present study, we were interested in determining the architecture of receptors containing the δ-subunit. To investigate this, we predefined the subunit arrangement by concatenation. We prepared five dual and three triple concatenated subunit constructs. These concatenated dual and triple constructs were used to predefine nine different GABA_A receptor pentamers. These pentamers composed of α₁-, β₃-, and δ-subunits were expressed in Xenopus oocytes and maximal currents elicited in response to 1 mm GABA were determined in the presence and absence of THDOC (3α, 21-dihydroxy-5α-pregnane-20-one). β₃-α₁-δ/α₁-β₃ and β₃-α₁-δ/β₃-α₁ resulted in the expression of large currents in response to GABA. Interestingly, the presence of the neurosteroid THDOC uncovered α₁-β₃-α₁/β₃-δ receptors, additionally. The functional receptors were characterized in detail using the agonist GABA, THDOC, Zn²⁺, and ethanol and their properties were compared with those of non-concatenated α₁β₃ and α₁β₃δ receptors. Each concatenated receptor isoform displayed a specific set of properties, but none of them responded to 30 mm ethanol. We conclude from the investigated receptors that δ can assume multiple positions in the receptor pentamer. The GABA dose-response properties of α₁-β₃-α₁/β₃-δ and β₃-α₁-δ/α₁-β₃ match most closely the properties of non-concatenated α₁β₃δ receptors. Furthermore, we show that the δ-subunit can contribute to the formation of an agonist site in α₁-β₃-α₁/β₃-δ receptors.

γ-Aminobutyric acid type A receptors (GABA_A receptors)2 mediate fast synaptic inhibitory neurotransmission in the mammalian brain. They belong to the family of ligand-gated ion channels that includes nicotinic acetylcholine, glycine, and serotonin type-3 receptors. GABA_A receptors are composed of pentameric combinations of α (1–6), β (1–4), γ (1–3), δ, ε, θ, π subunit subtypes (1–4). The five subunits are arranged pseudosymmetrically around a central Cl^- selective channel (1). Subunit composition confers specific physiological and pharmacological properties to GABA_A receptors (5, 6). The heterogeneity of GABA_A receptors potentially provides different targets for receptor subtype-selective drugs to improve the treatment of insomnia, anxiety, and epilepsy.

Synaptic receptors mediate phasic inhibition whereas extra-synaptic receptors mediate tonic inhibition (7–9). δ-subunit-containing GABA_A receptors occur exclusively extra-synaptically and have been shown to mediate tonic inhibition in dentate gyrus granule cells (10–13), cerebellar granule cells (11), thalamic neurons (14, 15), and in pyramidal neurons (16). The δ-subunit normally shows partnership with either α₄-or α₆-subunits (17, 18). Recently, α₁δ-subunit assemblies have been shown to be present in the hippocampal interneurons (19). GABA_A receptors containing the δ-subunit are activated by persistent and usually nonsaturating ambient GABA concentrations (0.5–1.0 μm) (20).

The homology of the δ-subunit to the β₃- and γ₂-subunits is 50 and 44%, respectively. Studies in various regions of rat brain suggest that δ- and γ₂-subunits do not coexist in the same receptor (21–23). Therefore, δ is generally considered as a substitute of the γ₂-subunit, although one study performed with rat brain extracts suggested that δ- and γ₂-subunits co-assemble to produce a receptor with novel pharmacology (24). αβδ forms functional receptors upon expression in heterologous expression systems (25–39). Several studies have shown that the δ-subunit is part of the GABA_A receptors that exhibit a unique pharmacology. Such receptor isoforms are Zn²⁺-sensitive (25, 26, 29, 36) and benzodiazepine-insensitive (25). It has also been demonstrated that extrasynaptic δ-subunit-containing GABA_A receptors are particularly sensitive to modulation by neurosteroids (30–32, 37–39). There is contradictory evidence on the effect of physiological concentrations of ethanol on δ-subunit-containing GABA_A receptors (32, 34, 35, 38).

In the absence of any method able to determine membrane protein architecture in situ in the nervous system, model systems have to be used. Recently, αβαδβ has been proposed to be the predominant subunit arrangement around the pore when viewed from the extracellular space for α₄β₃δ GABA_A receptors expressed in tsA 201 cells (40). This work was performed at the structural level. In the present study, we have focused on the architecture of α₁β₃δ GABA_A receptors expressed in Xenopus oocytes at the functional level. To investigate active channels, we used covalently linked α₁, β₃, and δ subunits to have a defined arrangement of different subunits in a pentamer (41–46). The concatenated receptors were characterized in detail using the agonist GABA, the neurosteroid THDOC, Zn²⁺, and ethanol, and their properties were compared with those of non-concatenated receptors. We provide evidence for the fact that: (a) the δ-subunit has a promiscuous role in receptor assembly, (b) the δ-subunit can form part of an agonist site, (c) neurosteroids uncover a receptor isoform, and (d) none of the functional receptors is modulated by ethanol.

EXPERIMENTAL PROCEDURES

Construction of Concatenated δ-Subunit-containing cDNAs— The approach used for subunit concatenation of GABA_A receptors has been described previously (41–45). The procedure is detailed here for the dual subunit construct α₁-10-δ (α₁-δ) and the triple subunit construct β₃-23-α₁-10-δ (β₃-α₁-δ). The number between two subunits describes the number of amino acid residues of the introduced synthetic linker. To prepare α₁-δ, the α₁- and δ-subunits were amplified by PCR. The forward primer for α₁ was complementary to the vector sequence preceding the α₁-subunit. The reverse primer was complementary to a PinA1 site followed by the first half of the linker and to the last nucleotides of α₁ before the stop codon. The forward primer for the δ-subunit was complementary to PinA1 site followed by the second half of the linker and the first 18 nucleotides of the sequence of the mature δ-subunit. The reverse primer was complementary to part of the vector sequence following the δ-subunit. The PCR products were purified using the PCR purification kit (Qiagen). The α₁-subunit construct was restricted with EcoRI and PinA1, and the δ-subunit was restricted using PinA1 and XbaI. The vector was restricted with EcoRI and XbaI and dephosphorylated with shrimp alkaline phosphatase (USB). Subsequently, the three fragments were ligated using the rapid DNA ligation kit (Roche Applied Science) to obtain α₁-δ. The other dual constructs with their respective linkers α₁-10-β₃ (α₁-β₃), β₃-23-α₁(β₃-α₁), δ-23-α₁ (δ-α₁), β₃-26-δ (β₃-δ) were prepared similarly where 10, 23, 26 corresponds to Q₄TGQ₄, Q₅A₃PTGQA₃PA₂Q₅, Q₅A₃PTGQ₂AQA₃PA₂Q₅, respectively. The triple subunit construct β₃-α₁-δ was prepared using two dual subunit constructs β₃-α₁ and α₁-δ. First, β₃-α₁ was cut with EcoNI in the α₁-subunit and with XbaI at a site near the 3′-end of the insert (in the vector behind the gene) to yield a fragment of 6.2 kb that contains the sequence of most of the vector, the β₃-subunit, and half of the α₁-subunit. The fragment was dephosphorylated as above. α₁-δ was also cut with EcoNI in the α₁-subunit and with XbaI in the vector behind the δ-subunit to yield a 3.2-kb fragment containing the sequence of half of the α₁-subunit, the δ-subunit and a small part of the vector. These two DNA fragments were ligated. In the same way, the triple constructs α₁-β₃-α₁ and β₃-δ-β₃ were prepared. Also two pentamers β₃-α₁-δ-α₁-β₃ and β₃-α₁-δ-β₃-α₁ were constructed as described in Ref. 45. Site-directed mutagenesis of β₃, Tyr-205 to Ser was done in the constructs β₃-δ and α₁-β₃-α₁ using the QuikChange XL site-directed mutagenesis kit (Stratagene).

Expression in Xenopus Oocytes—Capped cRNAs were synthesized (Ambion, Austin, TX) from the linearized vectors containing different non-concatenated and concatenated subunits. A poly-A tail of about 400 residues was added to each transcript using yeast poly-A polymerase (USB, Cleveland, Ohio). The concentration of the cRNA was quantified on a formaldehyde-agarose gel using Radiant Red stain (Bio-Rad) for visualization of the RNA with known concentrations of RNA ladder (GIBCO Invitrogen) as standard on the same gel. The cRNAs were dissolved in water and stored at -80 °C. Isolation of oocytes from the frogs, culturing of the oocytes, injection of cRNA, and defolliculation were done as described earlier (47). cRNA coding for each dual and triple subunit concatemer was injected either alone or in different combinations in oocytes resulting in a total of seven different concatenated receptors. Oocytes were injected with 50 nl of RNA solution containing each dual or triple subunit construct at 50 nm and pentameric constructs at 100 nm, unless indicated otherwise in Fig. 1. Combinations of α₁-, β₃-, and δ-subunits were expressed at a ratio of 10:10:10 nM or 10:10:50 nm. If the γ₂-subunit is used in place of δ, the latter ratio is required (48). Potentiation of currents by THDOC was similar, but current amplitudes were rather small in the former case (data not shown). Therefore, we used the second condition for detailed characterization. The injected oocytes were incubated in modified Barth's solution (47) at 18 °C for about 72 h for the determination of I_max and for at least 24 h before the measurements for detailed characterization of the functional receptors.

FIGURE 1.

Structure and functional expression in Xenopus oocytes of the GABA_A receptors investigated. The code for the subunits is given on the top line of the table (read subunit sequence of concatenated receptors anti-clockwise). The figure shows subunit composition and current amplitude (nA) evoked by 1 mm GABA in the absence and presence of 1 μm THDOC of non-concatenated and concatenated receptors 3 days after injection with RNA. Mean values with S.E. for each subunit combination are shown. n, number of oocytes.

Two-electrode Voltage Clamp Measurements—All measurements were done in medium containing 90 mm NaCl, 1 mm MgCl₂, 1 mm KCl, 1 mm CaCl₂, and 5 mm HEPES pH 7.4 at a holding potential of -80 mV. For the determination of maximal current amplitudes 1 mm GABA (Fluka) was applied in the absence and presence of 1 μm THDOC (Sigma) for 20 s. THDOC was prepared as a 10 mm stock solution in dimethyl sulfoxide (DMSO) and was dissolved in external solution resulting in a maximal final DMSO concentration of 0.5%. 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 0.4 mm from the surface of the oocyte (5). Non-concatenated and concatenated receptors containing the δ-subunit showed a pronounced decrease in response to GABA with time. This decrease amounted to about 30–70% and did not recover. The experiments were performed after the measured currents became constant. 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 the current amplitude, and n the Hill coefficient.

Relative current potentiation by THDOC was determined as (I_{1 μm THDOC + 1 mM GABA}/I_{1 mm GABA} - 1) × 100%. Inhibition curves for Zn²⁺ were fitted with the equation I(c) = I(0)/(1 + (IC₅₀/c)ⁿ), where I(0) is the control current in the absence of Zn²⁺ standardized to 100%, I is the relative current amplitude, c is the concentration of Zn²⁺, IC₅₀ the concentration of Zn²⁺ causing 50% inhibition of the current, and n the Hill coefficient. Zn²⁺ was pre-applied for a minimum of 1 min prior to co-application of GABA with Zn²⁺. Potentiation by ethanol was determined at EC₂₀ for GABA, using 30 mm ethanol. Relative current potentiation by ethanol was determined as (I_{30 μm ethanol + GABA EC20}/I_{GABA EC20} - 1) × 100%.

Data are given as mean ± S.E. for the I_max values for GABA with and without THDOC and as mean ± S.D. for analysis of properties of receptors using GABA, Zn²⁺, and ethanol. The perfusion system was cleaned between two experiments by washing with 100% dimethyl sulfoxide (DMSO) after application of THDOC and with 10 mm HCl for Zn²⁺ experiments to avoid contamination.

RESULTS

Preparation of Concatenated δ-Subunit-containing GABA_A Receptors—We used the subunit concatenation approach to determine the architecture of δ-subunit-containing GABA_A receptors. We assumed that the δ-subunit would either occupy the position of the γ₂-subunit, one of the two α-subunits, or one of the two β-subunits in the major isoform of GABA_A receptors that is arranged γ₂β₂α₁β₂α₁ counter-clockwise when viewed from the synaptic cleft (41, 42). We also analyzed receptors containing two δ-subunits in the same receptor with δ at γ position and one of the β positions. Five dual and three triple concatenated constructs were prepared to force the δ-subunit into defined positions to form nine different GABA_A receptor pentamers (Fig. 1). For the design of the linkers, we applied the rule that the sum of the predicted C-terminal protrusion of a preceding subunit and the artificial linker has to be minimally 23 residues in length. Shorter linkers do not result in receptor expression (41, 46).

Functional Expression of δ-Subunit-containing GABA_A Receptors—Concatenated receptors R1-R9 (Fig. 1) were expressed in Xenopus oocytes. The non-concatenated subunit combinations α₁δ, β₃δ, α₁β₃, α₁β₃δ and concatenated dual and triple subunit constructs were used as a control. GABA has been shown to be a partial agonist for δ-subunit-containing receptors (31, 32), and the maximal current evoked by GABA could be enhanced by a neurosteroid. Here we estimated receptor expression in the presence of the neurosteroid THDOC. Currents were determined at saturating concentration of GABA (1 mm) in the absence and presence of 1 μm THDOC (Fig. 1). The non-concatenated α₁δ and β₃δ receptors resulted in currents < 10 nA in either case. Both, α₁- and β₃-subunits were required to obtain robust expression of δ-subunit-containing receptors (Fig. 1). Further, non-concatenated α₁β₃ receptors were expressed to compare their properties with those of α₁β₃δ receptors to ensure that δ-subunit was being expressed in the latter receptors. α₁β₃ and α₁β₃δ displayed a different sensitivity toward THDOC. Current potentiation was 6- and 17-fold, respectively. This difference, together with the differential sensitivity to Zn²⁺ (see below), confirms that δ-subunit was indeed being incorporated into α₁β₃δ receptors, although we cannot completely rule out that a subpopulation of α₁β₃ receptors is expressed along with α₁β₃δ. To our surprise and for reasons we can only speculate (see “Discussion”) the concatenated β₃-α₁ construct in the absence and presence of THDOC and the β₃-α₁-δ construct in the presence of THDOC, themselves, resulted in substantial current expression, unlike all the other concatenated subunits (Fig. 1). For the β₃-α₁ construct this functional expression was analyzed further. When 50 nm construct were injected, the current was about 500 nA. Expression of 25 nm of β₃-α₁ resulted in about 30% of this current, and 10 nm gave currents less than 7% of the currents observed using 50 nm of cRNA (Fig. 1). So clearly this artifactual signal was only prominent at higher cRNA concentrations used. Unfortunately, high concentrations of cRNA were required for the δ-subunit-containing receptors to achieve significant expression. To exclude ambiguities in the interpretation of the properties observed for the receptors β₃-α₁-δ/α₁-β₃ (R1) and β₃-α₁-δ/β₃-α₁ (R5), we prepared the β₃-α₁-δ-α₁-β₃ (P1) and β₃-α₁-δ-β₃-α₁ (P5) pentamers. Notably, receptors containing the δ-subunit in different positions resulted in the current expression. The concatenated receptors with the subunit arrangement β₃-α₁-δ/α₁-β₃ (R1), β₃-α₁-δ-α₁-β₃ (P1), α₁-β₃-α₁/β₃-δ (R2), β₃-α₁-δ/β₃-α₁ (R5), pentamer β₃-α₁-δ-β₃-α₁ (P5), and β₃-δ-β₃/β₃-α₁ (R7) resulted in currents >240 nA, whereas α₁-β₃-α₁/δ-α₁ (R3), α₁-β₃-α₁/α₁-δ (R4), β₃-δ-β₃/α₁-β₃ (R6), β₃-α₁-δ/α₁-δ (R8), and β₃-α₁-δ/δ-α₁ (R9) resulted in currents <45 nA on co-application of GABA and THDOC. None of the functional receptors was directly activated by 1 μm THDOC alone (data not shown). With the exception of β₃-α₁-δ/α₁-β₃ (R1), THDOC significantly potentiated the maximal current amplitudes elicited by GABA. α₁-β₃-α₁/β₃-δ (R2) is especially remarkable in this respect as its expression was not strongly evident with GABA alone and was only uncovered in the presence of THDOC. Potentiation of maximal currents by THDOC amounted to about 22-fold in this case.

It was interesting to see if it was possible to place δ in one of the α positions. While β₃-δ-β₃/α₁-β₃ (R6) did not result in current expression, β₃-δ-β₃/β₃-α₁ (R7) resulted in currents of slightly larger amplitude as β₃-α₁. The current showed likewise relatively little stimulation by THDOC. The EC₅₀ was determined as 40 ± 18 μm and the Hill coefficient as 0.8 ± 0.1 (n = 5). Again these parameters are reminiscent of the current mediated by β₃-α₁. Therefore, we assume that R7 is probably not formed, but its existence cannot be fully excluded.

Pharmacological Properties of δ-Subunit-containing GABA_A Receptors—First, the functional receptors were characterized for their response to the natural agonist GABA. Current traces obtained with increasing concentrations of GABA in oocytes expressing β₃-α₁-δ/α₁-β₃ (R1) are shown in Fig. 2A. Averaged GABA concentration-response curves for the concatenated β₃-α₁-δ/α₁-β₃ (R1), β₃-α₁-δ/β₃-α₁ (R5), pentamer β₃-α₁-δ-β₃-α₁ (P5), and non-concatenated α₁β₃δ and α₁β₃ receptors are illustrated in Fig. 2B. The comparison between concatenated and non-concatenated receptors reveals that the corresponding EC₅₀ for concatenated β₃-α₁-δ/α₁-β₃ (R1) receptors is similar to non-concatenated α₁β₃δ receptors, while there was a 13-fold shift to the right for the concatenated pentamer β₃-α₁-δ-β₃-α₁ (P5), with δ at the γ position. A similar shift was observed for β₃-α₁-δ/β₃-α₁ (R5). The GABA dose-response properties of all studied receptors are summarized in Fig. 3.

FIGURE 2.

GABA concentration dependence. A, current traces from a GABA concentration response curve obtained from a Xenopus oocyte expressing β₃-α₁-δ/α₁-β₃ (R1) receptors. Bars indicate the time period of GABA perfusion. GABA concentrations are indicated above the bars. B, averaged GABA concentration response curves of β₃-α₁-δ/α₁-β₃ (R1), β₃-α₁-δ/β₃-α₁ (R5), pentamerβ₃-α₁-δ-β₃-α₁ (P5), α₁β₃δ, andα₁β₃ receptors. Because of overlap, the curve for β₃-α₁-δ-α₁-β₃ (P1) is omitted. Individual curves were first normalized to the observed maximal current amplitude and subsequently averaged. Mean ± S.D. of experiments carried out with 3–4 oocytes from two batches for each subunit combination are shown.

FIGURE 3.

Summary of the data from GABA concentration response curves and Zn²⁺ concentration inhibition curves. n, number of oocytes (from two different batches). A receptor was analyzed in the presence of 1 μm THDOC.

The currents elicited by 1 mm GABA in α₁-β₃-α₁/β₃-δ (R2) receptors were not large enough to determine the EC₅₀ of this receptor. THDOC potentiates the maximal GABA_A receptor currents without changing the GABA EC₅₀ significantly (30).3 Thus, for α₁β₃δ an EC₅₀ of 5.8 μm has been reported in the absence of THDOC and 6.6 μm in its presence (30). Therefore, we investigated α₁-β₃-α₁/β₃-δ (R2) receptors in the presence of 1 μm THDOC with increasing concentrations of GABA. Current traces are shown in Fig. 4A, and averaged results are illustrated in Fig. 4B. The EC₅₀ obtained for α₁-β₃-α₁/β₃-δ receptors was 8.3 ± 2.5 μm comparable to the EC₅₀ of non-concatenated α₁β₃δ receptors, and the Hill coefficient was 1.30 ± 0.13 (n = 4). Fig. 4C shows that already submicromolar concentrations of THDOC strongly stimulate α₁-β₃-α₁/β₃-δ (R2) receptors in the presence of 1 mm GABA.

FIGURE 4.

Properties of wild-type and mutant α₁-β₃-α₁/β₃-δ (R2) receptors. A, current traces from a GABA concentration response curve in the presence of 1 μm THDOC of α₁-β₃-α₁/β₃-δ (R2) receptors expressed in Xenopus oocytes. Bars indicate the time period of GABA perfusion. GABA concentrations are indicated above the bars. B, averaged GABA concentration response curves of α₁-β₃-α₁/β₃-δ (R2), α₁-β_m-α₁/β₃-δ, and α₁-β₃-α₁/β_m-δ receptors. For receptors containing no mutation, individual curves were first normalized to the observed maximal current amplitude and subsequently averaged. For mutated receptors, amplitudes were normalized to the average maximal response elicited by GABA inα₁-β₃-α₁/β₃-δ (R2) receptors. Mean ± S.D. of 3–4 oocytes from two batches for each subunit combination is shown for each experiment. C, averaged THDOC concentration response curve of α₁-β₃-α₁/β₃-δ (R2). Increasing concentrations of THDOC were applied in the presence of 1 mm GABA (mean ± S.D. of 3 oocytes from one batch).

The Hill coefficient >1 of α₁-β₃-α₁/β₃-δ (R2) receptors hinted at the presence of more than one binding site for the agonist GABA in this receptor. As the agonist site is usually located at the β/α interface, this suggests that the δ-subunit contributes part of an agonist site. A point mutation Y205S in the homologous β₂-subunit has been shown to strongly reduce GABA agonist properties of the affected site (43, 49). We introduced the point mutation Y205S into one of the two β₃-subunits individually i.e. α₁-β₃-α₁/β_3(m)-δ and α₁-β_3(m)-α₁/β₃-δ. Fig. 4B shows the dose-response curves in response to increasing concentrations of GABA in the presence of 1 μm THDOC for α₁-β₃-α₁/β₃-δ (R2), α₁-β₃-α₁/β_3(m)-δ, and α₁-β_3(m)-α₁/β₃-δ receptors. Mutant receptor currents were assumed to express equally well as wild-type α₁-β₃-α₁/β₃-δ receptors and were normalized to wild-type receptor current expression. The curves for wild-type and mutated receptors were fitted as has been described previously (43). In each case, currents observed at lower GABA concentrations were drastically reduced as expected for receptors that only gate efficiently upon occupation of both agonist sites (43).

Most of the receptors had a Hill coefficient ≤ 1. This cannot be taken as proof for the absence of a second agonist site in these receptors, because depending on the gating mechanism, the Hill coefficient may underestimate this number.

The concatenated β₃-α₁-δ/α₁-β₃ (R1), α₁-β₃-α₁/β₃-δ (R2), β₃-α₁-δ/β₃-α₁ (R5), pentamer β₃-α₁-δ-β₃-α₁ (P5), and non-concatenated α₁β₃δ and α₁β₃ receptors were investigated for their response to the divalent cation Zn²⁺. All receptors except for α₁-β₃-α₁/β₃-δ (R2) were analyzed in the absence of THDOC. The concentration-dependent inhibition of GABA-induced currents by the antagonist Zn²⁺ is shown in Fig. 5. Zn²⁺ leads to inhibition of currents elicited by GABA with an IC₅₀ in a similar range for the concatenated α₁-β₃-α₁/β₃-δ (R2), β₃-α₁-δ/β₃-α₁ (R5), pentamer β₃-α₁-δ-β₃-α₁ (P5), and non-concatenated α₁β₃δ receptors. The IC₅₀ for Zn²⁺ of β₃-α₁-δ/α₁-β₃ (R1) where δ-replaces the β₃-subunit located between the two α₁-subunits is about 7-fold lower than that for non-concatenated α₁β₃δ receptors. Fig. 3 summarizes the parameters describing the concentration response curves.

FIGURE 5.

Zinc concentration-inhibition curves of β₃-α₁-δ/α₁-β₃ (R1), α₁-β₃-α₁/β₃-δ (R2), β₃-α₁-δ/β₃-α₁ (R5)_, β₃-α₁-δ-β₃-α₁ (P5), α₁β₃δ, and α₁β₃ receptors. Experiments with R2 were carried out in the presence of 1 μm THDOC. Data obtained for inhibition by Zn²⁺ were standardized to the current amplitude elicited by GABA alone in the same oocyte and subsequently averaged from different experiments. Inhibition by Zn²⁺ was determined at EC₅₀ GABA. Mean ± S.D. of 3–4 oocytes from two batches for each subunit combination are shown.

Diazepam is a positive allosteric modulator that acts on receptors containing the γ₂-subunit. As expected β₃-α₁-δ/α₁-β₃ (R1) and β₃-α₁-δ/β₃-α₁ (R5) receptors lacking γ₂-subunit were insensitive to diazepam (data not shown).

Ethanol has been reported to stimulate currents elicited by α₁β₃δ receptors (34). We tested the effect of 30 mm ethanol on non-concatenated α₁β₃δ receptors and concatenated β₃-α₁-δ/α₁-β₃ (R1), α₁-β₃-α₁/β₃-δ (R2), β₃-α₁-δ/β₃-α₁ (R5), and β₃-α₁-δ-β₃-α₁ (P5) receptors. Results are summarized in Fig. 6. We did not observe any potentiation of the currents mediated by these receptors.

FIGURE 6.

Effect of ethanol on currents elicited by GABA. GABA was applied at a concentration eliciting currents amounting to EC₂₀ and ethanol at 30 mm. The figure shows the current stimulation in percent. The receptor was analyzed in the presence of 1 μm THDOC.

DISCUSSION

We investigated the architectural role of the δ-subunit in GABA_A receptor pentamers. Preliminary experiments showed that both α₁- and β₃-subunits were required to form a functional channel together with the δ-subunit. Therefore, we focused on the triple subunit combination α₁β₃δ. Functional expression of non-concatenated α₁β₃δ might theoretically result in multiple subunit arrangements. In this case, subunit concatenation (41–46) is a powerful approach to predefine receptor structure. Two or more subunits can be linked at the DNA level to control subunit composition and arrangement. To construct all possible subunit arrangements and of all studied receptors the pentameric concatenate clearly exceeded our work capacity. As discussed earlier the δ-subunit is thought to be a γ-subunit substitute, although it displays the highest degree of homology to the β-subunit. Nevertheless, we investigated all variants of the major GABA_A receptor isoform γβαβα, where the γ-subunit, one of the α-, or one of the β-subunits was replaced by the δ-subunit.

The δ-Subunit May Assume Several Positions in a Receptor Pentamer—First, we investigated the receptors composed of non-concatenated subunit combinations, concatenated dual and triple subunit constructs alone, or in different combinations for their functional expression. As it is well documented that neurosteroids may profoundly affect δ-subunit-containing receptors, we used THDOC in combination with GABA. In analogy with α₁β₂γ₂ receptors (41, 42) we expected only one single receptor combination to form a functional receptor. To our surprise four different receptor combinations, i.e. β₃-α₁-δ/α₁-β₃ (R1), α₁-β₃-α₁/β₃-δ (R2), β₃-α₁-δ/β₃-α₁ (R5), and β₃-δ-β₃/β₃-α₁ (R7) resulted in expression of sizeable currents. The observations with R7 should be taken with care (see below).

Formation of a δ-Subunit-containing Receptor Silent in the Absence of Neurosteroids—The application of 1 μm THDOC alone did not activate the δ-subunit-containing receptors (not shown), However, upon co-application with GABA, 1 μm THDOC produced a dramatic increase in the current amplitude in most of the non-concatenated and concatenated α₁-, β₃-, and δ-subunit-containing receptors. Potentiation by THDOC for non-concatenated α₁β₃δ receptors was about 17-fold. This is higher than that reported by Zheleznova et al. (39) who reported an about 3-fold potentiation of maximal GABA currents in α₁β₂δ receptors. Potentiation for β₃-α₁-δ/α₁-β₃ (R1), β₃-α₁-δ-α₁-β₃ (P1), α₁-β₃-α₁/β₃-δ (R2), β₃-α₁-δ/β₃-α₁ (R5), and pentamer β₃-α₁-δ-β₃-α₁ (P5) was about 1.3-fold, 5-fold, 22-fold, 5-fold, and 7-fold, respectively. We cannot explain the unusually low potentiation of R1 receptors. Especially interesting was α₁-β₃-α₁/β₃-δ (R2) that showed very small currents in response to GABA alone, but was uncovered upon co-application of 1 μm THDOC. This shows that in the absence of neurosteroid some of the αβδ GABA receptors may remain almost silent and therefore contribute little to inhibition. However, in the presence of THDOC these silent receptors get activated and thereby could exert a profound inhibitory influence on the neuronal activity. The in vivo concentration of neurosteroids in brain has been estimated 3–100 nm (37). At these concentrations we already observed strong potentiation of currents in α₁-β₃-α₁/β₃-δ (R2) receptors (Fig. 4C).

Properties of δ-Subunit-containing Receptors in Response to GABA, Zn²⁺, and Ethanol—The functional properties of the concatenated receptors were compared with those of non-concatenated receptors (Fig. 3). First, the EC₅₀ for channel opening by natural agonist GABA of concatenated and non-concatenated receptors were compared. Results show that concatenated β₃-α₁-δ/α₁-β₃ (R1) and α₁-β₃-α₁/β₃-δ (R2) receptors had a similar sensitivity to GABA as non-concatenated α₁β₃δ receptors, whereas β₃-α₁-δ-β₃-α₁ (P5) showed a 13-fold lower EC₅₀ than non-concatenated α₁β₃δ receptors. Our results obtained for the EC₅₀ of non-concatenated α₁β₃δ receptors expressed in Xenopus oocytes differ from a study by Hanchar et al. (34) that reported an EC₅₀ of 0.56 μm. In two other studies, an EC₅₀ for α₁β₃δ receptors expressed in L929 cells (28) and HEK293T cells (33) was determined to be 3.5 μm and 4.6 μm, respectively.

As mentioned earlier non-concatenated receptors lacking the γ-subunit have a high sensitivity to inhibition by Zn²⁺ (25, 26, 29, 36). Indeed, all the investigated receptors displayed a high sensitivity. α₁-β₃-α₁/β₃-δ (R2), β₃-α₁-δ/β₃-α₁ (R5), and pentamer β₃-α₁-δ-β₃-α₁ (P5) showed a similar sensitivity to inhibition by Zn²⁺ as non-concatenated α₁β₃δ receptors. For unknown reasons β₃-α₁-δ/α₁-β₃ (R1) receptors showed about 7-fold lower IC₅₀ for inhibition by Zn²⁺.

We investigated the effect of a relatively high, but still physiological concentration of ethanol on non-concatenated and concatenated receptors. 30 mm (corresponding to 1.38‰ (w/v) or 1.75‰ (v/v)) ethanol failed to affect currents mediated non-concatenated α₁β₃δ receptors and concatenated β₃-α₁-δ/α₁-β₃ (R1), α₁-β₃-α₁/β₃-δ (R2), β₃-α₁-δ/β₃-α₁ (R5), and β₃-α₁-δ-β₃-α₁ (P5) receptors. This is in contrast to the observation by Hanchar et al. (34) who reported a potentiation by the same concentration of ethanol of α₁β₃δ receptors expressed in Xenopus oocytes amounting to 88%. In principle different receptor pentamers could be formed upon injection of genetic information coding for α₁-, β₃- and δ-subunits, some of them ethanol-sensitive. With subunit concatenation, these receptors may be expressed individually. However, we observed that none of the three candidate receptors was potentiated by ethanol. The reason for these divergent observations is not known. A stimulation of about 160% by 30 mm ethanol of extra-synaptic currents in hippocampal interneurons was also observed (19). This current was attributed to GABA_A receptors containing α₁- and δ-subunits in combination with unknown β-subunits. In the latter case, a receptor-associated protein could be responsible for the observed stimulation.

Assembly of Receptors Following Injection of Individual Dual and Triple Subunit Constructs—Most of the dual and triple subunit constructs when injected alone did not result in current expression, with the exception of concatenated subunits β₃-α₁ and β₃-α₁-δ (Fig. 1). It is not clear whether these constructs are able to form tetramers or hexamers, or whether one of the subunits is hanging out, not being incorporated in the pentamer (46). Expression of β₃-α₁ alone resulted in a current that was potentiated less than 2-fold by THDOC. The current mediated by β₃-α₁-δ/β₃-α₁ (R5), which contains this dual subunit construct was potentiated about 7-fold. Also β₃-α₁-δ differed from β₃-α₁-δ/α₁-β₃ (R1) in this respect. These observations indicate that in the presence of suitable assembly partners, the mis-formation does not take place. To prove this, we constructed the pentamers β₃-α₁-δ/α₁-β₃ (P1) and β₃-α₁-δ-β₃-α₁ (P5). The functional properties of pentameric receptors were found to be similar as for the respective receptors composed of dual and triple subunit constructs with respect to sensitivity for GABA. In summary, we conclude that the assembly pathway is influenced by the co-expressed subunits.

The situation in the case of β₃-δ-β₃/β₃-α₁ (R7) is less clear. This receptor resulted in currents of slightly larger amplitude as β₃-α₁. The current showed likewise relatively little stimulation by THDOC. The EC₅₀ was determined as 40 ± 18 μm, and the Hill coefficient as 0.8 ± 0.1. Again these parameters are reminiscent of the current mediated by β₃-α₁. Therefore, we assume that R7 is probably not formed, but its formation with similar properties as β₃-α₁ cannot be fully excluded.

Abundance of the Different δ-Subunit-containing Receptors— Our functional study on α₁β₃δ GABA_A receptors should be compared with a structural study on α₄β₃δ GABA_A receptors. Using atomic force microscopy Barrera et al. (40) determined stoichiometry and subunit arrangement of these receptors expressed in tsA 201 cells. They showed that αβαδβ counter-clockwise is the predominant subunit arrangement around the pore when viewed from the extracellular space with 21% of the population exhibiting a distinct subunit arrangement of αβαβδ. Only a very small number of receptor entities were analyzed, and these numbers should therefore be taken with care. The above study was done at a structural level, whereas we focused on the function of δ-subunit-containing receptors. If it is assumed that α₁ is similar to α₄, αβαδβ receptors correspond to β₃-α₁-δ(/)β₃-α₁ (R5/P5) and αβαβδ to α₁-β₃-α₁/β₃-δ (R2) in our study. From the present experiments, it is difficult to conclude the relative abundance of the three expressing receptors. Subunit concatenation may affect expression levels. Although the β₃-α₁-δ-β₃-α₁ (P5) receptor with the δ-subunit in the γ-subunit position produces the largest current amplitudes, this receptor has an EC₅₀ for GABA about 12-fold higher than that of non-concatenated α₁β₃δ receptors. Nevertheless, active, non-concatenated α₁β₃δ receptors probably constitute a mixture of β₃-α₁-δ/α₁-β₃ (R1), α₁-β₃-α₁/β₃-δ (R2), and β₃-α₁-δ/β₃-α₁ (R5), where R2 is only active in the presence of neurosteroids. It should be noted that we cannot fully exclude that in addition β₃-δ-β₃/β₃-α₁ (R7) or other subunit arrangements that were not analyzed could also be formed. Evidence for the expression of multiple receptors has been obtained for another δ-subunit-containing receptor, namely α₆β₂δ (50). Taken together, our findings reveal a unique assembly profile for the δ-subunit that resembles that of the ε-subunit (51) with respect to the fact that both subunits can assume multiple positions in a receptor.

Ability of δ-Subunit to Contribute to the Formation of an Agonist Site—α₁-β₃-α₁/β₃-δ (R2) had a Hill coefficient greater than 1, hinting at the presence of more than one agonist site. The major isoform of GABA_A receptors has two different agonist binding sites located both at the interface of the β-and α-subunits (43). Assuming that the binding site is formed at the β₃-δ and β₃-α₁ interfaces in α₁-β₃-α₁/β₃-δ, we introduced a homologous point mutation β₂Y205S (49) into either of the β₃-subunits to disrupt both agonist binding sites selectively. Our results indicate the existence of two agonist sites involving the β₃-δ and β₃-α₁ interfaces in the α₁-β₃-α₁/β₃-δ (R2) receptor. Channel opening also occurs when the receptor is occupied with a single agonist molecule, but is promoted more than 30-fold if occupied by two agonists. Thus, the minus side of the δ-subunit may contribute to an agonist site, but we cannot exclude that the effect of the mutation in the β-subunit is allosterically propagated to the plus side of the δ-subunit (52). Whether or not the δ-subunits assume the role of the α-subunit in the agonist site is not clear from these data. It is however intriguing that the residue α₁F64 crucial in the agonist site of α₁β₂γ receptors (52) is conserved in the homologous position in δ-subunits. Mutation of this residue will clarify the question.

Summary—In summary, we have shown that GABA_A receptors containing the α₁-, β₃-, and δ-subunit have a stoichiometry of 2α₁:2β₃:1δ and that the δ-subunit exhibits the ability to promiscuously assemble into different ethanol-insensitive subunit arrangements at least in the Xenopus oocytes. Further, we show that at least one of these δ-subunit-containing receptors remains silent in the absence of neurosteroid. We have also found that the δ-subunit can contribute to the formation of an agonist site. In the future, it would be interesting to determine how the δ-subunit assembles in the brain. It is possible that the arrangement of δ-subunit-containing receptors in brain is controlled in a region-specific manner.