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Keywords:

  • GABA;
  • GABAA receptors;
  • receptor isoforms;
  • δ subunit

Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Delta (δ) subunit containing GABAA receptors are expressed extra-synaptically and mediate tonic inhibition. In cerebellar granule cells, they often form a receptor together with α6 subunits. We were interested to determine the architecture of these receptors. We predefined the subunit arrangement of 24 different GABAA receptor pentamers by subunit concatenation. These receptors (composed of α6, β3 and δ subunits) were expressed in Xenopus oocytes and their electrophysiological properties analyzed. Currents elicited in response to GABA were determined in presence and absence of 3α, 21-dihydroxy-5α-pregnan-20-one and to 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol. α636/δ receptors showed a substantial response to GABA alone. Three receptors, β36-δ/α63, α6363-δ and β3-δ-β363, were only uncovered in the combined presence of the neurosteroid 3α, 21-dihydroxy-5α-pregnan-20-one with GABA. All four receptors were activated by 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol. None of the functional receptors was modulated by physiological concentrations (up to 30 mM) of ethanol. GABA concentration response curves indicated that the δ subunit can contribute to the formation of an agonist site. We conclude from the investigated receptors that the δ subunit can assume multiple positions in a receptor pentamer composed of α6, β3 and δ subunits.

Abbreviations:
GABA

γ-aminobutyric acid

GABAA receptor

γ-aminobutyric acid type A receptor

THDOC

3α, 21-dihydroxy-5α-pregnan-20-one

THIP

4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol

GABAA receptors mediate inhibitory neurotransmission in the mammalian brain. They belong to the family of Cys-loop ligand-gated ion channels that includes nicotinic acetylcholine, glycine and serotonin type-3 receptors. GABAA receptors are composed of combinations of α (1–6), β (1–4), γ (1–3), δ, ε, θ, π subunits (Macdonald and Olsen 1994; Rabow et al. 1995; Barnard et al. 1998; Whiting et al. 1999). The five subunits are arranged pseudo-symmetrically around a central Cl− selective channel (Macdonald and Olsen 1994). Subunit composition confers specific physiological and pharmacological properties to GABAA receptors (Sigel et al. 1990; Sieghart and Sperk 2002).

Synaptic receptors mediate phasic inhibition whereas extra-synaptic receptors mediate tonic inhibition (Stell and Mody 2002; Mody and Pearce 2004;Farrant and Nusser 2005). δ subunit containing GABAA receptors occur exclusively extra-synaptically and have been shown to mediate tonic inhibition in many brain areas (for review, see Farrant and Nusser 2005) among them cerebellar granule cells (Stell et al. 2003). The δ subunit has assumed to be co-assembled with either α4 or α6 subunits (Jones et al. 1997; Sur et al. 1999), but recently, α1δ subunit assemblies have been demonstrated in hippocampal interneurons (Glykys et al. 2007).

Studies in various regions of rat brain suggest that δ and γ2 subunits do not coexist in the same receptor (Quirk et al. 1995; Araujo et al. 1998; Jechlinger et al. 1998). Therefore, δ is generally considered as a substitute of the γ2 subunit, although one paper claimed that δ and γ2 subunits co-assemble (Mertens et al. 1993). αβδ forms functional receptors upon expression in heterologous expression systems. Functional properties of δ subunit containing receptors and their sensitivity to neurosteroids have been characterized (Saxena and Macdonald 1994;Saxena and Macdonald 1996; Zheleznova et al. 2008). The neurosteroid 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP) elicits larger currents than GABA in α4/6β3 and α4/6β3δ receptors (Storustovu and Ebert 2006), indicating that GABA acts as a partial agonist at these receptors. δ subunit containing GABAA receptors have been implied in altered seizure susceptibility and anxiety during ovarian cycle (Maguire et al. 2005) and in postpartal depression (Maguire and Mody 2008).

α6 subunits are exclusively expressed in cerebellar granule cells (Sieghart and Sperk 2002). By immunogold staining, this subunit has been shown to be concentrated at Golgi synapses and at mossy fiber synapses and at a lower density in the extra-synaptic membrane (Nusser et al. 1996). In contrast, the δ subunit has been found exclusively in extrasynaptic locations, in the soma and on dendritic membranes (Nusser et al. 1998). Thus, both α6 and δ have been found in extrasynaptic membranes. In whole cerebellum, GABAA receptor subtypes have been quantified using sequential immunaffinity adsorption (Pöltl et al. 2003). The receptor composed of α6, β2/3 and δ subunits has been estimated to constitute 11% and 18% of all receptors, in rat and mouse cerebellum, respectively. As the α6 subunit is exclusively expressed in granule cells, the percentage of α6βxδ receptors is substantially higher in these cells. α6 less mice display a substantially reduced expression of δ (Jones et al. 1997), suggesting a direct association of these two subunits. This was actually demonstrated with co-immunoprecipitation (Khan et al. 1996).

It is unfortunately not possible to determine membrane protein architecture in situ in the neurons. Thus, model systems have to be used. In the present study, we have focused on the architecture of α6β3δ GABAA receptors expressed in Xenopus oocytes at the functional level. To investigate active channels we used covalently linked α6, β3 and δ subunits to have a defined arrangement of different subunits in a pentamer (Minier and Sigel 2004a). The concatenated receptors were characterized in detail using the agonist GABA, the neurosteroid 3α, 21-Dihydroxy-5α-pregnan-20-one (THDOC) and THIP and their properties were compared with those of non-concatenated receptors. In the present study, we provide evidence for the facts that (a) the δ subunit can assume different positions in the α6β3δ receptor pentamer, similarly to the previously described α1β3δ GABAA receptors (Kaur et al. 2009), (b) that the presence of neurosteroids strongly enhances or even uncovers currents mediated by functional receptors, and (c) that ethanol fails to modulate these receptors.

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Construction of concatenated δ subunit containing cDNAs

We thank Dr Lüddens for the cDNA coding for the rat δ subunit. The approach used for subunit concatenation of GABAA receptors has been described previously (Baumann et al. 2001, 2002, 2003; Minier and Sigel 2004b; Baur et al. 2006). We prepared the dual constructs α6-11-β3, α6-11-δ, β3-26-α6, β3-23-δ, δ-26-α6 and δ-26-β3, and the triple subunit constructs β3-23-δ-26-β3, β3-26-α6-11-δ and α6-11-β3-26-α6. 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 (Baumann et al. 2001, 2002). The linkers were Q5TGQ4 for α63 and α6-δ, Q5A3PTGQ2AQA3PA2Q5 for β36, δ-α6 and δ-β3, and Q5A3PTGQA3PA2Q5 for β3-δ and δ-β3.

Expression in Xenopus oocytes

Capped cRNAs were synthesized (Ambion, Austin, TX, USA) 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, OH, USA). The concentration of the cRNA was quantified on a formaldehyde agarose gel using Radiant Red stain (Biorad, Reinach, Switzerland) for visualization of the RNA with known concentrations of RNA ladder (Invitrogen, Basel, Switzerland) 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 (Sigel 1987). cRNA coding for each dual and triple subunit concatemer was injected either alone or in different combinations in oocytes resulting in a total of six different concatenated receptors. Oocytes were injected with 50 nL RNA solution containing each construct at 50 nM. Combinations of α6, β3 and δ subunits were expressed at a ratio of 50 : 50 : 50 nM or at a ratio of 10 : 10 : 50 nM. If the γ2 subunit is used in place of δ, the latter ratio is required (Boileau et al. 2002). Therefore, we used the second condition for the detailed characterization of the receptors. The injected oocytes were incubated in modified Barth’s solution (Sigel 1987) at 18°C for about 72 h for the determination of Imax and for at least 24 h before the measurements for the detailed characterization of the functional receptors.

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, Buchs, Switzerland) was applied in the absence and presence of 1 μM THDOC (Sigma, Buchs, Switzerland) for 20 s. THDOC was prepared as a 10 mM stock solution in dimethylsulfoxide (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 (Sigel et al. 1990). 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) = Imax/(1 + (EC50/c)n), where c is the concentration of GABA, EC50 the concentration of GABA eliciting half maximal current amplitude, Imax is the maximal current amplitude, I the current amplitude and n the Hill coefficient.

The current response to 1 μM THDOC, 100 μM THIP and stimulation of GABA currents by THDOC were determined on independent oocytes. Relative current potentiation by THDOC was determined as (I1 μM THDOC + 1 mM GABA/I1 mM GABA– 1) × 100%. Relative current potentation by 7.5 nM, 15 mM, 30 mM and 45 mM ethanol was determined similarly at a GABA concentration eliciting EC20. Where indicated 45 mM ethanol was pre-applied for a period of 30 s before addition of GABA.

Data are given as mean ± SEM for the Imax values for GABA with and without THDOC and as mean ± SD for analysis of properties of receptors using GABA. The perfusion system was cleaned between two experiments by washing with 100% DMSO after application of THDOC experiments to avoid contamination.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Preparation of concatenated δ subunit containing GABAA receptors

The subunit arrangement of α1β2γ2 GABAA receptors has been determined to be γ2β2α1β2α1 counter-clockwise when viewed from the synaptic cleft (Baumann et al. 2001, 2002; Baur et al. 2006). We assume that in α6β3γ2 receptors α6 assumes the position of α1. In a first approach, we investigated receptors R1–R5 where the δ subunit was forced either into the position of the γ2 subunit, or of one of the two α subunits, or of one of the two β subunits (Fig. 1b). In the latter receptors where δ occupies either the α or β position, γ was replaced by the β3 subunit (Baumann et al. 2001). This was achieved by subunit concatenation. In a second approach, we used the concatenated subunits to form 19 additional subunit configurations (Fig. 1c). Thus, six dual and three triple subunit constructs were prepared to form in total 24 different GABAA receptor pentamers. It would be desirable to construct concatenated pentamers (Baur et al. 2006) for all theoretically possible receptors. For reasons of time this was not done.

image

Figure 1.  (a–c) Structure, functional expression and pharmacological properties of the GABAA receptors investigated in Xenopus oocytes. The code for the subunits is given at the top line of the figures (read subunit sequence of concatenated receptors anti-clockwise). The figures shows subunit composition and current amplitude (nA) evoked by 1 mM GABA in the absence and presence of 1 μM THDOC, the EC50 for GABA, the current amplitude elicited by 100 μM THIP and by 1 μM THDOC of non-concatenated and concatenated receptors. Mean values with SEM for each subunit combination are shown. n, number of oocytes;–, not analyzed. (a) Non-concatenated receptors and individual non-concatenated and concatenated subunits, (b) the δ subunit was forced either into the position of the γ2 subunit, or of one of the two α subunits, or of one of the two β subunits, (c) concatenated constructs were combined with either other available concatenated constructs or loose subunits to form 19 additional receptors. aspontaneous currents amounting to about 1 μA were observed in the absence of GABA. bexperiments were performed in the presence of 1 μM THDOC. cα6363-δ (R2) receptors additionally displayed a low affinity component with 410 ± 260 μM (n = 3) GABA, amounting to about 40% of the total current. dβ3-δ-β363 (R3) receptors additionally displayed a low affinity component with 510 ± 290 μM (n = 3) GABA, amounting to about 14% of the total current. eThis subunit configuration might also assemble into β3-δ-β36 or β3-δ-β3/δ receptors. fThis subunit configuration might also assemble into β3-δ-β33 and β3-δ-β3/δ receptors. gThis subunit configuration might also assemble into α6366 and α636/δ receptors.

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Functional expression of δ subunit containing GABAA receptors

First, non-concatenated receptors were investigated. Injection of oocytes with 0.5 fmol poly(A+)RNA/subunit per Xenopus oocyte resulted in the expression of only small current amplitudes for α6β3δ receptors (not shown). Therefore, we increased the amount to 2.5 fmol/subunit. This increase resulted in a spontaneous current that was sensitive to picrotoxin (not shown). We then lowered the amounts of the RNAs coding for α6 and β3, respectively to 0.5 fmol for α6β3δ receptors and did not observe the spontaneous current anymore. All the other subunits and multi-subunit constructs were expressed at 2.5 fmol/subunit.

GABA has been shown to be a partial agonist for δ subunit containing receptors (Bianchi and Macdonald 2003; Wallner et al. 2003; Kaur et al. 2009) and the maximal current evoked by GABA could be enhanced by a neurosteroid. Therefore, currents were determined at saturating concentration of GABA (1 mM) in the absence and presence of 1 μM THDOC (Fig. 1a). δ, α6δ and β3δ receptors all resulted in currents <10 nA. Under our conditions, β3 did not form to a larger degree spontaneously open homomeric channels as evidenced by a normal membrane resistance. Both, α6 and β3 subunits were required to obtain robust expression of δ subunit containing receptors (Fig. 1a). Further, non-concatenated α6β3 receptors were expressed to compare their properties with those of non-concatenated α6β3δ receptors in order to ensure that δ subunit was being expressed in the latter receptors. The presence of the δ subunit enhanced the expressed current amplitudes significantly (Fig. 1a). This difference confirms that δ subunit was indeed being incorporated into α6β3δ receptors, although we cannot rule out that a sub-population of α6β3 receptors is expressed along with α6β3δ.

Some concatenated dual and triple subunit constructs expressed at high RNA concentration have been shown to result in current by themselves (Kaur et al. 2009). In the present study, only one of the seven constructs, namely α63 resulted in a small currents (Fig. 1a).

Second, concatenated receptors R1–R5 (Fig. 1b) were expressed in Xenopus oocytes. Receptors containing the δ subunit in different positions resulted in current expression. The concatenated receptors with the subunit arrangement β36-δ/α63 (R1), α6–β363-δ (R2) and β3-δ-β363 (R3) resulted in currents >300 nA, whereas β3-δ-β336 (R4) and β36-δ/β36 (R5) resulted in currents <20 nA on co-application of GABA and THDOC. None of the functional receptors was directly activated by 1 μM THDOC alone (Fig. 1b). In all of the cases where we found evidence for functional expression, GABA, in the absence of THDOC, elicited only small current responses (Fig. 1b).

Of the 19 additional subunit configurations (Fig. 1c) we investigated, only one receptor, namely α636/δ, in robust current expression (>200 nA) on co-application of GABA and THDOC. A significant current could also be activated by 1 mM GABA alone (Fig. 1c). Two more receptors, β3-δ-β36/δ and α6-δ/β3, resulted in the expression of smaller currents amounting to 100–200 nA. They were not further characterized.

Pharmacological properties of δ subunit containing GABAA receptors

The receptors that resulted in functional expression were characterized in detail. Current traces elicited in β36-δ/α63 (R1) receptors are illustrated in Fig. 2. While 1 mM GABA (Fig. 2a, b, c) or 1 μM THDOC (Fig. 2a) elicited only small current amplitudes, combination of 1 mM GABA with 1 μM THDOC (Fig. 2c) elicited large currents, thus showing that GABA acts as weak partial agonist at these channels. 100 μM THIP itself elicited much larger currents than GABA alone, but elicited smaller currents than the combination GABA/THDOC (Fig. 2b).

image

Figure 2.  Current traces recorded from oocytes expressing β36-δ/α63 (R1) receptors (a) Current traces recorded upon application of 1 μM THDOC followed by 1 mM GABA; (b) Current traces recorded upon application of 1 mM GABA followed by 100 μM THIP; (c) Current traces recorded upon application of 1 mM GABA followed by co-application of 1 mM GABA/1 μM THDOC. The bars indicate the time period of drug perfusion.

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100 μM THIP has previously been reported (Storustovu and Ebert 2006) to elicit about three-fold higher currents than maximal concentrations of GABA in non-concatenated α6β3δ receptors. In the latter case, RNAs coding for the subunits were injected at 32 ng : 3.2 ng : 32 ng/oocyte for α63:δ. In the present case 0.4 : 0.4 : 2 ng were used. The resulting receptors had similar properties in this respect.

The currents elicited by 1 mM GABA in R1, R2 and R3 receptors were not large enough to determine the EC50. THDOC potentiates the maximal GABAA receptor currents without affecting the GABA EC50 greatly (Wohlfarth et al. 2002; see also Fig. 3c). Therefore, we challenged these receptors in the presence of 1 μM THDOC with increasing concentrations of GABA. Current traces obtained from in oocytes expressing β36-δ/α63 (R1) are shown in Fig. 3a. Averaged GABA concentration–response curves for the concatenated β36-δ/α63 (R1), α6363-δ (R2) and β3-δ-β363 (R3) and non-concatenated α6β3δ receptors are illustrated in Fig. 3b, and for α636/δ receptors in Fig. 3c. As the latter receptor also resulted in sizeable currents in the absence of THDOC, the concentration response curve for GABA was also investigated in its absence. The concentration response curve for α636/δ was shifted about two-fold to the right in absence of THDOC (Fig. 3c). Respective EC50 values and Hill coefficients are summarized in Fig. 1b and c. In order to uncover possible low affinity components, the receptors were also investigated at GABA concentrations up to 10 mM concentration. While α636/δ receptors were characterized by a mono-phasic GABA concentration–response curve, α6363-δ (R2) receptors displayed a bi-phasic curve, the low affinity component amounting to about 40% of the maximal current amplitude (Fig. 3c). Thus, replacement of the β3 subunit adjacent to the δ subunit by a second δ subunit in α6363-δ (R2) receptors changes a bi-phasic to a mono-phasic concentration response curve. β36-δ/α63 (R1) and non-concatenated α6β3δ receptors showed a mono-phasic curve (not shown). β3-δ-β363 (R3) receptors additionally resulted in a bi-phasic behavior (not shown), the low affinity component amounting to about 14% of the maximal current amplitude.

image

Figure 3.  GABA concentration dependence. (a) Current traces from a GABA concentration response curve obtained from a Xenopus oocyte expressing β36-δ/α63 (R1) receptors. The bars indicate the time period of GABA/1 μM THDOC perfusion. GABA concentrations are indicated above the bars. (b) Averaged GABA concentration response curves of β36-δ/α63 (R1), α6363-δ (R2), β3-δ-β363 (R3) and α6β3δ receptors. Individual curves were first normalized to the observed maximal current amplitude and subsequently averaged. Concatenated receptors were analyzed in the presence of 1 μM THDOC. Mean ± SD of experiments carried out with 3–4 oocytes from two batches for each subunit combination is shown. (c) same as (b) for α6363-δ (R2) and α636/δ receptors except that the ratio between two subsequent GABA concentrations was 10-fold and the highest concentration of GABA was 10 mM to show the presence of low affinity components in α6363-δ. The concentration response curve for α636/δ receptors was also carried out in the absence of THDOC (open symbols).

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Figure 4 summarizes the effects of co-application 7.5 mM, 15 mM, 30 mM or 45 mM ethanol and 30 s pre-application of 45 mM ethanol before addition of GABA on the functional receptors. All measurements were carried out in the absence of THDOC at a GABA concentration eliciting EC20. Neither currents mediated by non-concatenated α6β3δ receptors nor currents mediated by the concatenated β36-δ/α63 (R1), α6363-δ (R2), β3-δ-β363 (R3) and α636/δ receptors showed any significant modulation. Similar results were obtained in the presence of THDOC using co-application of 30 mM ethanol (not shown).

image

Figure 4.  Lack of an effect by physiological concentrations of ethanol. GABAA receptors were expressed in Xenopus oocytes. The receptors were activated by a concentration of GABA eliciting EC20, followed by applications of the same concentration of GABA in combination with subsequently 7.5, 15, 30 and 45 mM ethanol. In some cases, only 30 mM ethanol were applied. The relative current amplitude of the responses in the presence of ethanol as compared to GABA alone is given. Data in the right column were obtained with 45 mM ethanol including a 30 s pre-application of ethanol before the combined application of GABA with ethanol. The experiments were carried out in the absence of THDOC. The only values that reach levels of statistical significance are the 45 mM values for α636/δ receptors.

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Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Our aim was to determine the architecture of α6β3δ GABAA receptors. Functional expression of non-concatenated α6β3δ might result in the formation of receptors with multiple subunit arrangements. In order to predefine the subunit arrangement, we used subunit concatenation (Baumann et al. 2001, 2002, 2003; Minier and Sigel 2004a; Baur et al. 2006). To construct all possible subunit arrangements as pentamers clearly exceeded our work capacity. Therefore, we chose to investigate all variants of the major GABAA receptor isoform γβαβα, where one of the α, or one of the β subunits or the γ subunit was replaced by the δ subunit. If a α or a β subunit was substituted, the γ subunit position was occupied by a β subunit, as is the case in αβ receptors. In addition, we used the constructs to form 19 alternative receptors.

Four of the δ subunit containing receptors studied show functional expression

First we analyzed the receptor in which the δ subunit was placed in all five possible positions in a pentamer composed of the consensus arrangement of α and β subunits. All receptors responded with current amplitudes <50 nA to the exposure of 1 mM GABA. If 1 μM of the neurosteroid THDOC was added to GABA, β36-δ/α63 (R1), α6363-δ (R2) and β3-δ-β363 (R3) receptors produced currents >300 nA (Fig. 1b). 1 μM THDOC alone elicited currents <10 nA in these receptors. 100 μM THIP resulted in much larger currents than observed on application of GABA, but this current amounted to only 25–40% of that induced by GABA + THDOC.

Secondly, we combined the concatenated constructs either with other available concatenated constructs or loose subunits to form 19 additional receptors. Only one of them, α636/δ, resulted in the expression of currents amounting to >200 nA. Interestingly this receptor was also activated by GABA alone, to an extent amounting to about 12%.

The expression of the concatenated dual subunit construct α63 alone resulted in about 180 nA current upon application of GABA + THDOC, but only in about 30 nA on application of THIP. It is not clear whether this construct is able to form tetramers or hexamers, or whether one of the subunits is hanging out, not being incorporated in the pentamer (Minier and Sigel 2004a). β36-δ/α63 (R1) and β3-δ-β363 (R3) receptors contain this dual subunit construct, but respond with 19- and fold-fold lager currents on application of THIP. In addition, the KD to GABA was significantly smaller (p < 0.025) in β36-δ/α63 (R1), and β3-δ-β363 (R3) receptors as compared with the dual subunit construct (Fig. 1a and b). Therefore, we conclude that these two receptors do not represent an artifact but are really formed. However, we cannot exclude that a fraction of current expressed from β3-δ-β363 (R3) receptors is due to α63.

Thus, the δ subunit can occupy both positions occupied by β subunits, the position of the α subunit between the two β subunits or both positions corresponding to the γ and the adjacent β subunit in the major adult isoform receptors, corresponding to γβαβα. The fact that the δ subunit can assume different positions is in contrast to our observations with the γ subunit that exclusively can only adopt a single specified position (Baumann et al. 2001, 2002).

The δ subunit contributes to the formation of an agonist site

The GABA concentration response curve for α6363-δ (R2) receptors is clearly bi-phasic (Fig. 3c). Replacement of the β3 subunit adjacent to the δ subunit by a δ subunit to form α636/δ receptors converts this bi-phasic curve into a mono-phasic curve with a Hill coefficient <1. The concentration response curve for α6363-δ (R2) receptors was carried out in the presence of THDOC. It could be argued that THDOC is responsible for the low affinity component. Therefore, we carried out the experiment with α636/δ receptors in the presence and absence of THDOC. In both cases, the Hill coefficient was <1. The presence of THDOC shifted the curve about two-fold to the left without resulting in a low affinity phase. Thus, at least in this receptor, THDOC does not induce a second phase.

Previously, we have presented evidence for the presence at the β3/δ subunit interface in α1313-δ receptors of an GABA agonist site (Kaur et al. 2009). The present observation strongly supports the proposition that the δ subunit is able to assume the role of the α subunit in an agonist site that is normally formed at the β/α subunit interface. While this agonist site has an EC50 of about 8 μM in α1313-δ receptors, its EC50 in α6363-δ (R2) is about 400 μM. Please note that the EC50 does not reflect the affinity of GABA to the site.

Comparison to currents mediated by non-concatenated α6β3δ receptors

It has previously been observed that the properties of α6β3δ receptors depend on the expression conditions, i.e., on the amount and the ratio of mRNA coding for the different subunits that is injected into Xenopus oocytes (Hadley and Amin 2007). Under certain expression conditions, bi-phasic GABA concentration response curves were obtained, supporting further a heterogeneity of expressed receptors. We also noticed here that injection of 2.5 fmol: 2.5 fmol: 2.5 fmol mRNA coding for α6, β3 and δ, respectively, resulted in spontaneous current in the absence of GABA and in current amounting to about 1100 nA upon application of 1 μM THDOC. In contrast, injection of 0.5 fmol : 0.5 fmol : 2.5 fmol mRNA did not result in spontaneous current and the induced current amounted to only about 50 nA on application of 1 μM THDOC. All these observations indicate that different expression conditions results in the formation of different receptors.

Thus, any comparison of non-concatenated with concatenated receptors should be done carefully. In spite of this difficulty, the properties of the non-concatenated receptors observed here were compared with those of concatenated β36-δ/α63 (R1), α6363-δ (R2), β3-δ-β363 (R3) and α636/δ receptors. Concatenated receptors resulted in lower current amplitudes elicited by 1 mM GABA than non-concatenated receptors. This phenomenon may be due to contamination of α6β3δ receptors by α6β3, and/or due to the additional formation of a receptor with a subunit arrangement not studied here. The dose–response curves for all concatenated receptors in respect to the major components were in a similar range as the one obtained for non-concatenated α6β3δ receptors. A low affinity component as observed in α6363-δ (R2) and β3-δ-β363 (R3) receptors was not evident in non-concatenated α6β3δ receptors, as expressed under our conditions. Two factors may contribute to this absence. First, properties of non-concatenated receptors depend strongly on the expression conditions and we may be chosen conditions that do not result in expression of low affinity receptors. Second, assuming equal contribution to the current of all four successfully expressed receptor configurations, the low affinity component would amount to only 14% of the total current. 100 μM THIP elicited similar current amplitudes as 1 mM GABA/1 μM THDOC in non-concatenated receptors. In concatenated receptors β36-δ/α63 (R1), α6363-δ (R2), β3-δ-β363 (R3) and α636/δ receptors, 100 μM THIP elicited 20–40% of the current elicited by 1 mM GABA/1 μM THDOC. This might be due to a small shift to the right in the THIP dose–response curves in concatenated receptors.

Ethanol action

Controversial observations have been reported on the action of physiological concentrations of ethanol on α6β3δ receptors. While one group reported positive allosteric modulation (Wallner et al. 2003), a consortium of several groups was unable to reproduce the findings (Borghese et al. 2006). It is well documented that injection of the subunits in different ratios affects the expressed current properties (Hadley and Amin 2007, this paper). In principle, the observed discrepancy in ethanol effects could be due to formation of different receptor pentamers in different laboratories upon injection of genetic information coding for α6, β3 and δ subunits. With subunit concatenation, these different types of receptors can be expressed individually. However, we observed that none of the four receptors β36-δ/α63 (R1), α6363-δ (R2), β3-δ-β363 (R3) and α636/δ was modulated by ethanol in the concentration range of 7.5–30 mM. The latter concentration corresponds to 1.38‰ (w/v). We cannot fully exclude formation of an ethanol sensitive receptor configuration not studied here, but we consider this unlikely.

Relative abundance of the different δ subunit containing receptors

Our results on a functional study on α6β3δ GABAA receptors should be compared with a structural study on α4β3δ GABAA receptors. Using atomic force microscopy Barrera et al. (2008) determined stoichiometry and subunit arrangement of receptors expressed in tsA 201 cells. They showed that αβαδβ is the predominant subunit arrangement around the pore when viewed from the extra-cellular 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. In addition, unlike in other atomic force microscopical studies, the receptors were depicted here as spots and not as expected as rings with a central hole (Müller et al. 2002) and antibodies as spots and not as heart-shaped entities (Fritz et al. 1997), respectively. The above study was done at a structural level including receptors retained in the endoplasmic reticulum, whereas we focused on the channel function of δ subunit containing receptors located in the surface membrane. If it is assumed that α6 is similar to α4, αβαδβ receptors correspond to β36-δ/β36 (R5) and αβαβδ to α6363-δ (R2) in our study. We did not observe any functional expression of β36-δ/β36 (R5). From the present experiments it is difficult to conclude on relative abundance of the three expressing receptors. Subunit concatenation may affect expression levels. In addition, single channel conductance and open probability could differ between different receptors. Nevertheless, active, non-concatenated α6β3δ receptors probably constitute a mixture of β36-δ/α63 (R1), α6363-δ (R2), β3-δ-β363 (R3) and α636/δ where R1–R3 receptors are only active in the presence of neurosteroids. As discussed above, evidence for the expression of multiple receptors resulting from injection into Xenopus oocytes of genetic information coding for α6, β3 and δ has been presented (Hadley and Amin 2007; this paper).

Our findings indicate that the assembly properties of the δ subunit resemble that of the ε subunit (Bollan et al. 2008) in respect to the fact that both subunits can assume multiple positions in a receptor. However, the ε subunit apparently prefers alternative positions as compared with the δ subunit, except that both subunits seem to be able to occupy the position of the β subunit adjacent to the γ subunit in the major adult isoform.

The assembly properties of α6β3δ GABAA receptors should be compared with those of α1β3δ receptors (Kaur et al. 2009). While both β3x-δ/αx3 (R1) and αx3x3-δ (R2) are formed with x = 1 and 6, β3x-δ/β3x (R5) receptors are only formed with x = 1, and β3-δ-β3x3 (R3) are only formed with x = 6. Whether or not αx3x/δ receptors, which are formed with x = 6, are also formed with x = 1 remains to be shown. As we assume, on the basis of functional properties that R5 receptors may only be formed with low efficiency upon delivery of genetic information coding for the α1, β3 and δ subunits (Kaur et al. 2009), there may only be subtle assembly differences between α1β3δ and α6β3δ receptors.

In summary, we have shown that in GABAA receptors containing the α6, β3 and δ subunits, the δ subunit exhibits the ability to promiscuously assemble into different subunit arrangements at least after expression in Xenopus oocytes. Further we show that most of the δ subunit containing receptors remain relatively silent in the absence of neurosteroid and that all are insensitive to ethanol. The architecture of α6β3δ GABAA receptors and their positioning in cerebellar granule cells needs to be established.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We thank Dr V. Niggli for carefully reading the manuscript. This work was supported by the Swiss National Science Foundation grant 3100A0-105272/2.

References

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References