Different α1β3 receptors are expressed by injecting Xenopus oocytes with different cRNA ratios

The assembly of Cys-loop receptors in Xenopus laevis oocytes into specific stoichiometries can be directed by injecting cRNA with variant subunit ratios34,36. To determine whether varying the injection ratios led to the expression of different receptors, we injected five mixtures of α1 and β3 cRNAs into oocytes (α1 + β3 in 1:1, 5:1, 10:1, 20:1 and 30:1 ratios). To allow for comparisons between injection ratios, all the cRNA was derived from a single stock. The total amount of cRNA injected ranged from 2.5 to 4.0 ng/oocyte and the functional properties of receptors including holding currents, GABA sensitivity and maximum peak current amplitudes were compared.

Initially, receptors expressed by the most extreme injection ratios 1:1 and 30:1, were compared. The holding currents of oocytes injected with α1 + β3 (1:1) cRNA were −150 ± 93 nA, n = 6 when voltage-clamped at −60 mV, suggesting constitutive receptor activity (Fig. 1A). In contrast, oocytes injected with α1 + β3 (30:1) had negligible holding current levels that averaged −13 ± 10 nA, n = 9 (Fig. 1A; p < 0.05).

Figure 1 GABA-evoked responses at α1β3 and α1β3γ2 GABA A receptors. Xenopus laevis oocytes were injected with cRNA and subjected to two-electrode voltage-clamp electrophysiology as described in the methods. For experimentation, oocytes were clamped at −60 mV and full GABA concentration response relationships were obtained on each oocyte. (A) Representative GABA-evoked traces from oocytes injected with the denoted cRNA mixtures. Bars above each trace indicate application periods and GABA concentrations and “/” a wash period. Dotted lines indicate a 0 nA baseline and holding currents were −150 ± 93 nA, n = 6 for α1 + β3 (1:1) and −13 ± 10 nA, n = 9 for α1 + β3 (30:1). (B,C) Baseline subtracted peak current amplitudes for full GABA concentration-response curves at oocytes injected with the indicated cRNA mixtures using free subunits (B) or a concatenated β3-α1 construct (C) were fitted to the Hill equation using non-linear regression (fixed bottom of 0 and slope of 1) and normalized to the maximal fitted value (I GABA_max_fit ). Averaged normalized data points are depicted as means ± S.E. as a function of the GABA concentration, fitted to the Hill equation and regression results are presented in Table 1. Each data point represents experiments from n = 5–9 oocytes from ≥2 batches. (D) α1β3 GABA A receptors can express in two stoichiometries of 2α1:3β3 (left) and 3α1:2β3 (right). The two binding sites for GABA at the β3(+)-α1(−) subunit interface are indicated by red arrowheads. Full size image

GABA activated receptors expressed from both injection ratios in a concentration-dependent manner, with maximum similar peak-current amplitudes ranging from 1200 to 4200 nA (p > 0.05). Fitting peak-current amplitudes as a function of the GABA concentration to the Hill equation revealed higher GABA sensitivity with receptors from the 1:1 cRNA ratio compared with those formed from the 30:1 ratio. The derived EC 50 values were significantly different (p < 0.0001) with a 10-fold difference between receptors at the 1:1 and 30:1 ratios (2.8 μM and 26 μM, respectively) (Fig. 1B; Table 1). The GABA sensitivity from the 30:1 injection ratio was similar to that of the ubiquitous α1β3γ2 receptor (Fig. 1B; Table 1).

Table 1 GABA concentration response relationships at various GABA A receptors. Full size table

In contrast, receptors formed from 5:1, 10:1 and 20:1 cRNA injection ratios resulted in less uniform receptor populations. This resulted in intermediate GABA sensitivities, concentration-response curves with shallow Hill slopes and comparatively high standard errors for individual data points (data not shown). Thus, receptors formed from different injection ratios had different functional properties that are likely the result of the expression of distinct receptor populations with altered subunit stoichiometries.

Expressing stoichiometry specific α1β3 receptors using concatenated constructs

To determine whether data obtained by varying cRNA ratios are from uniform receptor populations that consist of 2α:3β and 3α:2β stoichiometries, the functional properties of receptors expressed with a concatenated construct linking the β3 and α1 subunits were compared to receptors expressed with the 1:1 or 30:1 cRNA injection ratios. For this experiment, the N-terminal of the α1 subunit was linked to the C-terminal of the β3 subunit to generate the β3-α1 concatenated construct as previously described37. When cRNA transcribed from the β3-α1 construct was injected into oocytes (2.5 ng/oocyte) no GABA-elicited currents were observed (n = 10). Therefore, to express receptors in a 2α:3β and 3α:2β stoichiometry, β3-α1 was co-injected with either the β3 or α1 subunit, respectively.

First, the properties of 2α:3β receptors were compared to receptors formed by the 1:1 injection ratio. Both holding current levels and the GABA sensitivities of oocytes expressing receptors from the concatenated β3-α1 + β3 (1:2) construct were similar to oocytes injected with free α1 + β3 (1:1) subunits, with holding currents of −153 ± 65 nA, n = 6 compared to −150 ± 93 nA, n = 6 when voltage-clamped at −60 mV. The constitutive current observed with both α1 + β3 (1:1) and β3-α1 + β3 (1:2) could be the result of homomeric β3 receptors being expressed. To determine whether or not the observed holding currents are due to homomeric β3 receptors, we evaluated oocytes expressing α1 + β3 (1:1) and β3-α1 + β3 (1:2) using histamine (1 mM), an agonist that activates only homomeric β3 receptors38,39. We found that all cells that exhibited a significant holding current injected with either α1 + β3 (1:1) (4 out of 4 cells) or β3-α1 + β3 (1:2) (1 out of 4 cells) had some response to histamine (data not shown), indicating that homomeric β3 receptors are the major contributor to the holding current. Irrespectively, GABA activated expressed receptors in a concentration-dependent manner, with maximum peak-current amplitudes ranging from 1000 to 2000 nA. Indistinguishable EC 50 values of 1.4 μM and 2.8 μM were obtained from oocytes injected with β3-α1 + β3 (1:2) and α1 + β3 (1:1), respectively (p > 0.05, Fig. 1B,C, Table 1).

Next, properties of 3α:2β stoichiometry receptors were compared to receptors formed by the 30:1 injection ratio. Holding currents of oocytes injected with either β3-α1 + α1 (1:2) or free α1 + β3 (30:1) subunits were negligible with values of −22 ± 8 nA, n = 7 and −13 ± 10 nA, n = 9, respectively. GABA activated expressed receptors in a concentration-dependent manner, with maximum peak-current amplitudes ranging from 1400 to 2900 nA. The GABA EC 50 values of 41 μM and 26 μM from β3-α1 + α1 (1:2) and α1 + β3 (30:1) injections were similar (p > 0.05, Fig. 1B,C, Table 1).

Thus, there were no observable differences in the functional properties of α1β3 receptors in either the 2α:3β or 3α:2β stoichiometries between receptors expressed by different subunit ratios or a concatenated construct (Fig. 1D). Although the chosen cRNA ratios for the individual subunits appeared sufficient to ensure predominantly uniform receptor populations, the use of concatenated receptors may potentially yield more uniform populations. However, this methodology inherently carries a risk that the physical linkage of subunits could affect receptor pharmacology. Hence, for the remainder of the manuscript, conclusions will be drawn from data generated using both methodologies.

α1β3 receptors show stoichiometry-specific sensitivity to Zn2+ ions

Zn2+ ions are used to differentiate α1β3 from α1β3γ2 GABA A receptors25. Both α1-β3 and β3-β3 interfaces contribute to the binding pockets for Zn2+ 40. Since 3α1:2β3 receptors do not contain a β3-β3 interface, sensitivity to Zn2+ will likely be altered as a result of differing subunit stoichiometries. Therefore, Zn2+ inhibition of α1β3 receptor currents elicited by GABA (at EC 50 concentrations) was measured in oocytes.

Zn2+ (10 μM) inhibited 87 ± 10% (n = 5) of the GABA-induced current at 2α1:3β3 receptors formed by injection of α1 + β3 (1:1) cRNA (Fig. 2A). In contrast, only 13 ± 10% (n = 6) inhibition was observed at receptors formed by injecting α1 + β3 (30:1). Next, inhibitory concentration-response relationships were performed to ascertain the potency of Zn2+ at each stoichiometry. At the 2α1:3β3 stoichiometry, Zn2+ inhibited receptors from the 1:1 ratio with an IC 50 value of 0.84 μM (Fig. 2B). A similar IC 50 value of 1.6 μM for Zn2+ inhibition at concatenated receptors was observed with β3-α1 + β3 (1:2) injection (Fig. 2C). These two IC 50 values were not significantly different from each other (p > 0.05). At the highest concentration of Zn2+, both receptors were fully inhibited.

Figure 2 Zn2+ inhibition of GABA-evoked currents from α1β3 and α1β3γ2 GABA A receptors. Xenopus laevis oocytes were injected with cRNA and subjected to two-electrode voltage clamp electrophysiology as described in the methods. Control currents (I control ) were evoked using a GABA concentration corresponding to ~EC 50 and inhibition by Zn2+ was evaluated by co-applications with GABA control . (A) Representative GABA-evoked current traces from oocytes injected with the denoted cRNA mixtures. Bars above each trace indicate GABA and Zn2+ application periods. Dotted lines indicate the peak current amplitude by GABA control and “/” a wash period. (B,C) Concentration-response relationships of Zn2+ inhibition of GABA control -evoked currents at α1β3 or α1β3γ2 receptors stemming from injecting the indicated cRNA mixtures using free subunits (B) or a concatenated β3-α1 construct (C). Averaged Zn2+ inhibition values were depicted as means ± S.E.M as a function of the Zn2+ concentration and fitted to the Hill equation by non-linear regression. Regression results for α1 + β3 (1:1) were IC 50 = 0.84 (95% CI: 0.53–1.3), nH = −0.5 ± 0.32 and for β3-α1 + β3 (1:2) were IC 50 = 1.6 (95% CI: 1.1–2.4), nH = −0.6 ± 0.06. For the remaining cRNA mixtures, Zn2+ inhibition at the maximal tested concentration was too low to allow for meaningful fitting. Each data point represent experiments from n = 5–8 oocytes of ≥2 batches. (D) The depiction of α1β3 GABA A receptor stoichiometries from Fig. 1D was modified to indicate Zn2+ binding in the β3(+)-β3(−) subunit interface (red/orange arrowhead). Full size image

At the 3α1:2β3 receptors, the maximal tested concentration of Zn2+ (100 μM) only displayed partial inhibition (26 ± 17%, n = 6) at receptors from the 30:1 ratio (Fig. 2B). Similar partial inhibition was observed using the concatenated β3-α1 + α1 (1:2) construct (Fig. 2C). In both cases, the level of inhibition was too low to allow for meaningful fitting to the Hill equation. The partial inhibition of Zn2+ at 3α1:2β3 receptors mimicked observations at α1β3γ2 receptors (Fig. 2B,C).

These data demonstrate that receptors with a β3-β3 interface have high sensitivity to inhibition by Zn2+ (Fig. 2D). While it was previously suggested that residues located in the α1-β3 interface also contribute to Zn2+ sensitivity40 this appears to require substantially higher concentrations. In addition, α1β3γ2 receptors that also lack the β3-β3 interface, were likewise relatively insensitive to Zn2+ inhibition at concentrations below 100 μM.

Zolpidem is a positive modulator of α1β3 with a 3α1:2β3 subunit stoichiometry

The α1-α1 interface of 3α1:2β3 receptors is homologous to the α1-γ2 interface that binds benzodiazepines and non-benzodiazepines. Zolpidem has in vivo effects that are not related to binding in the classical α1-γ2 benzodiazepine site and we wanted to evaluate whether this α1 preferring modulator displayed any efficacy at α1β3 possessing an α1-α1 interface. Zolpidem (1 μM) was co-applied with a low GABA concentration (~EC 5–10 ) to evaluate potential modulation of control currents at 3α1:2β3 and compared with 2α1:3β3 and α1β3γ2 receptors.

As expected, zolpidem (1 μM) had no effect at 2α:3β receptors expressed by a 1:1 ratio of free subunits (Fig. 3A). In contrast, 3α1:2β3 receptors expressed from the 30:1 ratio were positively modulated by zolpidem. This resulted in an increase in current amplitudes of more than 100% compared with the current elicited by the GABA alone. This enhancement was inhibited by co-application of flumazenil (1 μM), a benzodiazepine site neutral antagonist (Fig. 3B). As expected, zolpidem (1 μM) also enhanced GABA-elicited currents at α1β3γ2 receptors, an enhancement that could be inhibited by flumazenil (Fig. 3C).

Figure 3 Zolpidem modulation of GABA-evoked currents from α1β3 and α1β3γ2 GABA A receptors. Xenopus laevis oocytes were injected with cRNA and subjected to two-electrode voltage clamp electrophysiology as described in the methods. Control currents (I control ) were evoked using a GABA concentration corresponding to ~EC 5–10 and modulation by zolpidem was evaluated by co-applications with GABA control . (A–C) Representative GABA-evoked current traces from oocytes injected with the denoted cRNA mixtures. Bars above each trace indicate GABA, zolpidem (Zolp) and flumazenil (Flu) concentrations and application periods. Dotted lines indicate the peak current amplitude by GABA control and “/” a wash period. For the specific traces, zolpidem had no robust effects at receptors from α1 + β3 (1:1) injection (A), but showed 130% modulation at receptors from α1 + β3 (30:1) injection which was inhibited 95% by co-application of flumazenil (B). Zolpidem likewise modulated receptors from injection of α1 + β3 + γ2 (1:1:5) by 160% which could be inhibited 85% by flumazenil (C). (D,E) Concentration-response relationships of zolpidem modulation of GABAcontrol-evoked currents at α1β3 or α1β3γ2 receptors stemming from injecting the indicated cRNA mixtures using free subunits (D) or a concatenated β3-α1 construct (E). Average modulatory values were depicted as means ± S.E.M as a function of the zolpidem concentration and fitted to the Hill equation by non-linear regression. Each data point represents experiments from n = 5–7 oocytes from ≥2 batches and regression results are presented in Table 1. Full size image

To determine the potency of zolpidem potentiation at 3α1:2β3 receptors, full concentration-response relationships were obtained and compared with those from the α1β3γ2 receptor. When injecting free subunits using α1 + β3 (30:1) and α1 + β3 + γ2 (1:1:5) cRNA, zolpidem had a significantly lower EC 50 value of 0.10 μM at α1β3 receptors compared to an EC 50 value of 0.48 μM at α1β3γ2 (p < 0.01) (Fig. 3D; Table 2). However, when injecting cRNA containing the concatenated construct using β3-α1 + α1 (1:2) and β3-α1 + γ2 (1:2), the EC 50 value of 0.030 μM at α1β3 receptors was not significantly different to the EC 50 value of 0.050 μM at α1β3γ2 receptors (p > 0.05) (Fig. 3E; Table 2). Efficacy levels at α1β3 receptors were 120% and 110% for the 30:1 ratio and concatenated receptors, respectively. Higher values were observed at α1β3γ2 receptors with 340% and 350% at α1 + β3 + γ2 (1:1:5) vs. β3-α1 + γ2 (1:2), respectively.

Table 2 Zolpidem modulation of GABA-evoked currents at various GABA A receptors. Full size table

Hence zolpidem modulated GABA-evoked currents at 3α1:2β3 receptors. Importantly, the potency of zolpidem at α1β3 (3α1:2β3) receptors was comparable with α1β3γ2 receptors regardless of whether free subunits or concatenated subunits were injected. While the potency of zolpidem was similar at both receptors, these experiments suggest that zolpidem enhanced α1β3γ2 receptors with greater efficacy. However, in this type of assay efficacy of a modulator is highly dependent on the utilized GABA concentration and should be treated with caution.

Modulatory mechanism of action of zolpidem at α1β3 receptors possessing a 3α1:2β3 subunit stoichiometry

The hallmark feature of allosteric modulation via the α1-γ2 benzodiazepine site by e.g. diazepam and zolpidem is an increase in the apparent potency of GABA. This causes a shift in the GABA concentration-response curve to the left in presence of zolpidem with little accompanying change in the maximal current amplitudes. In order to assess how zolpidem affects 3α1:2β3 stoichiometry, GABA concentration-response relationships were measured in the presence of zolpidem and compared to that in its absence.

At 3α1:2β3 receptors expressed by injecting α1 + β3 (30:1) cRNA, zolpidem (1 μM) left-shifted the GABA concentration-response curve by decreasing the EC 50 value from 26 μM to 5.6 μM (Fig. 4A, Table 1). Hence, zolpidem modulation caused a significant 5-fold change (p < 0.0001) of the GABA potency with no observed change in the maximum GABA-evoked peak current amplitudes. At receptors obtained by the concatenated β3-α1 + α1 (1:2) construct, zolpidem (1 μM) likewise caused a significant (p < 0.0001) 6-fold change in the potency of GABA, with an increase in the EC 50 value from 41 μM to 7.1 μM (Fig. 4B, Table 1). At α1β3γ2 receptors, zolpidem (10 μM) significantly decreased the EC 50 value from 53 μM to 13 μM (Fig. 4C; Table 1; p < 0.0001) with no change in the maximum peak current amplitudes.

Figure 4 Mechanism of zolpidem modulatory actions at α1β3 and α1β3γ2 GABA A receptors. Xenopus laevis oocytes were injected with cRNA and subjected to two-electrode voltage clamp electrophysiology as described in the methods. (A–C) Full GABA concentration-response relationships were obtained in presence of the indicated concentrations of zolpidem (Zolp) at receptors stemming from injection of cRNA of free subunits (A,C) or a concatenated construct (B). Baseline subtracted GABA + zolpidem peak current amplitudes were normalized to a maximal GABA control response (3 mM) in the same oocytes. Averaged normalized data points are depicted as means ± S.E.M. as a function of the GABA concentration and fitted to the Hill equation with regression results are presented in Table 1. Each data point represents experiments from n = 5–7 oocytes from ≥2 batches. GABA concentration response relationships in absence of zolpidem (from Fig. 1) are included for comparison. (D) The depiction of α1β3 GABA A receptor stoichiometries from Fig. 2D was modified to indicate zolpidem binding in the α1(+)-α1(−) subunit interface (red/green arrowhead). Full size image

These data demonstrate that the mechanism of modulatory action by zolpidem is increase of the GABA potency at both α1β3 and α1β3γ2 receptors. This increase ranged from 4 to 6 fold, which is in agreement with previous observations at α1β3γ2 receptors8. The modulatory effect was not accompanied by any change in maximal GABA-evoked peak current amplitudes and the similar magnitude of the changes in EC 50 values suggest that zolpidem has a similar efficacy at the two receptor types. Although zolpidem is not structurally a classical benzodiazepine, it binds at a similar site within the α1-γ2 of α1βγ2 receptors as benzodiazepines. Taken together, zolpidem is most likely binding at the α1-α1 interface of 3α:2β receptors to modulate receptor function analogous to the modulation of receptor function via binding at the α1-γ2 interface (Fig. 4D).

Diazepam enhances GABA currents at α1β3 receptors with a 3α1:2β3 subunit stoichiometry albeit with low efficacy

Finally, we determined whether α1β3 (3α1:2β3) receptors could be modulated by the classical benzodiazepine diazepam, which binds with equal affinity to GABA A receptors containing α1, α2, α3 and α5 subunits41,42. Like zolpidem, diazepam (1 μM) enhanced GABA-induced currents at α1β3 receptors obtained with α1 + β3 (30:1) cRNA and this effect was inhibited by co-application of flumazenil (Fig. 5A). However, the enhancement by diazepam was low in comparison with zolpidem (Fig. 5B). A full concentration-response relationship revealed an EC 50 value for diazepam of 0.040 μM (Fig. 5C). A similar EC 50 value of 0.020 μM was estimated for diazepam modulation of concatenated receptors and the observed potencies are similar to that reported for α1β3γ2 receptors8. From the concentration-response relationships, it is evident that the enhancement by diazepam displayed lower efficacy compared with zolpidem. When using free subunit cRNA diazepam enhanced GABA-elicited currents by a maximum of 40% whereas use of concatenated subunits resulted in 51% modulation and these were not significantly different from each other (Fig. 5C; p > 0.05). This represents half or less of the modulation observed with zolpidem (Table 2). Analogous to zolpidem, diazepam (1 μM) did not change the maximal GABA-evoked current amplitudes at 2α1:3β3 receptors obtained by injecting α1 + β3 (1:1) (data not shown).