Etomidate produces similar allosteric modulation in α1β3δ and α1β3γ2L GABA(A) receptors

Abstract

Background and purpose: Neuronal GABA(A) receptors are pentameric chloride ion channels, which include synaptic αβγ and extrasynaptic αβδ isoforms, mediating phasic and tonic inhibition respectively. Although the subunit arrangement of αβγ receptors is established as β-α-γ-β-α, that of αβδ receptors is uncertain and possibly variable. We compared receptors formed from free α1, β3 and δ or γ2L subunits and concatenated β3-α1-δ and β3-α1 subunit assemblies (placing δ in the established γ position) by investigating the effects of R-(+)-etomidate (ETO), an allosteric modulator that selectively binds to transmembrane interfacial sites between β3 and α1.

Experimental approach: GABA-activated receptor-mediated currents in Xenopus oocytes were measured electrophysiologically, and ETO-induced allosteric shifts were quantified using an established model.

Key results: ETO (3.2 μM) similarly enhanced maximal GABA (1 mM)-evoked currents in oocytes injected with 5 ng total mRNA and varying subunit ratios, for α1β3(1:1), α1β3δ(1:1:1) and α1β3δ(1:1:3), but this potentiation by ETO was significantly greater for β3-α1-δ/β3-α1(1:1) receptors. Reducing the amount of α1β3δ(1:1:3) mRNA mixture injected (0.5 ng) increased the modulatory effect of ETO, matching that seen with β3-α1-δ/β3-α1(1:1, 1 ng). ETO similarly reduced EC₅₀s and enhanced maxima of GABA concentration-response curves for both α1β3δ and β3-α1-δ/β3-α1 receptors. Allosteric shift parameters derived from these data depended on estimates of maximal GABA efficacy, and the calculated ranges overlap with allosteric shift values for α1β3γ2L receptors.

Conclusion and implications: Reducing total mRNA unexpectedly increased δ subunit incorporation into receptors on oocyte plasma membranes. Our results favour homologous locations for δ and γ2L subunits in α1β3γ2/δ GABA(A) receptors.

Keywords: GABAA receptors; allosteric modulation; concatenated subunit assemblies; electrophysiology; etomidate; general anaesthetics; δ subunits.

Figure 1

Modulation by THDOC of α1β3δ GABA_A receptors expressed from free or concatenated subunits with different molar ratios and total mRNA amounts. (A) Representative current traces evoked by saturating concentration of GABA (1 mM) as well as co-application of 1 mM GABA and 1 μM THDOC with THDOC pre-applied for 30 s. Receptors were expressed by injection of GABA_A receptor subunits into oocytes with different molar ratios and total mRNA amounts (see corresponding receptors in panel B). The horizontal bars above each current trace indicate the application of GABA and THDOC respectively. The timescale is the same, and the amplitude scale is 1 μA for all current traces. (B) The fold of potentiation by THDOC for α1β3 receptors as well as α1β3δ receptors formed with different molar ratios and total mRNA amounts injected into oocytes. The error bars represent SEMs. *Significantly different from α1β3δ(1:1:1, 0.5 ng) receptors at P < 0.001. #Significantly different from α1β3(1:1, 5 ng), α1β3δ(1:1:1, 5 ng) and α1β3δ(1:1:3, 5 ng) receptors at P < 0.01 or 0.001.

Figure 2

Modulation by ETO of α1β3δ GABA_A receptors expressed from free or concatenated subunits with different molar ratios and total mRNA amounts. (A) Representative current traces evoked by saturating concentration of GABA (1 mM) as well as co-application of 1 mM GABA and 3.2 μM ETO with ETO pre-applied for 30 s. Receptors were expressed by injection of GABA_A receptor subunits into oocytes with different molar ratios and total mRNA amounts (see corresponding receptors in panel B). The horizontal bars above each current trace indicate the application of GABA and ETO respectively. The timescale is the same, and the amplitude scale is 1 μA for all current traces. (B) The fold of potentiation by ETO for α1β3 receptors as well as α1β3δ receptors formed with different molar ratios and total mRNA amounts injected into oocytes. The error bars represent SEMs. *Significantly different from α1β3δ(1:1:1, 0.5 ng) receptors at P < 0.01 or 0.001. #Significantly different from α1β3(1:1, 5 ng), α1β3δ(1:1:1, 5 ng) and α1β3δ(1:1:3, 5 ng) receptors at P < 0.05 or 0.01.

Figure 3

ETO modulation of GABA concentration-response data for α1β3δ(1:1:3, 0.5 ng) receptors. (A) Examples of current traces evoked by increasing concentrations of GABA (in μM) as well as co-application of each concentration of GABA and ETO (3.2 μM) for α1β3δ(1:1:3, 0.5 ng) receptors. (B) The concentration–response curves for GABA alone (triangles) and co-application of GABA with 3.2 μM ETO (squares) were plotted for α1β3δ(1:1:3, 0.5 ng) receptors. ETO produced upward and leftward shifts of the concentration–response curve for the receptors. The horizontal bar above each current trace indicates GABA application or co-application of GABA and ETO. n = 4 cells for GABA or GABA + ETO concentration–response curve. The error bars represent SEMs.

Figure 4

ETO modulation of GABA concentration-response data for β3-α1-δ/β3-α1(1:1, 1 ng) receptors. (A) Examples of current traces evoked by increasing concentrations of GABA (in μM) as well as co-application of each concentration of GABA and ETO (3.2 μM) for β3-α1-δ/β3-α1(1:1, 1 ng) receptors. (B) The concentration–response curves for GABA alone (triangles) and co-application of GABA with 3.2 μM ETO (squares) were plotted for β3-α1-δ/β3-α1(1:1, 1 ng) receptors. ETO produced upward and leftward shifts of the concentration–response curve for the receptors. The horizontal bar above each current trace indicates GABA application or co-application of GABA and ETO. n = 7 cells for GABA or GABA + ETO concentration–response curve. The error bars represent SEMs.

Figure 5

ETO allosteric shift quantified in α1β3γ2L and α1β3δ receptors using MWC co-agonist fits with [ETO] as a binary parameter. (A) Normalized GABA-dependent responses (mean ± SD; n ≥ 5) for α1β3γ2L receptors are plotted. Lines underlying data points are fits to logistic functions. Open symbols are control GABA responses: max = 100 ± 2.5, EC₅₀ = 7.8 (5.0–10.3) μM, nH = 1.3 ± 0.18. Solid red symbols are GABA responses in the presence of 3.2 μM ETO: max = 124 ± 3.0, EC₅₀ = 0.79 (0.55–1.14) μM, nH = 1.2 ± 0.21. (B–D) Estimated P_open was calculated from average data for α1β3γ2L receptors from panel A (B), β3-α1-δ/β3-α1 receptors from Figure 4 (C) and α1β3δ receptors formed from diluted free subunits from Figure 3 (D), as described in Methods section. Simultaneous non-linear least squares fits to Equations and were performed as described in Methods section, using [ETO] as a binary parameter (either 0 or 1). (C) Shows β3-α1-δ/β3-α1 receptor results with GABA efficacy = 0.05, and (D) shows α1β3δ receptor results with GABA efficacy = 0.03. The allosteric shifts associated with 3.2 μM ETO are reported in Table 1 along with other fitted parameters.