Stoichiometry of a recombinant GABAA receptor

Abstract

GABA is the main inhibitory neurotransmitter in the mammalian brain. The postsynaptic GABAA receptor/pore complex is presumed to be a pentamer typically composed of a combination of alpha, beta, and gamma subunits, although the stoichiometry remains controversial. We probed the stoichiometry of the GABAA receptor by site-directed mutagenesis of a conserved leucine (to serine) in the putative second membrane-spanning domain of the rat alpha 1(alpha L263S), beta 2(alpha L259S), and gamma 2(alpha L274S) subunit isoforms. Coexpression of wild-type and mutant subunits of each class (e.g., alpha and alpha L263S), along with their wild-type counter-parts (e.g., beta and gamma), in Xenopus laevis oocytes resulted in mixed populations of receptors with distinct GABA sensitivities. This is consistent with the interpretation that the leucine mutation increased the GABA sensitivity in proportion to the number of incorporated mutant subunits. The apparent number of incorporated subunits for each class (alpha, beta, and gamma) could then be determined from the number of components comprising the compound GABA dose-response relationships. Using this approach, we conclude that the recombinant alpha 1 beta 2 gamma 2 GABAA receptor is a pentamer composed of two alpha subunits, two beta subunits, and one gamma subunit.

Fig. 5.

The stoichiometry does not depend on the αβγ cRNA injection ratio. The α_mβγ and αβγ_m cRNA combinations were injected into oocytes at ratios of 4:1:1 and 1:1:4, respectively. The GABA dose–response relationships were then fit by the Hill equation and compared with those determined with 1:1:1 cRNA injections. The EC₅₀values of the GABA dose–response relationships for α_mβγ cRNA ratios of 1:1:1 and 4:1:1 were (mean ± SD) 0.30 ± 0.13 μm (n = 9) and 0.30 ± 0.04 μm (n = 5), respectively. The EC₅₀ values of the GABA dose–response relationships for αβγ_m cRNA ratios of 1:1:1 and 1:1:4 were 0.99 ± 0.23 μm (n = 4) and 0.98 ± 0.062 μm (n = 4), respectively. These data indicate that at least at these cRNA ratios, the number of α and γ subunits in the GABA receptor is fixed.

Fig. 1.

Mutation of the conserved leucine in TM2 of the α1(L263S), β2(L259S), and γ2(L274S) subunit increases the GABA sensitivity of the GABA_A receptor. A, GABA-activated currents from oocytes expressing αβγ, α_mβγ, αβ_mγ, and αβγ_m subunit combinations. The subscript “m” indicates that the conserved leucine in TM2 was mutated to serine. GABA was bath-applied at the indicated concentrations. Note the increase in GABA sensitivity induced by the mutation in each subunit. Calibration: 100 sec; 350, 65, 35, and 500 nA for the four rows of traces, respectively. B, Average GABA dose–response relationships for each of the four combination of subunits (mean ± SEM). The continuous lines are the best fit of the Hill equation to the data points. The EC₅₀ values and Hill coefficients (mean ± SD) for the fits are αβγ: 45.8 ± 3.6 μm, 1.59 ± 0.09 (n = 5); α_mβγ: 0.30 ± 0.050 μm, 0.85 ± 0.10 (n = 9); αβ_mγ: 0.035 ± 0.004 μm, 1.12 ± 0.04 (n = 3); and αβγ_m: 0.99 ± 0.23 μm, 1.78 ± 0.36 (n = 4).

Fig. 2.

The number of components comprising the GABA dose–response relationship with αα_mβγ coexpression depends on the number of α subunits in the GABA receptor complex.A, With one α subunit in the GABA receptor complex, the GABA dose–response relationship from oocytes coexpressing α, α_m, β, and γ subunits would be composed of two components (continuous line): one component from activation of αβγ receptors and one component from activation of α_mβγ receptors (dashed lines, scaled to facilitate comparison with the continuous line). The EC₅₀ values of these two components would be ≈0.30 and ≈45.8 μm (indicated on the abscissa) as demonstrated by the data in Figure 1. The mutant α subunit is shownshaded. B, With two α subunits in the GABA receptor complex, the GABA dose–response relationship from oocytes coexpressing α, α_m, β, and γ subunits would be composed of three components (continuous line): one component from activation of αβγ receptors, one component from activation of α_mβγ receptors, and an intermediate component from activation of receptors containing both an α and an α_m subunit (dashed lines). The EC₅₀ values of the first and third components would be ≈0.30 and ≈45.8 μm, and the predicted EC₅₀ of the intermediate component, assuming the two α_m subunits contribute equally to the shift, would be 3.7 μm. A similar logic could be applied to αββ_mγ and αβγγ_m coexpression to determine the number of β and γ subunits, respectively.

Fig. 3.

GABA dose–response relationships from coexpression of αα_mβγ, αββ_mγ, and αβγγ_m subunits. A, cRNA encoding for α, α_m, β, and γ subunits was coinjected into oocytes. The dose–response relationship for GABA (mean ± SEM,n = 4) was well described by the sum of three Hill equations (continuous line), suggesting, in terms of GABA sensitivity, three receptor subtypes. The EC₅₀ values of the three components were 0.26 ± 0.05, 2.2 ± 0.1, and 36.3 ± 8.1 μm (α-to-α_m cRNA injection ratio = 1:3). The dashed linesrepresenting the first component and the combination of the first and second component are shown to delineate the individual components. The three components suggest that there are two α subunits in the GABA_A receptor complex. The inset is a GABA dose–response relationship from an oocyte expressing primarily αβγ and α_mβγ receptors. This was achieved by injecting αβγ cRNA, waiting 5 d, and then injecting α_mβγ cRNA. This ensures that α and α_mcRNAs do not coexist to any appreciable extent in the oocyte. In this case there was no intermediate component, and the GABA dose–response relationship was described by the sum of two Hill equations with EC₅₀ values of 0.34 and 33.4 μm (indicated byarrows). The EC₅₀ values from three such experiments (mean ± SD) were 0.42 ± 0.17 and 30.0 ± 7.5 μm for the first and second components, respectively.B, cRNA encoding for α, β, β_m, and γ subunits was coinjected into oocytes. The dose–response relationship for GABA (mean ± SEM, n = 3) was also well described by the sum of three Hill equations (continuous line). The EC₅₀ values of the three components were 0.025 ± 0.01, 0.94 ± 0.07, and 39.2 ± 7.9 μm (β-to-β_m cRNA injection ratio = 1:1). The three components suggest that there are two β subunits in the GABA_A receptor complex. C, cRNA encoding for α, β, γ, and γ_m subunits was coinjected into oocytes. The dose–response relationship for GABA (mean ± SEM,n = 3) was described by the sum of two Hill equations (continuous line) with EC₅₀ values of 1.09 ± 0.12 and 40.9 ± 4.8 μm(γ-to-γ_m cRNA injection ratio = 1:1). Thedashed line represents the first component. Two components suggest that there is one γ subunit in the GABA_A receptor complex.

Fig. 4.

The EC₅₀ values of the components with αα_mβγ, αββ_mγ, and αβγγ_m coexpression do not depend on the relative amplitudes of the components. A, Plot of the EC₅₀ values of the three components as a function of the fraction of the wild-type component for αα_mβγ coexpression. The fraction of the wild-type component is the amplitude of the wild-type component divided by the total amplitude and is determined from the fits to the individual compound dose–response relationships. In these experiments, GABA dose–response relationships were constructed from oocytes in which the α-to-α_m cRNA injection ratio was varied (1:3, 1:1, and 3:1) to shift the relative amplitudes of the different components. The total amount of α and α_m cRNA remained fixed with respect to β and γ. For all α-to-α_m ratios tested, the GABA dose–response relationships were described by the sum of three Hill equations. Thedashed lines represent the mean EC₅₀ values determined from αβγ and α_mβγ coexpression (Fig.1). The continuous line is a linear regression to the EC₅₀ of the intermediate component and demonstrates a slight but significant (p < 0.05) negative dependence that is in the opposite direction from that predicted if there were multiple, indistinguishable, intermediate components (see text). These data indicate three components and hence two α subunits in the GABA receptor complex. B, Similar plot as inA, but for αββ_mγ coexpression at different ratios of β to β_m (1:1 and 3:1). Thedashed lines represent the mean EC₅₀ values determined from αβγ and αβ_mγ coexpression (Fig.1). These data indicate three components and hence two β subunits in the GABA receptor complex. C, Similar plot as inA, but for αβγγ_m coexpression at different ratios of γ to γ_m (1:3 and 1:1). These data indicate two components and hence one γ subunit in the GABA receptor complex.