Functional genomics of epilepsy-associated mutations in the GABAA receptor subunits reveal that one mutation impairs function and two are catastrophic

Abstract

A number of epilepsy-causing mutations have recently been identified in the genes of the α1, β3, and γ2 subunits comprising the γ-aminobutyric acid type A (GABA_A) receptor. These mutations are typically dominant, and in certain cases, such as the α1 and β3 subunits, they may lead to a mix of receptors at the cell surface that contain no mutant subunits, a single mutated subunit, or two mutated subunits. To determine the effects of mutations in a single subunit or in two subunits on receptor activation, we created a concatenated protein assembly that links all five subunits of the α1β3γ2 receptor and expresses them in the correct orientation. We created nine separate receptor variants with a single-mutant subunit and four receptors containing two subunits of the γ2^R323Q, β3^D120N, β3^T157M, β3^Y302C, and β3^S254F epilepsy-causing mutations. We found that the singly mutated γ2^R323Q subunit impairs GABA activation of the receptor by reducing GABA potency. A single β3^D120N, β3^T157M, or β3^Y302C mutation also substantially impaired receptor activation, and two copies of these mutants within a receptor were catastrophic. Of note, an effect of the β3^S254F mutation on GABA potency depended on the location of this mutant subunit within the receptor, possibly because of the membrane environment surrounding the transmembrane region of the receptor. Our results highlight that precise functional genomic analyses of GABA_A receptor mutations using concatenated constructs can identify receptors with an intermediate phenotype that contribute to epileptic phenotypes and that are potential drug targets for precision medicine approaches.

Keywords: GABA receptor; brain disorder; channel activation; concatemer; cryo-electron microscopy; epilepsy; heterozygous; missense; neurotransmission; neurotransmitter; synaptic transmission.

Figure 1.

A, pentameric structure of the GABA and diazepam-bound α1β3γ2 receptor (PDB code 6HUP) from above showing the orientation of the subunits. The subunits are colored with respect to their order in the pentamer: first γ2 (green), second β3 (maroon), third α1 (blue), fourth β3 (red), and fifth α1 (dark blue). B, side view showing the first γ2 subunit adjacent to the second β3 subunit. The γ2^R323 residue on the M2-M3 loop is depicted with the side chain in black in a transparent sphere. C, side view showing the fourth β3 subunit adjacent to the fifth α1 subunit, with the GABA-binding site highlighted. The side chains of the β3^D120N residue at the GABA-binding site, the β3^T157M residue within an internal β-sheet, the β3^Y302C residue in the M2-M3 loop, and the β3^S254F residue in the M1 region are shown in black.

Figure 2.

A, schematic of the coding region of concatenated receptor containing the DNA construct. Linker lengths are 15 amino acids ((AGS)₅), 27 amino acids ((AGS)₅LGS(AGS)₃), 18 amino acids (AGT(AGS)₅), and 27 amino acids ((AGS)₄AGT(AGS)₄). B, schematic of the expected arrangement of the concatenated receptor where the subunits arrange in a counter-clockwise orientation. GABA- and clobazam-binding sites are shown. C, representative data (above) from a single two-electrode voltage clamp experiment where different concentrations of GABA (open bars) were applied to construct a concentration–response curve to GABA (below). Filled bars, reference 3 mm GABA applications; open bars, GABA applications at concentrations shown. Peak currents were measured, and the mean ± S.E. (error bars) was plotted (open circles) and fitted to the Hill equation (below). D, representative data (above) from a single two-electrode voltage clamp experiment constructing a modulation curve to clobazam (below). Three pulses of reference 10 μm GABA (closed bars) were applied prior to co-application of 10 μm GABA and clobazam at concentrations shown (closed bars). Percent modulation of the control GABA response was calculated, and the mean ± S.E. was plotted and fitted to the Hill equation (below). The fitted EC₅₀ of clobazam was 86 nm (log EC₅₀ = −4.03 ± 0.06, mean ± S.E., n = 10), and the fitted E_max was 306% (320 ± 32, mean ± S.E., n = 10).

Figure 3.

A, schematic of concatenated receptor indicating the location of mutations when they are introduced into the γ2 or distinct β3 subunits. Red circles indicate the location of mutations on the first γ2 subunit, and red circles, purple squares, and blue circles indicate location of mutations on the second, fourth, or both second and fourth β3 subunits, respectively. B, representative traces of WT and γ2^R323Q, β3^D120N, β3^T157M, β3^S254F, and β3^Y302C mutant receptors with mutation(s) in the labeled locations after application of reference 3 mm GABA (filled bars). Scale bars, 500 nA and 100 s. C, absolute current elicited by 3 mm GABA after injection of 2 ng of RNA. Individual data points are depicted as either open circles or squares with WT as black bars and gray circles and a color and pattern scheme identical to that in A. Bars and error bars represent mean ± S.D. of 10–13 individual cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with WT, one-way ANOVA with Tukey's post hoc test.

Figure 4.

A, schematic of concatenated receptor indicating the location of the γ2^R323Q mutation (red circle) within the concatenated construct (left). Shown are representative data (right) from a single two-electrode voltage clamp experiment where different concentrations of GABA (open bars) were applied to construct a concentration–response curve to GABA at γ2^R323Q-β3-α1-β3-α1 receptors. Filled dark red bars and traces represent reference 3 mm GABA applications, and open red bars and traces represent GABA applications at the concentrations shown. Shown is a concentration–response curve to GABA (below) of WT γ2-β3-α1-β3-α1 (○) and γ2^R323Q-β3-α1-β3-α1 (○) receptors normalized to the Est. P_o(max) and fitted to the Hill equation. Dots, mean ± S.E. (error bars) of 10–13 individual experiments. The EC₅₀ of the mutant receptor derived from the curve fit is shown. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with WT, one-way ANOVA with Tukey's post hoc test. B, representative traces of WT (black) and γ2^R323Q (red) mutant receptors after application of reference 3 mm GABA (filled bars) and 10 mm GABA, 1 μm diazepam, and 3 μm etomidate (open bars), respectively. Scale bars, 500 nA and 100 s. The Est. GABA P_o(max) of WT (○) and γ2^R323Q (○) mutant receptors (below) was determined by dividing the current elicited by 3 mm GABA by the current elicited by 10 mm GABA, 1 μm diazepam, and 3 μm etomidate and corrected for the reference 3 mm GABA current. Lines and error bars represent the mean ± S.D. of 10 individual cells.

Figure 5.

A, schematic of concatenated receptor indicating the location of β3^D120N and β3^T157M mutations introduced within the second (closed red circle), fourth (closed purple square) or the second and fourth (closed blue circles) subunits within the resulting pentameric receptor. B, concentration–response curve to GABA of WT γ2-β3-α1-β3-α1 (open black circle), γ2-β3^D120N-α1-β3-α1 (open red circle), γ2-β3-α1-β3^D120N-α1 (open purple square), and γ2-β3^D120N-α1-β3^D120N-α1 (closed blue circle) receptors; C, Concentration–response curve to GABA of WT γ2-β3-α1-β3-α1 (open black circle), γ2-β3^T157M-α1-β3-α1 (open red circle), γ2-β3-α1-β3^T157M-α1 (open purple square), and γ2-β3^T157M-α1-β3^T157M-α1 (closed blue circle) receptors normalized to the Est. P_o(max) and fitted to the Hill equation. The EC₅₀ of the mutant receptor derived from the curve fit is shown. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with WT, one-way ANOVA with Tukey's post hoc test. D, representative traces of β3^D120N and β3^T157M receptors after application of 3 mm GABA (filled bars) and 10 mm GABA, 1 μm diazepam, and 3 μm etomidate (open bars), respectively. Red traces indicate mutation at the second subunit location, and purple traces indicate mutation at the fourth. Scale bars, 500 nA and 100 s. E, estimated GABA P_o(max) of WT, β3^D120N, and β3^T157M receptors was determined by dividing the current elicited by 3 mm GABA by the current elicited by 10 mm GABA, 1 μm diazepam, and 3 μm etomidate and corrected for the reference 3 mm GABA. Open gray circles, WT; open red circles, receptors with a mutation in the second position; open purple squares, receptors with a mutation in the fourth position. Lines and error bars represent mean ± S.D. of 10 individual cells.

Figure 6.

A, concentration–response curve to GABA of WT γ2-β3-α1-β3-α1 (open black circles), γ2-β3^Y302C-α1-β3-α1 (open red circles), γ2-β3-α1-β3^Y302C-α1 (open purple squares) and γ2-β3^Y302C-α1-β3^Y302C-α1 (closed blue circles) receptors normalized to the Est. P_o(max) and fitted to the Hill equation. Dots, mean ± S.E. (error bars) of 10–13 individual experiments. The EC₅₀ of the mutant receptor derived from the curve fit is shown. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with WT, one-way ANOVA with Tukey's post hoc test. B, representative traces of β3^Y302C mutant receptors after application of reference 3 mm GABA (filled bars) and 10 mm GABA, 1 μm diazepam, and 3 μm etomidate (open bars), respectively. Red traces indicate mutation at the second subunit location, purple indicates mutation at the fourth subunit location, and blue indicates mutation at both the second and fourth locations. Scale bars, 500 nA and 100 s. C, estimated GABA P_o(max) of WT γ2-β3-α1-β3-α1 (open black circles), γ2-β3^Y302C-α1-β3-α1 (open red circles), γ2-β3-α1-β3^Y302C-α1 (open purple squares) and γ2-β3^Y302C-α1-β3^Y302C-α1 (closed blue circles) mutant receptors. Est. P_o(max) was determined by dividing the current elicited by 3 mm GABA by the current elicited by 10 mm GABA, 1 μm diazepam, and 3 μm etomidate and corrected where 3 mm GABA was not at the maximum of the concentration–response curves. Lines and bars, mean ± S.D. of 10 individual cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with WT, one-way ANOVA with Tukey's post hoc test. D, concentration–response curve to GABA of WT γ2-β3-α1-β3-α1 (open black circles), γ2-β3^S254F-α1-β3-α1 (open red circles), γ2-β3-α1-β3^S254F-α1 (open purple squares), and γ2-β3^S254F-α1-β3^S254F-α1 (closed blue circles) receptors normalized to the Est. P_o(max) and fitted to the Hill equation. Dots, mean ± S.E. of 10–13 individual experiments. The EC₅₀ of the mutant receptor derived from the curve fit is shown. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with WT, one-way ANOVA with Tukey's post hoc test. E, representative traces of β3^Y302C mutant receptors after application of reference 3 mm GABA (filled bars) and 10 mm GABA, 1 μm diazepam, and 3 μm etomidate (open bars), respectively. Red traces, mutation at the second subunit location; purple traces, mutation at the fourth subunit location; blue traces, mutation at both the second and fourth locations. Scale bars, 500 nA and 100 s. F, estimated GABA P_o(max) of WT γ2-β3-α1-β3-α1 (open gray circles), γ2-β3^S254F-α1-β3-α1 (open red circles), γ2-β3-α1-β3^S254F-α1 (open purple squares), and γ2-β3^S254F-α1-β3^S254F-α1 (closed blue circles) mutant receptors. Est. P_o(max) was determined by dividing the current elicited by 3 mm GABA by the current elicited by 10 mm GABA, 1 μm diazepam, and 3 μm etomidate and corrected where 3 mm GABA was not at the maximum of the concentration–response curves. Lines and error bars, mean ± S.D. of 10 individual cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with WT, one-way ANOVA with Tukey's post hoc test.

Figure 7.

A–C, enlarged view of the α1β3γ2 cryo-EM structure (PDB code 6HUP) (41) showing the β3 mutant residues β3^D120 (A), β3^T157 (B), and β3^Y302 (C) in the two different subunit locations of the second β3 subunit (left) and fourth β3 subunit (right). The subunits are colored with the first γ2 subunit in green, the second β3 subunit in maroon, the third α1 subunit in light blue, the fourth β3 subunit in red, and the fifth α1 subunit in dark blue and the GABA molecule in blue, red, and green. Residues from the adjacent α1 or γ2 subunits are indicated. At the β1^D120 (A), β1^T1577 (B), and β1^Y302 (C) residues, the interacting partners are identical residues either on the adjacent subunit or within the β3 subunit itself.

Figure 8.

A–C, enlarged view of the GABA and diazepam-bound α1β3γ2 cryo-EM structure (PDB code 6HUP) (A), apo-α1β3γ2 (PDB 6i53) (B), and α1β1γ2 (PDB code 6DW0) (C) (40, 41) showing the β3^S254 or β1^S254 mutant residues in the two different subunit locations of the second β3 subunit (left) and fourth β3 subunit (right). The subunits are colored with the first γ2 subunit in green, the second β3 subunit in maroon, the third α1 subunit in light blue, the fourth β3 subunit in red, and the fifth α1 subunit in dark blue. Residues from adjacent α1 or γ2 subunits are indicated. Although the interacting partners of the Ser-254 residue are within the β subunit, the increased volume of the phenylalanine residue that substitutes for the serine at position 254 will cause the helix to occupy the space closer to the M3 helix of the adjacent subunit. When in the second position in the apo or α1β1γ2 structures, the M3 helix of the γ2 helix is kinked rather than parallel to the M1 helix of the β subunit. The residues of the M1 and M3 that face each other are the β1^M253, β1^L256, and β1^I259 residues for both subunits; the γ2^V341, γ2^I344, γ2^F345, and γ2^S348 residues of the first subunit (left); and the α1^Y321, α1^F323, and α1^Y325 residues of the third subunit (right). At the locations of all of these mutations, the sequence between β1 and β3 is identical.