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Figure 1. A, pentameric structure of the GABA and diazepam-bound 132 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 2 (green), second 3 (maroon), third 1 (blue), fourth 3 (red), and fifth 1 (dark blue).
B, side view showing the first 2 subunit adjacent to the second 3 subunit. The 2R323 residue on the M2-M3 loop is depicted with the side chain in black in a
transparent sphere. C, side view showing the fourth 3 subunit adjacent to the fifth 1 subunit, with the GABA-binding site highlighted. The side chains of the
3D120N residue at the GABA-binding site, the 3T157M residue within an internal -sheet, the 3Y302C residue in the M2-M3 loop, and the 3S254F residue in the
M1 region are shown in black.
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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 M GABA (closed bars) were applied prior to co-application of 10 M 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 Emax was 306% (320 32, mean S.E., n 10).
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Figure 3. A, schematic of concatenated receptor indicating the location of mutations when they are introduced into the 2 or distinct 3 subunits. Red circles
indicate the location of mutations on the first 2 subunit, and red circles, purple squares, and blue circles indicate location of mutations on the second, fourth,
or both second and fourth 3 subunits, respectively. B, representative traces of WT and 2R323Q, 3D120N, 3T157M, 3S254F, and 3Y302C 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.
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Figure 4. A,schematic of concatenated receptor indicating the location of the 2R323Q 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 2R323Q-3-1-3-1 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 WT2-3-1-3-1 (E) and 2R323Q-3-1-3-1 (E) receptors normalized to the Est. Po(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) and2R323Q (red) mutant receptors after application of reference 3 mM GABA (filled bars) and 10 mM GABA, 1 M diazepam, and 3 M etomidate (open bars), respectively. Scale bars, 500 nA and 100 s. The Est. GABA Po(max) of WT (E) and 2R323Q (E) mutant receptors (below) was determined by dividing the current elicited by 3 mM GABA by the current elicited by 10 mM GABA, 1 M diazepam, and 3 M etomidate and corrected for the reference 3 mM GABA current. Lines and error bars represent the mean S.D. of 10 individual cells.
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Figure 5. A, schematic of concatenated receptor indicating the location of 3D120N and 3T157M 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 WT2-3-1-3-1 (open black circle), 2-3D120N-1-3-1 (open red circle), 2-3-1-3D120N-1 (open purple square), and 2-3D120N-1-3D120N-1 (closed blue circle) receptors; C, Concentration–response curve to GABA of WT 2-3-1-3-1 (open black circle), 2-3T157M-1-3-1 (open red circle), 2-3-1 3T157M-1 (open purple square), and 2-3T157M-1-3T157M-1 (closed blue circle) receptors normalized to the Est. Po(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 of3D120N and3T157M receptors after application of 3mM GABA(filled bars) and 10mM GABA, 1M diazepam, and 3M 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, estimatedGABAPo(max)ofWT, 3D120N, and 3T157M receptors was determined by dividing the current elicited by 3 mM GABA by the current elicited by 10 mM GABA, 1 M diazepam, and 3 M 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.
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Figure 6. A, concentration–response curve to GABA of WT 2-3-1-3-1 (open black circles), 2-3Y302C-1-3-1 (open red circles), 2-3-1-3Y302C-1 (open purple squares) and 2-3Y302C-1-3Y302C-1 (closed blue circles) receptors normalized to the Est. Po(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 3Y302C mutant receptors after application of reference 3 mM GABA
(filled bars) and 10 mM GABA, 1 M diazepam, and 3 M etomidate (open bars), respectively. Red tracesindicate 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 Po(max) of WT 2-3-1-3-1 (open black circles), 2-3Y302C-1-3-1 (open red circles), 2-3-1-3Y302C-1 (open purple squares) and2-3Y302C-1-3Y302C-1 (closed blue circles) mutant receptors. Est. Po(max) was determined by dividing the current elicited by 3 mM GABA by the current elicited by 10 mM GABA, 1M diazepam, and 3M 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 2-3-1-3-1 (open black circles), 2-3S254F-1-3-1 (open red circles), 2-3-1-3S254F-1 (open purple squares), and 2-3S254F-1-3S254F-1 (closed blue circles) receptors normalized to the Est. Po(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 3Y302C mutant receptors after application of reference 3 mM GABA (filled bars) and 10 mM GABA, 1M diazepam, and 3M 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 GABAPo(max)of WT 2-3-1-3-1 (open gray circles), 2-3S254F-1-3-1 (open red circles), 2-3-1-3S254F-1 (open purple squares), and 2-3S254F-1-3S254F-1 (closed blue circles) mutant receptors. Est. Po(max) was determined by dividing the current elicited by 3 mM GABA by the current elicited by 10 mM GABA, 1 M diazepam, and 3 M 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.
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Figure 7. A–C, enlarged view of the 132 cryo-EM structure (PDB code 6HUP) (41) showing the 3 mutant residues 3D120 (A), 3T157 (B), and 3Y302 (C) in the two
different subunit locations of the second3 subunit(left) and fourth 3 subunit (right).The subunits are colored with the first2 subunit in green, the second 3 subunit in maroon, the third 1 subunit in light blue, the fourth 3 subunit in red, and the fifth 1 subunit in dark blue and the GABA molecule in blue, red, and green. Residues from the adjacent 1 or2 subunits are indicated. At the 1D120 (A), 1T1577 (B), and 1Y302 (C) residues, the interacting partners are identical residues either on the adjacent subunit or within the 3 subunit itself.
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SI 1. (A) RNA gel of concatenated receptor showing single RNA band at the expected size of ~7.5 kb. (B) Western blot with uninjected oocytes, oocytes injected with loose subunits or the concatenated construct. Concatenated receptor is translated at the expected size without any additional fragments.
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