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Figure 1-Concentration response curve for GABA at β2γ2 GABAA receptors. Receptors were expressed in Xenopus oocytes and exposed to subsequently higher concentrations of GABA and the elicited current amplitude was determined. Individual curves were first normalized to the fitted maximal current amplitude and subsequently averaged. Data are expressed as mean ± S.E.M., n = 5 from two batches of oocytes. (a) Original current traces. GABA applications are indicated by a bar. The numbers indicate the concentration of GABA in μM. (b) Averaged concentration-response curve for β2γ2 GABAA receptors. The dotted line shows for comparison corresponding data on α1β2γ2 GABAA receptors.
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Figure 2-Concentration response curve for diazepam at β2γ2 GABAA receptors. Receptors were expressed in Xenopus oocytes and exposed to either GABA alone or GABA in the presence of subsequently higher concentrations of diazepam and the elicited current amplitude was determined. At each concentration of diazepam current potentiation was calculated. Individual curves for potentation were first normalized to the fitted maximal current amplitude and subsequently averaged. Data are expressed as mean ± S.E.M., n = 3 from two batches of oocytes. (a) Original current traces. (b) Averaged concentration-response curve for β2γ2 GABAA receptors. The dotted line shows for comparison corresponding data on α1β2γ2 GABAA receptors.
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Figure 3-Sequence alignment of α1, β2 and γ2 subunits of the rat GABAA receptor. Mutated residues of β2 and γ2 subunits are indicated with numbers. Homologous positions of the α1 subunit are also highlighted.
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Figure 4-Influence of different point mutations on the concentration dependence of GABA. Wild type or mutant β2γ2 GABAA receptors were expressed in Xenopus oocytes and exposed to subsequently higher concentrations of GABA and the elicited current amplitude was determined. Individual curves for each subunit combination were first normalized to the fitted maximal current amplitude and subsequently averaged. Averaged concentration-response curve are shown. Data are expressed as mean ± S.E.M., n = 3–7 from two batches of oocytes.
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Figure 5-Influence of different point mutations on the potentiation by diazepam. Receptors were expressed in Xenopus oocytes and first exposed to 7 µM GABA alone or the same concentration of GABA in the presence of 1 µM diazepam and the elicited current amplitude was determined. Current potentiation by diazepam was calculated and averaged for each subunit combination. Data are expressed as mean ± S.E.M., n = 4–15 from two batches of oocytes.
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Figure 6-Structural hypothesis for diazepam binding at the extracellular β2+/γ2 interface. Panel (a) shows the reference binding pose from our previous studies17 at the α1+ (orange)/ γ2− (cyan) interface. Panel (b) shows the most closely corresponding binding pose from the computational docking at the β2+ (red)/γ2− (cyan) interface. The homologous key amino acids in the binding pockets, as well as diazepam, are rendered in stick representation. While sidechain rotamers show some differences, ligand position, binding mode and key interations are very similar.
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Figure 7-Schematic representation of β2γ2 GABAA receptors. The location of amino acid residues of interest is indicated. Point mutations resulting in disrupting assembly are not shown. The binding site for GABA is concluded to locate at the β2+/ β2− interface, that for diazepam at the β2+/γ2− interface. Please note that the subunit arrangement was not addressed in this study.
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Figure 1. Concentration response curve for GABA at β2γ2 GABAA receptors. Receptors were expressed in Xenopus oocytes and exposed to subsequently higher concentrations of GABA and the elicited current amplitude was determined. Individual curves were first normalized to the fitted maximal current amplitude and subsequently averaged. Data are expressed as mean ± S.E.M., n = 5 from two batches of oocytes. (a) Original current traces. GABA applications are indicated by a bar. The numbers indicate the concentration of GABA in μM. (b) Averaged concentration-response curve for β2γ2 GABAA receptors. The dotted line shows for comparison corresponding data on α1β2γ2 GABAA receptors.
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Figure 2. Concentration response curve for diazepam at β2γ2 GABAA receptors. Receptors were expressed in Xenopus oocytes and exposed to either GABA alone or GABA in the presence of subsequently higher concentrations of diazepam and the elicited current amplitude was determined. At each concentration of diazepam current potentiation was calculated. Individual curves for potentation were first normalized to the fitted maximal current amplitude and subsequently averaged. Data are expressed as mean ± S.E.M., n = 3 from two batches of oocytes. (a) Original current traces. (b) Averaged concentration-response curve for β2γ2 GABAA receptors. The dotted line shows for comparison corresponding data on α1β2γ2 GABAA receptors.
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Figure 3. Sequence alignment of α1, β2 and γ2 subunits of the rat GABAA receptor. Mutated residues of β2 and γ2 subunits are indicated with numbers. Homologous positions of the α1 subunit are also highlighted.
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Figure 4. Influence of different point mutations on the concentration dependence of GABA. Wild type or mutant β2γ2 GABAA receptors were expressed in Xenopus oocytes and exposed to subsequently higher concentrations of GABA and the elicited current amplitude was determined. Individual curves for each subunit combination were first normalized to the fitted maximal current amplitude and subsequently averaged. Averaged concentration-response curve are shown. Data are expressed as mean ± S.E.M., n = 3–7 from two batches of oocytes.
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Figure 5. Influence of different point mutations on the potentiation by diazepam. Receptors were expressed in Xenopus oocytes and first exposed to 7 µM GABA alone or the same concentration of GABA in the presence of 1 µM diazepam and the elicited current amplitude was determined. Current potentiation by diazepam was calculated and averaged for each subunit combination. Data are expressed as mean ± S.E.M., n = 4–15 from two batches of oocytes.
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Figure 6. Structural hypothesis for diazepam binding at the extracellular β2+/γ2 interface. Panel (a) shows the reference binding pose from our previous studies17 at the α1+ (orange)/ γ2− (cyan) interface. Panel (b) shows the most closely corresponding binding pose from the computational docking at the β2+ (red)/γ2− (cyan) interface. The homologous key amino acids in the binding pockets, as well as diazepam, are rendered in stick representation. While sidechain rotamers show some differences, ligand position, binding mode and key interations are very similar.
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Figure 7. Schematic representation of β2γ2 GABAA receptors. The location of amino acid residues of interest is indicated. Point mutations resulting in disrupting assembly are not shown. The binding site for GABA is concluded to locate at the β2+/ β2− interface, that for diazepam at the β2+/γ2− interface. Please note that the subunit arrangement was not addressed in this study.
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