Figure 1.
Chemical structures of diazepam, 3-NCS and Ro15–1788.
Figure 2.
Alignment of Loop C of the α subunits.
The underlined residues were individually mutated to cysteine. The bold residues react covalently with 3-NCS. α4 was not investigated and is only shown for comparison.
Figure 3.
Covalent reaction at the binding level.
Extent of covalent reaction of 3-NCS at α1S204Cβ2γ2, α1S205Cβ2γ2, α1T206Cβ2γ2 mutant GABAA receptors and homologous receptors formed with α2, α3, and α6. α5 was expressed together with β1 and γ2 subunits for reasons mentioned in the results section. Receptors were expressed in HEK-293 cells, membranes harvested and exposed to 10 µM 3-NCS. After incubation, the residual 3-NCS was removed by filtration. [3H]Ro15–1788 was used as radioactive ligand to determine the residual binding. Covalent binding is expressed as 100% minus % residual binding. Data are shown as mean ±SD for three experiments each (triplicates of each point in each experiment).
Figure 4.
Concentration dependence at the binding level.
Concentration dependence of the covalent reaction of the 3-NCS at α6T205Cβ2γ2 mutant GABAA receptors. Extent of covalent reaction was determined at different concentrations of 3-NCS as indicated below Fig. 3. Data are shown as mean ±SD for three experiments each (triplicates of each point in each experiment).
Table 1.
Concentration dependence of the covalent reaction at mutant receptors at the binding level.
Figure 5.
Covalent reaction at the functional level.
Exposure of mutated α1S205Cβ2γ2 receptors to 3-NCS results in a covalent reaction. a) Receptors were exposed twice to 0.5 µM GABA followed by a washing period and a 3 min incubation in 10 µM 3-NCS. After this treatment GABA was applied twice with a 3 min interval. In some experiments GABA was applied up to six times. Thus, the current elicited by GABA was stimulated irreversibly. Application of 0.5 µM GABA in combination with 1 µM diazepam results in a further stimulation of the partially irreversibly reacted receptors that was smaller than that seen upon b) direct exposure to diazepam in independent experiments. Exposure of α1T204Cβ2γ2 receptors 3-NCS does not result in a covalent reaction. c) Receptors were exposed twice to 0.5 µM GABA followed by a 3 min incubation in 10 µM 3-NCS. Application of 0.5 µM GABA in combination with 1 µM diazepam results in stimulation similar to the stimulation seen in an oocyte that had not been previously exposed to 3-NCS (Fig. 4d).
Table 2.
Covalent reaction at the functional level.
Figure 6.
The results obtained with NCS [33] and the results obtained with 3-NCS described here are summarized. For each receptor isoform αxβ1/2γ2 (x = 1, 2, 3, 5, 6) amino acid residues homologous to 101 in α1 are shown in black, those homologous to 204 are shown in red, those homologous to 205 are shown in blue and those homologous to 206 are shown in green. Residue 101 is located near to the C atom carrying the Cl atom in diazepam, and 204, 205 and 206 are near to C atom adjacent to the keto group. The residues showing covalent reaction are shown in full color and those showing no reaction coloured in reduced saturation. The residues in larger font size react with a high apparent affinity. The residues marked with * and (*) indicate an antagonistic effect and a partial modulatory effect by 3-NCS on the corresponding α5mβ2γ2 receptors, respectively. The residues marked with + indicate that the corresponding α3mβ2γ2 receptors were only investigated at the functional level.