An intersubunit electrostatic interaction in the GABAA receptor facilitates its responses to benzodiazepines

Abstract

Benzodiazepines are positive allosteric modulators of the GABA_A receptor (GABA_AR), acting at the α-γ subunit interface to enhance GABA_AR function. GABA or benzodiazepine binding induces distinct conformational changes in the GABA_AR. The molecular rearrangements in the GABA_AR following benzodiazepine binding remain to be fully elucidated. Using two molecular models of the GABA_AR, we identified electrostatic interactions between specific amino acids at the α-γ subunit interface that were broken by, or formed after, benzodiazepine binding. Using two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes, we investigated these interactions by substituting one or both amino acids of each potential pair. We found that Lys¹⁰⁴ in the α₁ subunit forms an electrostatic bond with Asp⁷⁵ of the γ₂ subunit after benzodiazepine binding and that this bond stabilizes the positively modified state of the receptor. Substitution of these two residues to cysteine and subsequent covalent linkage between them increased the receptor's sensitivity to low GABA concentrations and decreased its response to benzodiazepines, producing a GABA_AR that resembles a benzodiazepine-bound WT GABA_AR. Breaking this bond restored sensitivity to GABA to WT levels and increased the receptor's response to benzodiazepines. The α₁ Lys¹⁰⁴ and γ₂ Asp⁷⁵ interaction did not play a role in ethanol or neurosteroid modulation of GABA_AR, suggesting that different modulators induce different conformational changes in the receptor. These findings may help explain the additive or synergistic effects of modulators acting at the GABA_AR.

Keywords: Cys-loop receptor; GABA receptor; Xenopus; allosteric regulation; benzodiazepines; cysteine-mediated cross-linking; electrophysiology; electrostatics; ionotropic receptor; sedative.

Figure 1.

Two different homology models of the α₁ (orange)–γ₂ (green) interface of the GABA_AR. The model on the left is based on a modified ivermectin-unbound GluCl crystal structure (17) and represents the GABA-unbound closed state of the channel. The model on the right is based on the glutamate-bound GluCl crystal structure with a contribution from the ELIC crystal structure (18) and represents the diazepam (in red)-bound receptor. Both models depict the inside of the interface. Labeled interactions represent putative electrostatic interactions of residues 6 Å or less apart that are predicted to occur between residues in the α₁ and γ₂ subunits before (A–F) or after (H–K) diazepam binding. A, α₁ Arg²⁸–γ₂ Asp²⁶; 5 Å. B, α₁ Glu¹⁶⁵–γ₂ Arg⁹⁷; 4 Å. C, α₁ Glu¹³⁷–γ₂ Arg¹⁹⁴; 5 Å. D, α₁ Glu⁵⁸–γ₂ Arg¹⁹⁷; 5 Å. E, α₁ Asp⁵⁶–γ₂ Arg¹⁹⁷; 5 Å. F, α₁ Lys²⁷⁸–γ₂ Asp¹⁶¹; 5 Å. G, α₁ Lys³¹¹–γ₂ Asp²⁶⁰; 3 Å. H, α₁ Lys¹⁰⁵–γ₂ Asp¹²⁰; 5 Å. I, α₁ Lys¹⁰⁴–γ₂ Asp⁷⁵; 5 Å. J, α₁ Glu⁵⁸–γ₂ Arg¹⁹⁷; 5 Å. K, α₁ Asp⁵⁶–γ₂ Arg¹⁹⁷; 6 Å. Black dashed lines represent intersubunit bonds.

Figure 2.

Diazepam enhancement of GABA_AR function is altered in some cysteine mutations of residues predicted to form electrostatic interactions at the α₁–γ₂ subunit interface. EC_5–10 GABA was applied alone as well as in the presence of 1 μm diazepam to WT and multiple cysteine-substituted receptors. The horizontal dashed line indicates the level of potentiation produced by diazepam in WT receptors. A one-way ANOVA showed a significant effect of cysteine substitution on receptor enhancement by 1 μm diazepam (F(11,60) = 26.310, p < 0.001). A post hoc Tukey's test showed a significant change (p < 0.001) in potentiation by 1 μm diazepam in α₁(E58C, α₁(K104C)-, α₁(E137C)-, γ₂(D75C)-, and γ₂(R197C)-containing GABA_AR. Each symbol represents the percent potentiation of the GABA EC_5–10 seen in one oocyte, and each bar represents the mean percent potentiation. Error bars represent the S.E.

Figure 3.

Homology models of the α₁ (orange)–γ₂ (green) interface inside the GABA_A receptor in both the GABA-unbound closed state of the channel and the diazepam-bound open state of the channel. These models predict that the nitrogen atom of α₁ lysine 104 (orange residue) and oxygen atom of γ₂ aspartic acid 75 (green residue) are within 9 Å of each other before GABA and diazepam bind (A and enlarged in B) but move to within 5 Å of each other after GABA and diazepam (in red) bind to the receptor (C).

Figure 4.

Formation and breakage of the disulfide bond between α₁(K104C) and γ₂(D75C) affects responses to GABA. A, GABA concentration-response curves were generated in WT α₁β₂γ₂, single mutants α₁(K104C)β₂γ₂ and α₁β₂γ₂(D75C), and double mutant α₁(K104C)β₂γ₂(D75C) GABA_A receptors. A repeated-measures ANOVA revealed no difference in the concentration-response curve between WT and mutant receptors. However, one-way ANOVAs showed significant effects of mutation at 3 μm (F(3,26) = 15.504, p < 0.001) and 10 μm GABA (F(3,26) = 18.163, p < 0.001) with a Tukey's post hoc test at both concentrations showing a significant increase in response in α₁(K104C)β₂γ₂(D75C) receptors compared with the other three receptors (***, p < 0.001). Some symbols are hidden behind other symbols. B, DTT (2 mm; dark symbols and bars) increased the absolute concentration of GABA required to produce an EC₅ response in α₁(K104C)β₂γ₂(D75C) but not WT receptors. A two-way ANOVA followed by a Tukey's post hoc test revealed a significant effect of DTT treatment on α₁(K104C)β₂γ₂(D75C) receptors (***, p < 0.001). Each symbol represents the GABA EC₅ of one oocyte, and each bar represents the mean GABA EC₅. Error bars represent the S.E.

Figure 5.

α₁(K104C)β₂γ₂(D75C) receptors spontaneously cross-link and reform this cross-link after DTT application. A, illustration depicting the disulfide bond that spontaneously cross-links the α₁ and γ₂ subunits of the α₁(K104C)β₂γ₂(D75C) GABA_AR, is broken after DTT application, and slowly reforms over time. B, effect of PMTS application on currents elicited by the GABA EC_5–10 of WT, α₁(K104C)β₂γ₂, α₁β₂γ₂(D75C), and α₁(K104C)β₂γ₂(D75C) GABA_A receptors. The change in GABA EC_5–10 currents by PMTS was decreased in single mutant receptors both before (F(3,18) = 14.56, p < 0.05) and after (F(3,18) = 45.45, p < 0.001) DTT application compared with those seen in WT receptors. The change in EC_5–10 currents produced by PMTS was not significantly altered after DTT application to WT or single mutant receptors but did significantly change in double mutant receptors (F(3,37) = 41.698, p < 0.001)). A one-way ANOVA showed a significant effect of time after DTT treatment (pre-DTT treatment, 5 min after, and 60 min after) on α₁(K104C)β₂γ₂(D75C) GABA_AR (F(2,13) = 108.363, p < 0.001), and a Tukey's post hoc test showed a significant difference between pre-DTT and 5 min after DTT application and a significant difference between 5 min after DTT and 60 min after DTT (***, p < 0.001; each symbol represents an oocyte, and each bar represents the mean response. Error bars represent the S.E.). C, sample tracing showing spontaneous reformation of the α₁–γ₂ intersubunit disulfide bond in the α₁(K104C)β₂γ₂(D75C) GABA_AR. The GABA EC₅ measured in the oocyte before DTT application was 3 μm GABA, but after DTT application 3 μm GABA elicited a much smaller response. After ∼60 min, the response to 3 μm GABA had returned to pre-DTT levels. D, time courses of EC₅ values plotted for five oocytes expressing α₁(K104C)β₂γ₂(D75C) receptors returning to their pre-DTT values. This can also be interpreted as the time required to reform the disulfide bond after DTT application. The average time to return to half of the pre-DTT EC₅ was 26.9 ± 2.5 min.

Figure 6.

Benzodiazepine responses of WT and mutant GABA_A receptors before (white symbols and bars) and after (dark symbols and bars) DTT application. Bar graphs show the percent potentiation of GABA EC_5–10 in WT, α₁(K104C)β₂γ₂, α₁β₂γ₂(D75C), and α₁(K104C)β₂γ₂(D75C) GABA_A receptors produced by 1 μm diazepam (A), flunitrazepam (B), Ro 15-4513 (C), and zolpidem (D). A two-way ANOVA followed by a Tukey's multiple comparison post hoc test showed a significant increase in all benzodiazepine-site responses after DTT application to α₁(K104C)β₂γ₂(D75C) GABA_A receptors but not WT or single mutant receptors (*, p < 0.05; ***, p < 0.001 with each symbol representing the percent potentiation of the GABA EC_5–10 seen in one oocyte, and each bar representing the mean percent potentiation. Error bars represent the S.E.).

Figure 7.

Sample tracings showing the effects of DTT and H₂O₂ treatment on potentiation by 1 μm diazepam and 1 μm flunitrazepam. The top panels show tracings obtained from oocytes expressing WT receptors, and the bottom panels show tracings of oocytes expressing α₁(K104C)β₂γ₂(D75C) GABA_A receptors. DTT application to α₁(K104C)β₂γ₂(D75C) receptors increased both diazepam (A) and flunitrazepam (B) potentiation, and hydrogen peroxide application reversed this increase back to pre-DTT levels.

Figure 8.

Modulators acting at sites other than the benzodiazepine site at WT and cysteine-substituted GABA_A receptors are unaffected by DTT treatment. Before modulator effects were tested, the EC_5–10 concentration of GABA was determined in each oocyte. Effects of 200 mm ethanol and 100 nm allopregnanolone were measured in WT and α₁(K104C)β₂γ₂(D75C) GABA_A receptors before (white symbols and bars) and after (dark symbols and bars) application of DTT with no significant changes being observed (two-way ANOVAs; 200 mm ethanol (F(3,43) = 0.031); 100 nm allopregnanolone (F(3,45) = 0.176). Each symbol represents the percent potentiation of the GABA EC_5–10 seen in one oocyte, and each bar represents the mean percent potentiation. Error bars represent the S.E.

Figure 9.

Effect of alanine substitution at α₁ Lys¹⁰⁴ and γ₂ Asp⁷⁵ on GABA sensitivity and benzodiazepine responses. A, GABA concentration-response curves of WT, α₁(K104A)β₂γ₂, α₁β₂γ₂(D75A), and α₁(K104A)β₂γ₂(D75A) receptors. The concentration-response curves were significantly different (F(21,132) = 1.937, p < 0.05). Each symbol represents the data from three to six oocytes, and error bars represent the S.E. In some cases, error bars fall within symbols. B, bar graph comparing levels of diazepam and flunitrazepam potentiation between WT and alanine-substituted receptors. Potentiation of GABA EC_5–10 by 1 μm diazepam and 1 μm flunitrazepam was decreased for single but not double alanine substitution mutants compared with WT receptors. A one-way ANOVA revealed a significant effect of mutant on receptor potentiation by diazepam (F(3,23) = 51.407, p < 0.001) and flunitrazepam (F(3,19) = 26.926, p < 0.001). Each symbol represents the percent potentiation observed in one oocyte, and each bar represents the mean percent potentiation. Error bars represent the S.E.

Figure 10.

Charge reversal at α₁ Lys¹⁰⁴ and γ₂ Asp⁷⁵ does not rescue GABA sensitivity or benzodiazepine responses to WT responses. A, the α₁(K104D)β₂γ₂(D75K) receptor GABA concentration-response curve is significantly right-shifted compared with WT receptors (F(8,105) = 2.8, p < 0.01). The EC₅₀ for WT receptors was 86.8 ± 16.5 μm, increasing to 146.3 ± 23.1 μm for the α₁(K104D)β₂γ₂(D75K) GABA_AR. Each symbol represents the mean from five to six oocytes, and error bars represent the S.E. B, bar graph comparing levels of benzodiazepine enhancement between α₁(K104D)β₂γ₂(D75K) and WT receptors. The α₁(K104D)β₂γ₂(D75K) GABA_AR was unable to fully rescue responses to WT levels of potentiation by 1 μm diazepam and Ro 15-4513 but was able to rescue the responses to 1 μm flunitrazepam and zolpidem. Each symbol represents the percent potentiation of the GABA EC_5–10 seen in one oocyte, and each bar represents the mean potentiation observed. Error bars represent the S.E.