High-Dose Benzodiazepines Positively Modulate GABAA Receptors via a Flumazenil-Insensitive Mechanism

Abstract

Benzodiazepines (BZDs) produce versatile pharmacological actions through positive modulation of GABA_A receptors (GABA_ARs). A previous study has demonstrated that high concentrations of diazepam potentiate GABA currents on the α₁β₂γ₂ and α₁β₂ GABA_ARs in a flumazenil-insensitive manner. In this study, the high-concentration effects of BZDs and their sensitivity to flumazenil were determined on synaptic (α₁β₂γ₂, α₂β₂γ₂, α₅β₂γ₂) and extra-synaptic (α₄β₂δ) GABA_ARs using the voltage-clamp electrophysiology technique. The in vivo evaluation of flumazenil-insensitive BZD effects was conducted in mice via the loss of righting reflex (LORR) test. Diazepam induced biphasic potentiation on the α₁β₂γ₂, α₂β₂γ₂ and α₅β₂γ₂ GABA_ARs, but did not affect the α₄β₂δ receptor. In contrast to the nanomolar component of potentiation, the second potentiation elicited by micromolar diazepam was insensitive to flumazenil. Midazolam, clonazepam, and lorazepam at 200 µM exhibited similar flumazenil-insensitive effects on the α₁β₂γ₂, α₂β₂γ₂ and α₅β₂γ₂ receptors, whereas the potentiation induced by 200 µM zolpidem or triazolam was abolished by flumazenil. Both the GABA_AR antagonist pentylenetetrazol and Fa173, a proposed transmembrane site antagonist, abolished the potentiation induced by 200 µM diazepam. Consistent with the in vitro results, flumazenil antagonized the zolpidem-induced LORR, but not that induced by diazepam or midazolam. Pentylenetetrazol and Fa173 antagonized the diazepam-induced LORR. These findings support the existence of non-classical BZD binding sites on certain GABA_AR subtypes and indicate that the flumazenil-insensitive effects depend on the chemical structures of BZD ligands.

Keywords: GABAA receptors; benzodiazepine; loss of righting reflex; non-classical binding site; voltage-clamp electrophysiology.

Figure 1

Diazepam modulated the α₁β₂γ₂, α₂β₂γ₂ and α₅β₂γ₂ receptors in flumazenil-sensitive and flumazenil-insensitive manners. The concentration-response relationships of diazepam for modulating the α₁β₂γ₂ (A), α₂β₂γ₂ (B), α₅β₂γ₂ (C), and α₄β₂δ (D) receptors were determined in the absence or presence of flumazenil. The GABA concentrations were 1, 0.1, 1, and 0.1 μM for α₁β₂γ₂, α₂β₂γ₂, α₅β₂γ₂, and α₄β₂δ receptors, respectively. Representative current traces are shown on the top; data summary is shown on the bottom. Data represent mean ± SEM, n = 4–5.

Figure 2

Different BZD ligands exhibited distinct modulation of ternary GABA_ARs. Effects of lorazepam (LOR), clonazepam (CLO), midazolam (MID), zolpidem (ZOL) and triazolam (TRI) in potentiating the GABA-elicited currents were evaluated at low (10 μM) and high (200 μM) concentrations in the absence or presence of flumazenil (100 μM) on the α₁β₂γ₂ (A), α₂β₂γ₂ (B), α₅β₂γ₂ (C) and α₄β₂δ (D) receptors. The dotted lines indicate the basal levels without drug treatment. Data represent mean ± SEM, n = 4, * p < 0.05, ** p < 0.01, vs control group, via t-test.

Figure 3

Pentylenetetrazol and Fa173 abolished the flumazenil-insensitive diazepam effects on ternary GABA_ARs. Effects of diazepam (DZP) in potentiating the GABA-elicited currents were evaluated at low (10 μM) and high (200 μM) concentrations in the absence or presence of pentylenetetrazol (PTZ, 100 μM) or Fa173 (100 μM) on the α₁β₂γ₂ (A), α₂β₂γ₂ (B) and α₅β₂γ₂ (C) receptors. The dotted lines indicate the basal levels without drug treatment. Data represent mean ± SEM, n = 4, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group, via t-test.

Figure 4

Flumazenil antagonized LORR induced by zolpidem, but not by diazepam or midazolam. A series of doses of diazepam (DZP), midazolam (MID) and zolpidem (ZOL) were administered intraperitoneally, and percentage of LORR (A,D,G) and latency to and duration of LORR (B,E,H) were recorded. Flumazenil (FLU) was intravenously injected immediately before BZD administration, and its effects on LORR induced by 50 mg kg⁻¹ diazepam, 100 mg kg⁻¹ midazolam or 50 mg kg⁻¹ zolpidem were evaluated (C,F,I). Data represent mean ± SEM, n = 9−10. Figure on top of each bar is the number of mice that lost the righting reflex over the total number of the mice tested. One-way ANOVA; * p < 0.05, ** p < 0.01 vs control group, according to post hoc analysis with Dunnett’s multiple comparison test.

Figure 5

Pentylenetetrazol and Fa173 antagonized diazepam-induced LORR. Pentylenetetrazol (PTZ, 20 mg kg⁻¹, i.v.) or Fa173 (1.2 μmol/mouse, i.c.v.) was injected immediately before diazepam (DZP, 50 mg kg⁻¹, i.p.) administration. The effect of pentylenetetrazol (A) or Fa173 (B) on latency to and duration of diazepam-induced LORR were recorded. Data represent mean ± SEM, n = 10. Figure on top of each bar is the number of mice that lost the righting reflex over the total number of the mice tested. * p < 0.05, ** p < 0.01, *** p < 0.001 vs control group, via t-test.