High-Dose Benzodiazepines Positively Modulate GABA(A) Receptors via a Flumazenil-Insensitive Mechanism

Benzodiazepines (BZDs) produce versatile pharmacological actions through positive modulation of GABA(A) receptors (GABA(A)Rs). A previous study has demonstrated that high concentrations of diazepam potentiate GABA currents on the α(1)β(2)γ(2) and α(1)β(2) GABA(A)Rs in a flumazenil-insensitive manner. In this study, the high-concentration effects of BZDs and their sensitivity to flumazenil were determined on synaptic (α(1)β(2)γ(2), α(2)β(2)γ(2), α(5)β(2)γ(2)) and extra-synaptic (α(4)β(2)δ) GABA(A)Rs 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 α(1)β(2)γ(2), α(2)β(2)γ(2) and α(5)β(2)γ(2) GABA(A)Rs, but did not affect the α(4)β(2)δ 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 α(1)β(2)γ(2), α(2)β(2)γ(2) and α(5)β(2)γ(2) receptors, whereas the potentiation induced by 200 µM zolpidem or triazolam was abolished by flumazenil. Both the GABA(A)R 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(A)R subtypes and indicate that the flumazenil-insensitive effects depend on the chemical structures of BZD ligands.