Flumazenil-Insensitive Benzodiazepine Effects in Recombinant alpha beta and Neuronal GABA(A) Receptors

Gamma-aminobutyric acid, type A (GABA(A)) receptors are complex heterogeneous pentamers with various drug binding sites. Several lines of evidence suggest that benzodiazepines modulate certain GABA(A) receptors in a flumazenil-insensitive manner, possibly via binding sites other than the classical ones. However, GABA(A) receptor subtypes that contain non-classical benzodiazepine binding sites are not systemically studied. The present study investigated the high-concentration effects of three benzodiazepines and their sensitivity to flumazenil on different recombinant (alpha 1 beta 2, alpha 2 beta 2, alpha 3 beta 2, alpha 4 beta 2, alpha 5 beta 2 and alpha 1 beta 3) and native neuronal GABA(A) receptors using the whole-cell patch-clamp electrophysiology technique. The classical benzodiazepine diazepam (200 mu mol/L) and midazolam (200 mu mol/L) produced flumazenil-insensitive effects on alpha 1 beta 2 receptor, whereas the imidazopyridine zolpidem failed to modulate the receptor. Flumazenil-insensitive effects of diazepam were also observed on the alpha 2 beta 2, alpha 3 beta 2 and alpha 5 beta 2, but not alpha 4 beta 2 receptors. Unlike beta 2-containing receptors, the alpha 1 beta 3 receptor was insensitive to diazepam. Moreover, the diazepam (200 mu mol/L) effects on some cortical neurons could not be fully antagonized by flumazenil (200 mu mol/L). These findings suggested that the non-classical (flumazenil-insensitive) benzodiazepine effects depended on certain receptor subtypes and benzodiazepine structures and may be important for designing of subtype- or binding site- specific drugs.