Covalent modification of GABAA receptor isoforms by a diazepam analogue provides evidence for a novel benzodiazepine binding site that prevents modulation by these drugs

Abstract

Classical benzodiazepines, for example diazepam, interact with αxβ2γ2 GABAA receptors, x = 1, 2, 3, 5. Little is known about effects of α subunits on the structure of the binding pocket. We studied here the interaction of the covalently reacting diazepam analog 7-Isothiocyanato-5-phenyl-1,3-dihydro-2H-1, 4-benzodiazepin-2-one (NCS compound) with α1H101Cβ 2γ2 and with receptors containing the homologous mutation, α2H101Cβ2γ2, α3H126Cβ2γ2 and α5H105Cβ2γ2. This comparison was extended to α6R100Cβ2γ2 receptors as this mutation conveys to these receptors high affinity towards classical benzodiazepines. The interaction was studied at the ligand binding level and at the functional level using electrophysiological techniques. Results indicate that the geometry of α6R100Cβ2γ 2 enables best interaction with NCS compound, followed by α3H126Cβ2γ2, α1H101Cβ2γ2 and α2H101Cβ2γ2, while α5H105Cβ2γ2 receptors show little interaction. Our results allow conclusions about the relative apposition of α1H101 and homologous positions in α2, α3, α5 and α6 with the position occupied by -Cl in diazepam. During this study we found evidence for the presence of a novel site for benzodiazepines that prevents modulation of GABAA receptors via the classical benzodiazepine site. The novel site potentially contributes to the high degree of safety to some of these drugs. Our results indicate that this site may be located at the α/β subunit interface pseudo-symmetrically to the site for classical benzodiazepines located at the α/γ interface. © 2008 The Authors.