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

Classical benzodiazepines, for example diazepam, interact with α x β 2 γ 2 GABA A 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 α 1 H101Cβ 2 γ 2 and with receptors containing the homologous mutation, α 2 H101Cβ 2 γ 2 , α 3 H126Cβ 2 γ 2 and α 5 H105Cβ 2 γ 2 . This comparison was extended to α 6 R100Cβ 2 γ 2 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 α 6 R100Cβ 2 γ 2 enables best interaction with NCS compound, followed by α 3 H126Cβ 2 γ 2 , α 1 H101Cβ 2 γ 2 and α 2 H101Cβ 2 γ 2 , while α 5 H105Cβ 2 γ 2 receptors show little interaction. Our results allow conclusions about the relative apposition of α 1 H101 and homologous positions in α 2 , α 3 , α 5 and α 6 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 GABA A 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.