Relative positioning of classical benzodiazepines to the γ₂-subunit of GABA_A receptors

GABA_A receptors are the major inhibitory neurotransmitter receptors in the brain. Benzodiazepine exert their action via a high affinity-binding site at the α/γ subunit interface on some of these receptors. Diazepam has sedative, hypnotic, anxiolytic, muscle relaxant, and anticonvulsant effects. It acts by potentiating the current evoked by the agonist GABA. Understanding specific interaction of benzodiazepines in the binding pocket of different GABA_A receptor isoforms might help to separate these divergent effects. As a first step, we characterized the interaction between diazepam and the major GABA_A receptor isoform α₁β₂γ₂. We mutated several amino acid residues on the γ₂-subunit assumed to be located near or in the benzodiazepine binding pocket individually to cysteine and studied the interaction with three ligands that are modified with a cysteine-reactive isothiocyanate group (-NCS). When the reactive NCS group is in apposition to the cysteine residue this leads to a covalent reaction. In this way, three amino acid residues, γ₂Tyr58, γ ₂Asn60, and γ₂Val190 were located relative to classical benzodiazepines in their binding pocket on GABA_A receptors.