Identification of a residue in the γ‐aminobutyric acid type A receptor α subunit that differentially affects diazepam‐sensitive and ‐insensitive benzodiazepine site binding

GABA A receptors that contain either the α4‐ or α6‐subunit isoform do not recognize classical 1,4‐benzodiazepines (BZDs). However, other classes of BZD site ligands, including β‐carbolines, bind to these diazepam‐insensitive receptor subtypes. Some β‐carbolines [e.g. ethyl β‐carboline‐3‐carboxylate (β‐CCE) and methyl 6,7‐dimethoxy‐4‐ethyl‐β‐carboline‐3‐carboxylate (DMCM)] display a higher affinity for α4‐ compared to α6‐containing receptors. In order to identify the structural determinants that underlie these affinity differences, we constructed chimeric α6/α4 subunits and co‐expressed these with wild‐type rat β2 and γ2 L subunits in tsA201 cells for radioligand binding analysis. After identification of candidate regions, site‐directed mutagenesis was used to narrow the ligand selectivity to a single amino acid residue (α6N204/α4I203). Substitutions at α6N204 did not alter the affinity of the imidazobenzodiazepine Ro15‐4513. A homologous mutation in the diazepam‐sensitive α1 subunit (S205N) resulted in a 7–8‐fold reduction in affinity for the β‐carbolines examined. Although the binding of the classical agonist flunitrazepam was relatively unaffected by this mutation in the α1 subunit, the affinity for Ro15‐1788 and Ro15‐4513 was decreased by ∼19‐fold and ∼38‐fold respectively. The importance of this residue, located in the Loop C region of the extracellular N‐terminus of the subunit protein, emphasizes the differential interaction of ligands with the α subunit in diazepam‐sensitive and ‐insensitive receptors.