Two Conserved Arginines in the Extracellular N‐Terminal Domain of the GABAA Receptor α5 Subunit Are Crucial for Receptor Function

Abstract: The γ‐aminobutyric acid (GABA) binding pocket within the GABAA receptor complex has been suggested to contain arginine residues. The aim of this study was to test this hypothesis by mutating arginine residues potentially contributing to the formation of a GABA binding pocket. Thus, six arginines conserved in human GABAA receptor α subunits (arginine 34, 70, 77, 123, 135, and 224) as well as two nonconserved arginines (79 and 190), all located in the extracellular N‐terminal segment of the α5 subunit, were substituted by lysines. The individual α5 subunit mutants were coexpressed with human β2 and γ2s GABAA receptor subunits in Chinese hamster ovary cells by transient transfection. Electrophysiological whole‐cell patch‐clamp recordings show that, of the eight arginine residues tested, the two arginines at positions 70 and 123 appear to be essential for the GABA‐gated chloride current because the EC50 values of the two mutant constructs increase >100‐fold compared with the wild‐type α5,β2,γ2s GABAA receptor. However, diazepam and allopregnanolone modulation and pentobarbital stimulation properties are unaffected by the introduction of lysines at positions 70 and 123. A double mutant carrying lysine substitutions at positions 70 and 123 is virtually insensitive to GABA, suggesting alterations of one or more GABA binding sites.