Two conserved arginines in the extracellular N-terminal domain of the GABA(A) receptor alpha(5) subunit are crucial for receptor function.

The gamma-aminobutyric acid (GABA) binding pocket within the GABA(A) 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 GABA(A) receptor alpha 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 alpha(5) subunit, were substituted by lysines. The individual alpha(5) subunit mutants were coexpressed with human beta(2) and gamma(2s) GABA(A) 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 EC(50) values of the two mutant constructs increase >100-fold compared with the wild-type alpha(5),beta(2), gamma(2s) GABA(A) 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.