Abstract:The ␥-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 ␣ 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 ␣ 5 subunit, were substituted by lysines. The individual ␣ 5 subunit mutants were coexpressed with human ␤ 2 and ␥ 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 Ͼ100-fold compared with the wild-type ␣ 5 ,␤ 2 ,␥ 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.

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