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, receptor complex has been suggested to contain arginine residues. The aim of this s tudy 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, 1 23, 135, and 224) as well as two nonconserved arginines (79 and 190), all l ocated in the extracellular N-terminal segment of the alpha(5) subunit, wer e substituted by lysines. The individual alpha(5) subunit mutants were coex pressed with human beta(2) and gamma(2s) GABA(A) receptor subunits in Chine se hamster ovary cells by transient transfection. Electrophysiological whol e-cell patch-clamp recordings show that, of the eight arginine residues tes ted, the two arginines at positions 70 and 123 appear to be essential for t he GABA-gated chloride current because the EC50 values of the two mutant co nstructs increase >100-fold compared with the wild-type alpha(5),beta(2),ga mma(2S) GABA(A) receptor. However, diazepam and allopregnanolone modulation and pentobarbital stimulation properties are unaffected by the introductio n of lysines at positions 70 and 123. A double mutant carrying lysine subst itutions at positions 70 and 123 is virtually insensitive to GABA, suggesti ng alterations of one or more GABA binding sites.