GABAA receptors, along with the receptors for acetylcholine, glycine, and serotonin, are members of a ligand-gated ion channel superfamily (Ortells and Lunt, 1995). Because of the paucity of crystallographic information for these ligand-gated channels, little is known about the structure of their binding sites or how agonist binding is transduced into channel gating. We used the substituted cysteine accessibility method to obtain secondary structural information about the GABA binding site and to systematically identify residues that line its surface. Each residue from alpha1 Y59 to K70 was mutated to cysteine and expressed with wild-type beta2 subunits in Xenopus oocytes or HEK 293 cells. The sulfhydryl-specific reagent N-biotinylaminoethyl methanethiosulfonate (MTSEA-Biotin) was used to covalently modify the cysteine-substituted residues. Receptors with cysteines substituted at positions alpha1 T60, D62, F64, R66, and S68 reacted with MTSEA-Biotin, and alpha1 F64C, R66C, and S68C were protected from reaction by agonist. We conclude that alpha1 F64, R66, and S68 line part of the GABA binding site. The alternating pattern of accessibility of consecutive engineered cysteines to reaction with MTSEA-Biotin indicates that the region from alpha1 Y59 to S68 is a beta-strand.

Although gamma-aminobutyric acid (GABA)A receptor alpha subunits are important for benzodiazepine (BZD) binding and GABA-current potentiation by BZDs, the presence of a gamma subunit is required for high affinity BZD effects. To determine which regions unique to the gamma2S subunit confer BZD binding and potentiation, we generated chimeric protein combinations of rat gamma2S and alpha1 subunits using a modified protocol to target crossover events to the amino-terminal extracellular region of the subunits. Several chimeras with full open reading frames were constructed and placed into vectors for either voltage-clamp experiments in Xenopus laevis oocytes or radioligand binding experiments in human embryonic kidney 293 cells. Chimeras (chi) containing at least the amino-terminal 161 amino acids of gamma2S bound BZDs with wild-type affinity when coexpressed with alpha1 and beta2 subunits. Further analysis of the gamma2S binding site region uncovered two areas, gamma2S K41-W82 and gamma2S R114-D161, that together are necessary and sufficient for high affinity BZD binding. Surprisingly, although the 161-amino acid residue amino terminus of the gamma2S subunit is sufficient for high affinity BZD binding, it is not sufficient for efficient allosteric coupling of the GABA and BZD binding sites, as demonstrated by reduced diazepam potentiation of the GABA-gated current and GABA potentiation of [3H]flunitrazepam binding. Thus, by using gamma/alpha chimeras, we identified two gamma2 subunit regions required for BZD binding that are distinct from domain or domains responsible for allosteric coupling of the BZD and GABA binding sites.

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