||Structural investigation of GABAA receptors has been limited by difficulties imposed by its transmembrane-complex nature. In the present study, the topology of a membrane-proximal β-rich (MPB) domain in the C139-L269 segment of the receptor α1, subunit was probed by mapping the benzodiazepine (BZ)-binding and epitopic sites, as well as fluorescence resonance energy transfer (FRET) analysis. Ala-scanning and semi-conservative substitutions within this segment revealed the contribution of the phenyl rings of Y160 and Y210, the hydroxy group of S186 and the positive charge on R187 to BZ-binding. FRET with the bound BZ ligand indicated the proximity of Y160, S186, R187 and S206 to the BZ-binding site. On the other hand, epitope-mapping using the monoclonal antibodies (mAbs) against the MPB domain established a clustering of T172, R173, E174, Q196 and T197. Based on the lack of FRET between Trp substitutionally placed at R173 or V198 and bound BZ, this epitope-mapped cluster is located on a separate end of the folded protein from the BZ-binding site. Mutations of the five conserved Cys and Trp residues in the MPB domain gave rise to synergistic and rescuing effects on protein secondary structures and unfolding stability that point to a CCWCW-pentad, reminiscent to the CWC-triad 'pin' of immunoglobulin (Ig)-like domains, important for the structural maintenance. These findings, together with secondary structure and fold predictions suggest an anti-parallel β-strand topology with resemblance to Ig-like fold, having the BZ-binding and the epitopic residues being clustered at two different ends of the fold. Baicalin, a naturally occurring flavonoid, was previously reported to have moderate affinity toward the BZ-binding site of GABAA receptor, and exert anxiolytic-like effects in the Vogel conflict test. In the present study, the anxiolytic effects of baicalin alone and in combination with other anxiolytics were tested in mice using the elevated plus-maze (EPM). Baicalin treatment (7.5 -30 mg/kg) significantly increased entries into and time spent in open arms, indicative of an anxiolytic-like effect. Motor-depressive and myorelaxant side effects commonly associated with anxiolytics were not observed with baicalin at effective anxiolytic doses in the holeboard and horizontal wire tests, respectively. Co-administration of baicalin (3.75 mg/kg) with dl-tetrahydropalmatine (dl-THP; 0.25 mg/kg), an anxiolytic-hypnotic alkaloid, both at sub-effective doses induced an additive effect resulting in considerable anxiolysis. Similarly, an additive anxiolytic-like effect was observed with baicalin (3.75 mg/kg) and diazepam (DZ; 0.5 mg/kg). Results obtained from this study demonstrated the potential of baicalin as a candidate anxiolytics and its possible application in multidrug therapy.