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Several naturally occuring monoflavonoids, isolated from Scutellaria baicalensis Georgi using silica gel and LH-20 chromatography, inhibited [3H] flunitrazepam binding to the central benzodiazepine receptors (BDZ-Rs) with high to moderately low binding affinity. The most potent one is 5,7,2’-trihydroxy-6,8-dimethoxyflavone (K36) with a Ki of 7.7 nM and the weakest one is baicalin with a Ki of 77.1 μM. The functional properties of wogonin (Ki=1.06 μM) and K36 were determined by electrophysiological studies, employing dorsal root ganglion (DRG) neurons and recombinant GABAA receptor functionally expressed in Xenopus laevis oocytes. Wogonin and K36 brought about an enhancement of the GABA-activated current, which could be reversed by the co-application of BDZ-R antagonist Ro15-1788. Threshold of stimulation is 0.3-1 μM for wogonin and 1-3 nM for K36. Both wogonin and K36 act as partial positive allosteric modulator at the preference BDZ-R. The pharmacological effects of wogonin and dichloromethane (DCM) extract were examined in mice. For the large-scale preparation of wogonin, it was crystallized directly from the DCM extract instead of using silica gel chromatography. The purity of the crystal (wogonin) was up to 95 %, which was determined by TLC and HPLC. The acute toxicity of wogonin and DCM extract was low with a LD50 of 3.9 g/kg and 5.9 g/kg, respectively. In acute treatment, wogonin (7.5-30 mg/kg), DCM extract (4-16 mg/kg) and diazepam (1 mg/kg) had a significant anxiolytic effect in the elevated plus-maze test for anxiety. In chronic treatment (5 days pre-treatment), the anxiolytic effect of wogonin (15-30 mg/kg), DCM extract (8 mg/kg) and diazepam (1 mg/kg) were significantly reduced. The anxiolytic effect of wogonin (15 mg/kg ) and DCM extract (8 mg/kg ) was abolished by the BDZ-R antagonist Ro15-1788 (1.25 mg/kg). Wogonin (3.75-30 mg/kg), DCM extract (4-16 mg/kg) and diazepam (1 mg/kg) did not exert any significant effect on locomotor activity acutely or chro

GABAA receptors are GABA (γ-aminobutyric acid)-gated chloride channels, which are major mediators of neuronal inhibition in the brain and are modulated by benzodiazepines, barbiturates, alcohol, and other important centrally acting drugs. A membrane-proximal β-rich fragment of GABAA receptor containing key ligand-binding residues has been successfully overexpressed in E. coli in our lab (Hong Xue,et al.,1997). Binding assay using the method of fluorescence anisotropy shows that this fragment (C166-L296) bear moderate affinity to benzodiazepine ligand. Alanine scanning combined with CD and fluorescent assay method was carrried out on this fragment to investigate the individual contribution of each residue to both the secondary structure and the binding affinity and to further study the structure-function relationship of this fragment. Present study manifests that substitution by alanine on residues R191, S213, R214, N216, F244, Y252, F253, F272 and L291 result in considerably reduced binding affinity. Among these, the loss of binding affinity on muants R191A, S213A, R214A, N216A are directly as a result of loss of side chain interaction when binding to the ligand, which means residues R191, S213, R214 and N216 may possibly involved in binding site. Nevertheless, the binding affinity loss of mutants F244A, Y252A, F272A and L291A are due to indirect change of structure rather than direct loss of side chain interaction.

GABAA receptors mediate the major inhibitory synaptic transmission in the central nervous system, and form a number of binding sites for clinically important drugs such as benzodiazepines. Alterations in GABAergic neurotransmission have been implicated in the pathogenetics of a number of neuropsychiatric diseases and disorders. GABAA receptor αl subunit is the most widely expressed in the brain among at least 17 known subunits. This single fact implies that αl subunit may play important roles in the brain. Although it has been suggested by in vitro studies that αl may involve in functions such as the benzodiazepine binding, the function of this subunit has not been addressed directly in vivo. Gene targeting is by far the most advanced technique for studies of gene functions in vivo. The present thesis work aimed at the construction of a replacement type of vector for a targeted disruption of the GABAA receptor αl subunit in mice. The success of this construction will facilitate the ablation of αl subunit gene and provide a mouse model for the in vivo studies on GABAA receptor αl subunit. For the construction of the targeting vector, a 13-kb DNA fragment containing the first two exons of mouse GABAA receptor al subunit was obtained from screening of a λ2001 based 129/Sv mouse genomic library. After characterizations by restriction digestions and DNA sequencing, a 6 kb fragment corresponding to part of the 5' non-coding sequence of the gene and a 1.8 kb fragment harboring Exon 2 of the gene were inserted into the targeting vector flanking a neomycin resistant gene (neoR). A copy of thymidine kinase (PGK-TK) gene was incorporated into the vector adjacent to the 1.8 kb fragment. The vector was linearized and electroporated inito cultured Rl mouse embryonic stem (ES) cells, which were maintained at the undifferentiated state by the inclusion of leukaemia inhibitory factor (LIF) into the culture media. ES cells carrying neo but not PGK-TK were selected with medium contain