Benzodiazepine/ GABAA receptor characteristics

Extract

[...] PCR and gel electrophoresis Reverse transcriptase polymerase chain reaction (RTPCR) was performed to detect the mRNA for the alpha-1 subunit (245bp) of GABAA receptor μL of cDNA was added to 24 μl PCR master mix and the thermocycler was set to denature at 94(C, anneal at 54(C, and extend at 72(C, and then terminate at 4(C. The PCR products were run on a agarose gel then were visualized with Ethidium Bromide under UV light. Data Analysis Microsoft Excel was used to calculate all data and GraphPad Prism 5 was used to plot the graphs and used for all data analyses. [...]

[...] The concentration of protein in each homogenate was determined using a modified Lowry procedure. Tissue/Sample Preparation for PCR H101R and CD 57 mice were sacrificed by cervical dislocation. Brains were removed and homogenized in Trizol Reagent (Invitrogen, Inc.) and RNA was extracted per the manufacturer's protocol and quantified by spectrophotometry. Briefly mL chloroform was added to 1mL Trizol homogenate and samples were centrifuged at 13,000 rpm for 15 minutes at 4oC. The aqueous (upper) phase was removed to a fresh tube and 0.5 mL isopropanol were added to precipitate the RNA. [...]

[...] Our finding that BZ binding is mediated by specific GABAA receptor subtypes may further enhance the design of drugs with increased clinical specificity. For example, the α1 subtype of the GABAA receptor was mainly responsible for the sedative and motor effects of nonselective BZs such as diazepam, whereas other subtypes were primarily responsible for the anxiolytic action. Furthermore, the α1 H101R mice allowed us to delineate the GABAA subtypes responsible for the actions of BZ. Thus, these finding suggests could help to develop drugs for other neurological disorders based on their specificity for GABAA receptor subtypes in distinct neuronal circuits. [...]

[...] Klausberger, Sarto, Ehya, Fuchs, Furtmuller, Mayer, Huck, Sieghart, W (2001) Alternate Use of Distinct Intersubunit Contacts Controls GABAA Receptor Assembly and Stoichiometry. Journal of Neuroscience 21(23):9124-9133. Lüddens, Korpi, E.R, Seeburg, P.H. (1994) GABAA/Benzodiazepine Receptor Heterogeneity: Neurophysiological Implications. Neuropharmacology 34 245-254. McKernan RM, Farrar Collins Emms Asuni Quirk Broughton H (1998) Photoaffinity labeling of the benzodiazepine binding site of alpha1beta3gamma2 gamma-aminobutyric acidA receptors with flunitrazepam identifies a subset of ligands that interact directly with His102 of the alpha subunit and predicts orientation of these within the benzodiazepine pharmacophore. [...]

[...] The classical benzodiazepines such as diazepam and clonazepam interact indiscriminately with the ( subunit of GABAA receptor (Rudolph et al., 1999). The amino acid at position 101 for the and 5 subunits is a conserved histidine residue, which seems critical for BZ binding. In contrast, the benzodiazepine insensitive receptor subtypes in the brain and have an arginine in the corresponding position. The pharmacological significance of GABAA receptor subtype, which contains the subunits, was evaluated by introducing the point mutation into the knock-in mice, such that the conserved histidine at position 101 (H101) is changed to an arginine (H101R). [...]