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Benzodiazepine/ GABAA receptor characteristics

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  1. Introduction.
  2. Methods.
    1. Tissue preparation.
    2. BZ saturation binding.
  3. Displacement experiment.
  4. Mutant Mouse Binding.
  5. PCR and gel electrophoresis.
  6. Results.
    1. Saturation analysis of 3H-Flu binding.
    2. Displacement experiment.
    3. Mutant mouse binding.
  7. Discussion.
    1. Displacement study.
    2. Mutant mouse binding.
  8. Conclusion.

GABAA (?-aminobutyric acidA) receptors are recognized as the chief inhibitory neurotransmitter receptors in the mammalian central nervous system. The GABAA receptors are pentameric membrane proteins consisting of five subunits. The subunits can combine in different ways to form GABAA receptors, but the ?1?2?2 receptor subtype is found in most brain areas. The GABAA receptor is a membrane-spanning receptor that changes conformation when bound to GABA. This conformational change allows Cl? ions to pass down an electrochemical gradient, causing hyperpolarization of the neuron. The subunits can combine in different ways to form GABAA receptors, but the ?1?2?2 receptor subtype is found in most brain areas

[...] 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 ?L of cDNA was added to 24 ?l 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. [...]

[...] DISCUSSION Our results clearly indicate that benzodiazepine compounds bind to a specific receptor molecule and that the binding of these compounds depends on the composition of select proteins, specifically H101 on the alpha-1 subunit of the GABAA receptor. Saturation Analysis of 3H-Flu binding The present study has demonstrated BDZs bind to the GABAA receptor with high affinity and saturability, and the non-homogenous regional distribution of BZ binding sites match the regional distribution of endogenous transmitter. All three brain regions examined were saturable and there were high affinity receptors since BZs for the sites were all in the nM range. [...]

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