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Browsing by Subject "GABAA-reseptori"

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  • Taina, Kaisa (2013)
    γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain and it's activity is mediated via metabotropic GABAB - and ionotropic GABAA receptors. Receptors containing αβγ subunit combination are localized in both synaptic and extrasynaptic sites and they mediate fast synaptic phasic activity. αβδ- and α5βγ-containing receptors reside only in extrasynaptic space where they regulate tonic inhibition of the nerve cell. Disruption in tonic inhibition may cause several diseases. Drugs that selectively affect extrasynaptic GABAA receptors are believed to help treatment of diseases like sleep disorder, neuropsychiatry disorders, epilepsy, cognition impairment and recovery from stroke. Drug development of δ- selective GABAA agonists and positive modulators that enhance tonic inhibition as well as α5βγ selective inverse agonists that reduce tonic inhibition and enhance cognition are under investigation right now. Muscimol is a psychoactive molecule which activates all GABAA receptors but has higher affinity to cerebellar granule cell-specific receptor subtypes α6β2γ2 and extra synaptic α6β2δ than to the most common receptor subtype α1β2γ2. However, the binding of [3H]muscimol has produced contradictory results in former studies. Binding to membrane homogenates results to binding levels of the same magnitude in most brain regions, while in receptor autoradiography the high affinity binding is δ-subunit dependent. The affinity of muscimol to GABAAR subtypes has thus far been determined using saturation analysis, i.e. by measuring concentration-dependent binding of [3H]muscimol at equilibrium and by determining KD, the dissociation constant of the ligand. However, this value expresses the affinity of the ligand to the receptor, but does not give any information on the rate of the association and dissociation. In this master's thesis I have investigated association and dissociation rate of [3H]muscimol from recombinant GABAA receptors and from native GABAA receptors present in wild-type (WT) and δ-subunit knock-out (δKO) mice forebrain and cerebellar membranes. We concluded from binding assays that [3H]muscimol dissociates extremely slowly from δ-receptors. This explains further contradictory results: in membrane/filtration assay the washing procedure is generally much faster resulting in only low dissociation from αβγ receptors, while in the long washing procedure of receptor autoradiography only αβδ-binding is retained. The high affinity of muscimol to αβδ receptors is suggested to be due to its extremely slow dissociation from these receptors. Also association of [3H]muscimol to αβδ receptors seems to be slower, but this needs confirming studies. The exceptional binding properties of muscimol make it an interesting leading molecule in development of drugs which act via extrasynaptic GABAA receptors.