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Functional characterisation of recombinant GABAA receptors

Krishek, Belinda Janine; (1995) Functional characterisation of recombinant GABAA receptors. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

γ-Aminobutyric acidA (GABAA) receptors are ligand-gated ion channels that mediate inhibitory synaptic transmission in the central nervous system. Cloning studies have revealed that GABAA receptors are hetero-oligomeric proteins composed of several distinct subunit proteins (α, β, γ and δ). The aim of this study was to examine the functional role(s) of some individual subunits within the GABAA receptor complex using the Xenopus laevis oocyte expression system. The physiological and pharmacological properties of expressed GABAA receptors were characterised using two-microelectrode current and voltage clamp recording techniques. The functional role(s) of some of the individual subunits were studied by applying a selection of agonists, antagonists and potentiators to GABAA receptors of differing subunit compositions. Inhibitions of GABA responses by picrotoxin and bicuculline, or enhancements by pentobarbitone and pregnanolone were independent of the subunit composition. In contrast, the modulation of the GABA response by benzodiazepines was dependent on the presence of a γ2 subunit, whereas for zinc- induced inhibition the presence of γ2 and/or δ subunits were influential. The β1 subunit was important in the expression and formation of GABAA receptors. To further assess the functional properties of the β1 subunit, homomeric murine and bovine pi receptors were expressed and compared. The effect of changing the extracellular pH on ion channel function was studied as an alternative method of effecting modulation on GABAA receptors comprising various subunit compositions. pH also affected the inhibition of the GABA response by zinc. The role of protein kinase C (PKC) phosphorylation in modulating GABAA receptor function was ascertained using wild-type receptors of different subunit compositions and receptors incorporating selected site-directed mutants of serine residues involved in known consensus sequences for PKC in the large intracellular domains of the β1 and γ2 subunits. This study provides evidence of distinct functional role(s) of individual subunits within the GABAA receptor and indicates for the very first time a novel pharmacology for δ subunit containing receptors. Furthermore, two novel sites on the GABAA receptor have been uncovered which can modulate receptor function. These sites are sensitive to H+ or PKC-induced phosphorylation and may be significant in affecting the pharmacological and physiological profile of the GABAA receptor.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Functional characterisation of recombinant GABAA receptors
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Health and environmental sciences
URI: https://discovery.ucl.ac.uk/id/eprint/10104906
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