Laverty, DC; (2016) Structural and functional studies of a chimeric GABA-A receptor. Doctoral thesis , UCL (University College London).

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Abstract

GABA-A receptors are ligand-gated ion channels principally responsible for inhibitory neurotransmission in the mammalian CNS. GABA binding initiates a series of conformational changes causing the receptor to transition from inactive (shut/closed) to active (open) ion channel states; and during prolonged agonist exposure, to a desensitized (closed) state. Critical to the fine-tuning of inhibitory responses in vivo is the allosteric modulation of GABA-A receptors by an array of compounds, many of which impart their effect through binding within the receptor’s transmembrane domain. Beyond the importance of GABA-mediated inhibition in maintaining nervous system function, GABA-A receptors are established therapeutic targets for psychiatric and neurodevelopmental disorders. Despite this, an understanding of the structure of these receptors at atomic resolution is crucially lacking; particularly with regards to the structural elements underpinning channel gating and allosteric modulation. Therefore, GABA-A receptor ion channels were subjected to atomic-resolution structural analyses using chimeric receptors, in addition to comparative studies with bacterial ion channel homologues. A functional receptor was formed from chimeras between the extracellular domain of the prokaryotic ion channel GLIC and the transmembrane domain of GABA-A receptor α1 subunits. These receptors exhibited GABA-A receptor-like properties with respect to their response to brain neurosteroids. The amenability of this receptor to high-level expression and purification was assessed. The baculovirus-insect cell expression system was identified as an appropriate system for generating receptor of sufficient quantity and purity to generate diffracting protein crystals. Additional studies of GABA-A receptor modulators at the bacterial homologs GLIC and ELIC identified previously unreported effects prompting further structural investigation using X-ray crystallography, cryo-electron microscopy and native mass spectrometry. In conclusion, these studies reveal a new system for atomic structural resolution investigation of GABA-A receptor subunits, likely to be applicable to other receptors. These receptors are potentially powerful tools for understanding the mechanism of GABA-A receptor allosteric modulation.

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