Abstract
dc:description.abstractGABA<sub>A</sub> receptors are pentameric ligand-gated ion channels (pLGICs) that control multiple aspects of animal physiology, including the rapid inhibitory neurotransmission within the vertebrate central nervous system. They activate and desensitize in a few milliseconds, which is a crucial requirement for accurate synaptic signalling. Such fast conformational transitions, however, have proven impervious to investigation via conventional structural biology methods. Moreover, GABA<sub>A</sub> receptors operate in cellular membranes with diverse lipid compositions and biophysical properties. These are thought to play essential modulatory functions, however, a molecular understanding of such impacts is lacking. As a result, the mechanisms of GABA-ergic signalling, in physiologically meaningful environments and time scales, remain among the most important unknowns in molecular neuroscience. To address these issues, I first sought to identify optimal reconstitution environments for recombinantly expressed β3 homomeric and synaptic α1β3γ2L GABA<sub>A</sub> receptors by testing multiple nanodisc scaffolds and lipid compositions. Subsequently, with the optimal condition established, I proceeded to investigate a series of GABA<sub>A</sub> receptor structures bound to β-lactam antibiotics that are postulated to act as open channel pore blockers. Surprisingly, complexes with the penicillin derivative nafcillin suggested that the GABA<sub>A</sub> receptor gating motions might include conformations that diverge considerably from the pseudo-symmetric arrangements observed previously. To test whether the asymmetry observed is physiologically meaningful, and to achieve a comprehensive characterisation of the GABA<sub>A</sub> receptor gating cycle, I employed a time-resolved cryo-EM strategy that enables millisecond-scale time delays between agonist application and sample freezing. This approach allowed the visualization of novel and complex asymmetric gating motions involved in the activation and desensitization of the receptor, ranging from side chain to major secondary structure rearrangements. Remarkably, the asymmetric gating cycle is conserved among homomeric and heteromeric GABA<sub>A</sub> receptors, with lipids such as cholesterol playing a crucial role in stabilizing the newly formed interfaces. Moreover, these results have further revealed a novel inhibitory mechanism driven by the lipid PIP<sub>2</sub>, which directly controls the opening of α1β3 and α1β3γ2L receptors. Lastly, I will also demonstrate how positive allosteric modulators alter the gating landscape and how the general anaesthetic propofol can directly activate the receptor. This work provides a new structural and mechanistic framework to explore and interpret the action of pharmacological modulators and to design novel molecules with improved therapeutic properties.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2024
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Mihaylov, Daniel
- Advisor dc:contributor.advisor
-
- Aricescu, A Radu
Subjects
dc:subject × 5Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.110545
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/371323