A Structural and Computational Study of GABAA Receptor Diversity

GABAA receptors (GABAARs) are pentameric ligand-gated ion channels that modulate the activity of neural circuits in response to binding of the neurotransmitter γ-aminobutyric acid (GABA) or small molecule therapeutics, such as benzodiazepines or general anaesthetics. GABAARs assemble from a pool of 19 paralogous genes, each conferring the receptor unique properties, such as ligand binding, gating kinetics or subcellular localization. Yet, the landscape of possible receptor assemblies, their signalling properties and the principles governing assembly remain elusive. In this thesis, electron cryo-microscopy (cryo-EM) was used to structurally characterize synaptic and extrasynaptic receptors isolated from cells expressing various combinations of α1, α4, β3, γ2 and δ subunits. I discovered that, from a pool of two or more subunits, individual cells can assemble more than one receptor subtype. The subtypes differ in relative subunit stoichiometry or arrangement and have unique signaling properties because most ligands bind at inter-subunit interfaces. Structural evidence is provided to show that differential assembly diversifies receptor responses to physiological and synthetic ligands by creating or eliminating ligand-binding pockets. For example, the α4β3δ receptor can simultaneously bind two neurotransmitters, GABA and histamine. Finally, I show that subtype stoichiometry depends on the identity of the α subunit and that, when co-expressed, α1 and α4 segregate into distinct subcomplexes. A computational model of receptor assembly was developed and combined with published single-cell RNA sequencing data to calculate the upper boundary for receptor diversity in recombinant systems and in vivo. These findings suggest that differential assembly of GABAARs may be a pervasive mechanism for regulating cellular responses to physiological and synthetic ligands. The rich diversity discovered in the GABAARs system may have facilitated the evolution of complex developmental programs and intelligence in animals.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2022

Author dc:creator

• Sente, Andrija

Advisor dc:contributor.advisor

• Aricescu, Alexandru Radu

• electron cryo-microscopy
• cryoEM
• GABA
• GABAA receptors

Licence

• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.86673

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/339265