Mechanism of anaesthetic activation, combination and antagonism

The type A γ-aminobutyric acid receptors (GABAAR) – pentameric ligand gated ion channels (pLGICs) – are the main mediators of fast inhibitory neurotransmission in the human brain. Malfunctions in these receptors are a common cause for neurological disorders such as anxiety, epilepsy, schizophrenia and insomnia. Fortunately, due to numerous binding sites and decades of dedicated research, a large library of GABAAR targeting drugs is available to treat such conditions. The most prominent group of ligands targeting GABAA receptors are general anaesthetics. The effects of anaesthetics on the human body such as immobility, analgesia, amnesia and muscle relaxation are exploited during surgery and are indispensable in modern medicine to allow invasive procedures. Although the binding sites of several anaesthetics have been identified through structural and functional experiments, the underlying mechanism of such drugs remains unclear. In the first chapter of this thesis, I will use a series of high resolution cryo-EM structures to elucidate the mechanism of general anaesthetics on the human α1β3γ2L GABAA receptor. Etomidate and propofol were chosen as examples due to their clinical importance. I will further compare the ligand’s impact on the receptor with the physiological GABA-triggered activation mechanism. I will address concerns regarding different receptor reconstitution systems such as membrane-scaffold protein (MSP)-, saposin-, and SMALP nanodiscs. Antibody fragments have been used in the past to support particle alignment and distribution in cryo-EM specimen. On the example of several cryo-EM structures, I will discuss the impact and necessity of such fiducials. Anaesthetics are thought to target the inter-subunit clefts of several pLGICs in the TMD. In the second chapter of this work, I will present several structures of the α1β3γ2L GABAA receptor with ligands occupying unexpected and previously missed binding sites including the historically labelled ‘orphan’ interface which was thought to be excluded from anaesthetic binding. In modern medicine, anaesthetics are often applied in cocktails. Thus, it is crucial to understand the rules of additivity and synergy between anaesthetics. Having identified ligands binding to all GABAA interfaces, I will discuss this concept based on my structural findings. While we have access to antidotes of sedatives such as benzodiazepines and opioids, well-functioning antagonists for anaesthetic action are missing. The relatively low pharmacological index of anaesthetics made them once one of the most dangerous drugs used in clinics. While the safety of anaesthetics has improved over several decades, unwanted potentially lethal side effects remain. In the last chapter of this thesis, I will structurally characterize a novel anaesthetic antidote and I will elucidate its mechanism through a yet undescribed ligand binding pocket in the receptor.

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

• Miehling, Jonas

Advisor dc:contributor.advisor

• Aricescu, Radu

• GABA receptor
• GABAA
• GABAAR
• Anaesthetics
• Etomidate
• Antidote
• Propofol
• Barbiturate
• Alphaxalone
• structural biology
• cryo EM
• cryo electron microscopy
• pharmacology

Licence

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

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.87926

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/340490