| Summary: | <p>The GABA<sub>A</sub>Rs is the main inhibitory neurotransmitter receptor in the brain, and is primarily
responsible for maintaining the excitatory-inhibitory balance in neurons on which normal
function of the nervous system depends. This receptor is subject to modulation by several
endogenous and pharmacological agents, which can have dramatic effects on the brain and
behaviour. Common examples of drugs acting GABA<sub>A</sub>Rs include anaesthetics, anxiolytics
and sedatives. In particular, there has been great interest in developing drugs against
the α2- and α3-containing GABA<sub>A</sub>Rs because of their roles in pain and anxiety. The aim
of the work described in this thesis was to investigate modulation of GABA<sub>A</sub>Rs by positive
modulators. In particular, because nanobodies raised against GABAARs have previously
been shown to positively modulate the α1β3γ2L receptor, new nanobodies were raised
against the α2β3 and α3β3 GABA<sub>A</sub>Rs, in the hope of obtaining specific modulators. These
were characterised for affinity and modulatory activity at their respective GABA<sub>A</sub>Rs, and
several high-affinity nanobodies that act as positive allosteric modulators were identified.
In order to investigate the structural effect of these nanobodies, methods for expression and
purification of full-length heteromeric GABA<sub>A</sub>Rs were then investigated. A comparison of
transient transfection, BacMam and stable cell lines indicated that the highest expression
was obtained from HEK293 cells stably transduced with triheteromeric GABA<sub>A</sub>Rs. After
developing a method for purification of these receptors, the interaction of the α3β3γ2L
GABA<sub>A</sub>R with one nanobody, α3β3nb173, was investigated by single-particle cryo-EM.
Several datasets were collected, and the best map had a nominal resolution of 3.8 Å. This
map revealed a novel modulatory site on the GABA<sub>A</sub>R, at the top of the β+/α- interfaces
and above the GABA binding site. These findings have implications for the discovery of
novel drugs against the GABA<sub>A</sub>R, potentially of major clinical importance, either by using
nanobodies directly or developing compounds to mimic their activity at this novel site.
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