ABSTRACT
Mutations in glycine receptor alpha 2 (GlyRa2), an anionic-selective pentameric ligand-gated ion channel, have
been implicated in neurodevelopmental disorders such as autism spectrum disorder and epilepsy. This
receptor plays an important role in neurodevelopment and mediates tonic inhibition in specific regions of the
adult brain. Functional studies have shown that GlyRa2 has slower activation and desensitization kinetics
compared to other GlyR subtypes. However, the precise mechanistic differences between GlyRa2 and other
GlyR subtypes remain unknown. Furthermore, while there is increasing evidence of diverse mechanisms of
lipidic modulation on ion channels, there is a lack of information regarding the lipidic profile of GlyRa2. Given
the importance of lipids in regulating the function of membrane proteins, it is important to investigate the
specific lipidic profile of GlyRa2 and how lipidic modulators affect channel function. The overarching goal of this
study is to examine the structure-function relationship and investigate the allosteric modulation of GlyRa2. The
study is divided into two aims. Aim 1 will test the hypothesis that GlyRa2 conformational changes differ from
GlyRa1 upon glycine binding, channel opening, and desensitization. This aim will be assessed using cryogenic
electron microscopy (cryo-EM) and patch-clamp electrophysiology with site-directed mutagenesis. Preliminary
results have shown high resolution structures in distinct conformational states with various ligands. Aim 2 will
test the hypothesis that GlyRa2-specific modulators alter the conformation of the receptor, and that their
specificity is attributed to site-specific or globally distinct features of GlyRa2. These modulators are expected to
induce conformational differences that correspond to changes in the channel's function. Additionally, Aim 2 will
test the hypothesis that GlyRa2 preferentially interacts with different lipid species than GlyRa1, corresponding
to the distinct membrane environments these two channels are found in. This aim will expand our knowledge of
GlyRa2-specific lipidic modulation and the effect lipids have on channel function. Native mass spectrometry will
be used to investigate lipidic interactions, and these findings will be correlated to observable lipid densities
from our cryo-EM structures. Overall, the findings from this study will provide a better understanding of the role
of GlyRa2 in neurogenesis and adult physiology and pathology. In addition, this work will set the stage for the
future development of pharmacologic therapies targeting GlyRa2.