Investigating the Structural Basis of Human Glycine Receptor Modulation for Neurodevelopmental Disorders - ABSTRACT Mutations in glycine receptor alpha 2 (GlyRα2), 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 GlyRα2 has slower activation and desensitization kinetics compared to other GlyR subtypes. However, the precise mechanistic differences between GlyRα2 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 GlyRα2. Given the importance of lipids in regulating the function of membrane proteins, it is important to investigate the specific lipidic profile of GlyRα2 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 GlyRα2. The study is divided into two aims. Aim 1 will test the hypothesis that GlyRα2 conformational changes differ from GlyRα1 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 GlyRα2-specific modulators alter the conformation of the receptor, and that their specificity is attributed to site-specific or globally distinct features of GlyRα2. 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 GlyRα2 preferentially interacts with different lipid species than GlyRα1, corresponding to the distinct membrane environments these two channels are found in. This aim will expand our knowledge of GlyRα2-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 GlyRα2 in neurogenesis and adult physiology and pathology. In addition, this work will set the stage for the future development of pharmacologic therapies targeting GlyRα2.
