Cryo-Electron Microscopy of Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptors are pentameric ligand-gated cation channels. These neurotransmitter-gated cation channels facilitate excitatory neurotransmission in the central and peripheral nervous systems. The heteropentameric α4β2 and homopentameric α7 subtypes are the two most abundant nicotinic acetylcholine receptors found in the human brain and are the focus of my dissertation. These receptors are intricately involved in learning and memory, reward, sensory processing, pain and neuroprotection. Dysregulation of these receptors is linked to neurodegenerative diseases and mental illnesses, including epilepsy, Alzheimer's disease, Parkinson's disease and schizophrenia. The properties of these receptor subtypes are determined both by the receptor subunits that compose them and the stoichiometry of subunits. Given the emergent properties of different receptor assemblies and the roles of these receptors in both neurotransmission and disease states motivated my dissertation studies. I sought to understand how different assemblies of subunits give rise to differences in ligand recognition, ion permeation and ion selectivity, and what principles govern subunit assembly. Through the course of my dissertation work I utilized cryo-electron microscopy to investigate the structural properties of the α7 and α4β2 receptors. I developed optimized sample preparation procedures to obtain high densities of receptor molecules in random orientations over the sample holes of a cryo-EM grid. I also developed a Fab labeling strategy to facilitate the determination of both structures of the α4β2 receptors from a mixed population. Success in these goals simultaneously overcame problems imposed by the pseudo-symmetric nature of heteromeric proteins and having a compositionally heterogeneous sample. This strategy is broadly applicable to other heteromeric proteins that form from different combinations of subunits. α7 served as a model system to learn and develop the skills required to independently perform all aspects of a cryo-EM experiment but has proven refractory to structural characterization due to a disordered transmembrane domain. The skills and procedures developed from working on the α7 receptor, combined with the Fab labeling strategy, allowed me to determine the high resolution structures of both physiologically relevant stoichiometries of the α4β2 nicotinic receptor from a single sample. Comparison of these structures revealed principles governing subunit assembly and why there are only two possible arrangements of α4 and β2 subunits, structural features that govern ion conductance and permeation properties, differences in agonist binding at high and low sensitivity binding sites, and identified putative cholesterol binding sites.