Browsing by Subject "Receptors, Nicotinic"
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Item Cryo-Electron Microscopy of Nicotinic Acetylcholine Receptors(2018-08-30) Walsh, Richard Michael, Jr.; Rice, Luke M.; Hibbs, Ryan E.; Jiang, Youxing; Monteggia, LisaNicotinic 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.Item Structure and Function of the Alpha3Beta4 Nicotinic Acetylcholine Receptor(2020-12-01T06:00:00.000Z) Gharpure, Anant Vishwanath; Jiang, Youxing; Zhang, Xuewu; Bai, Xiaochen; Hibbs, Ryan E.Nicotinic acetylcholine receptors are pentameric ligand-gated ion channels that are essential for the proper function of the central and peripheral nervous systems. The α3β4 subtype is highly expressed in the autonomic ganglia, where it contributes to signal transduction from the central nervous system to the periphery. Moreover, α3β4 receptors are found in key brain regions that modulate reward circuits and have therefore been identified as potential targets for anti-addiction therapeutics. Given the physiological importance of this protein, I sought to understand the molecular mechanisms underlying ligand recognition, channel gating, and ion permeation in the α3β4 nicotinic acetylcholine receptor. Paramount to this goal was the pursuit of a high-resolution structure of the α3β4 subtype. I initially attempted to determine a crystal structure of this receptor before taking advantage of recent technological advances in cryo-electron microscopy. Using this method, I solved the first structure of the α3β4 nicotinic receptor, which was also the first high-resolution structure of any nicotinic acetylcholine receptor in a lipidic environment. By obtaining structural information of the protein bound to a non-selective nicotinic agonist as well as an α3β4-selective ligand, I was able to draw conclusions regarding ligand-selectivity in the nicotinic receptor family. Furthermore, these structures provided a detailed view of the ordered regions of the intracellular domain for the first time, giving insight into the full ion permeation pathway of these channels. This work also provided a blueprint to examine other outstanding questions in the field. Specifically, I used structural and functional approaches to begin to understand the consequences of accessory subunit incorporation, the role of multivalent cations in the desensitization of nicotinic receptors, and the role of the intracellular domain in ion selectivity and rectification.Item Understanding the Atomic-Scale Mechanisms of the Human [alpha]4[beta]2 Nicotinic Acetylcholine Receptor(2018-04-10) Morales-Perez, Claudio Luis; Jiang, Youxing; Hibbs, Ryan E.; Kavalali, Ege T.; Thomas, Philip J.Nicotinic acetylcholine receptors are pentameric ligand-gated ion channels. These receptors are present in the central and peripheral nervous systems where they mediate fast synaptic transmission by allowing the flux of cations through the plasma membrane. The heteropentameric α4β2 subtype is the most abundant nicotinic receptor in the brain and it's the focus of my dissertation project. This receptor is involved in learning, memory formation, mood, attention and reward. Its dysfunction has been linked to neurodegenerative diseases and mental illnesses including schizophrenia, Alzheimer's disease, epilepsy, Parkinson's disease and nicotine addiction. Because of its key role in the brain and connection to diseases, I sought to understand the basic principles underlying gating, subunit assembly, ligand recognition and ion selectivity of the human α4β2 nicotinic acetylcholine receptor. I developed multiple biochemical and biophysical methods that enabled the crystallization of the α4β2 receptor. The expression system and assay for stoichiometry I developed are applicable to a broad range of soluble and membrane proteins. I leveraged these methods to obtain crystals of the receptor that, after extensive optimization, diffracted X-rays to beyond 4 Å resolution. This result provided the first high-resolution structure of a nicotinic acetylcholine receptor. Co-crystallization with the agonist nicotine revealed principles of ligand selectivity among the different classes of subunit interfaces; specifically, I was able to explain high-affinity nicotine binding to the α-β subunit interfaces and its exclusion at β-β and β-α interfaces. Nicotine stabilized the receptor in a non-conducting, desensitized conformation. I showed that the constriction point in the permeation pathway was formed at the selectivity filter located at the cytosolic end of the pore. In addition, I used the high-resolution structure as a template to perform site-directed mutagenesis to examine the mechanisms of ligand recognition and channel gating. I elucidated the ligand exclusion mechanism at the β-β and β-α interfaces and proposed a potential role for these interfaces in the allosteric gating mechanism. In summary, these structural and functional studies have provided information on the basic principles of the high-affinity nicotine interactions, the architecture of allosteric sites and the permeation pathway, principles of subunit assembly, and increase our understanding of the mechanism of channel desensitization.