Understanding the Atomic-Scale Mechanisms of the Human [alpha]4[beta]2 Nicotinic Acetylcholine Receptor
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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.