Browsing by Subject "Protein Subunits"
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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 Slow Inactivation of Sodium Channels: Structural Clues and Disease Associations(2009-06-19) Webb, Jadon Ray; Cannon, Stephen C.Voltage gated sodium channels underlie the rapid upstroke of action potentials in electrically excitable mammalian tissues. A cardinal feature of Na+ channels is their ability to rapidly inactivate to a refractory state during membrane depolarization, in a process known as 'fast inactivation'. During sustained membrane depolarization or prolonged busts of discharges, channels can further inactivate to non-conducting states collectively referred to as 'slow inactivation'. Fast inactivation occurs by occlusion of the inner pore by the intracellular III-IV Loop, and defects in fast inactivation gating are known to underlie certain forms of myotonia, periodic paralysis, epilepsy, and cardiac arrhythmias. The mechanism of slow inactivation and its relevance to human disease, on the other hand, are much less understood. The primary aim of this thesis was to characterize the mechanism of sodium channel slow inactivation, and also to further define its role in disease. In Chapter 1, an overview of sodium channel structure and gating is provided as background for understanding the rational and interpretation of the experimental studies. The experiments in Chapter 2 characterized the gating of a sodium channel mutation (P1158S) associated with temperature-sensitive periodic paralysis. This disease mutation caused a robust defect in slow inactivation, in accordance with an emerging model that associates defective slow inactivation with increased susceptibility to paralytic attacks. Additionally, the slow inactivation gating defects were elicited by cold temperature, analogous to the temperature-dependent provocation of paralysis. This finding further strengthens the association between defective slow inactivation gating and a specific disease phenotype. Chapter 3 explores the interaction of the sodium channel Beta-1 subunit and slow inactivation, which is incompletely characterized especially in mammalian cell expression systems. I found that co-expression of wild-type Beta-1 significantly depolarized the voltage-dependence of steady-state slow inactivation and also reduced the number of channels occupying the slow state (IS) after a long depolarizing conditioning pulse, but did not affect the kinetics of slow inactivation.. To understand which region(s) of Beta-1 are important for modulation of slow inactivation, two mutant constructs were tested. A point mutation in the extracellular N-terminus associated with epilepsy (C121W) disrupts a critical disulfide bond in an Ig-like fold and abolished the ability of Beta-1 to modulate slow inactivation. Conversely, truncation of the short cytoplasmic C-terminus did not alter the effects of Beta-1 on slow inactivation. These observations parallel the structure-function relations that have been established for Beta-1 modulation of fast inactivation. Interestingly, however, I used a mutant fast-inactivation deficient alpha-subunit to show that the Beta-1 effect on slow inactivation was independent of coupling to fast inactivation. In Chapter 4, the interaction of slow inactivation and alkali metal cations is explored. External cations have been shown to influence slow inactivation, but little is known about the location and mechanism of this interaction. To address this, I examined the interaction of Group IA alkali metal cations with slow inactivation in rat Nav1.4 channels expressed in HEK293t cells. Slow inactivation was significantly impeded by external, but not internal Na+ and Li+ cations in the buffer solutions. External K+, Rb+, and Cs+, on the other hand, caused little effect compared to sucrose (cation-free) buffer. Cation effects on slow inactivation were found to be very low affinity and were not dependent on the ability of cations to permeate deep into the channel. Indeed, Na+ interaction occurred at a shallow apparent electrical distance of 0.15 relative to the outside of the channel, and was affected by mutagenesis in the outer