2026-09-23T16:28:29Z https://www.ideals.illinois.edu/oai-pmh

oai:www.ideals.illinois.edu:2142/122222 2024-03-02 col_2142_5131 col_2142_16508 com_2142_5130 com_2142_16507 com_2142_14793 com_2142_8903

Grosman, Claudio Grosman, Claudio Anakk, Sayeepriyadarshini Llano, Daniel A Lingle, Christopher J 2023-12 application/pdf en text Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2025-12-01 The student, Nicole Godellas, accepted the attached license on 2023-11-15 at 16:38. The student, Nicole Godellas, submitted this Dissertation for approval on 2023-11-15 at 17:07. This Dissertation was approved for publication on 2023-11-29 at 11:52. DSpace SAF Submission Ingestion Package generated from Vireo submission #19932 on 2024-03-01 at 13:48:28 Development and application of quantitative ligand-binding studies to elucidate mechanistic aspects of pentameric ligand-gated ion channels Godellas, Nicole E Ligand-binding Assays Cys-loop Receptors Pentameric Ligand-gated Ion Channels Function Nicotinic Receptors Ion Channels Mechanisms 2023-11-29 Mechanistic studies of ligand-gated ion channels are not only an indispensable component of our understanding of Biology, but also, a fundamental step for the rational design of targeted therapeutics. For several decades, ion-channel mechanisms have been elucidated by means of electrophysiological studies of mutants; however, studying the effect of loss-of-function mutations (that is, mutations that render the channel “electrically silent”) requires an alternative method. Here, we have developed, optimized, and applied an equilibrium-type ligand-binding assay to a number of questions in the field of ion-channel physiology that cannot be answered by measuring ion transport. The application of ligand-binding assays to probe function in ion channels is, by no means, new; this classical assay emerged decades ago. However, its application to the mechanistic study of receptor-channels received little attention. We began with both a practical and theoretical study of this approach and optimized the methodological conditions for a competition ligand-binding assay using the human homomeric α7 AChR, radiolabeled α-bungarotoxin (α-BgTx), unlabeled small-molecule cholinergic ligands, and calculations in the framework of a kinetic reaction scheme modeling our pentameric receptor of interest. We, then, applied this approach to the elucidation of mechanistic aspects of these ligand-gated ion channels. We concluded that: 1) Ligand-binding affinities are insensitive to binding-site occupancy; 2) Mutations that are distant from the orthosteric-binding sites (say, in the transmembrane domain) have little to no effect on the channel’s affinity for orthosteric ligand; 3) The binding of the SARS-CoV-2 spike protein to the orthosteric-binding sites of the human α7 nAChR (and its subsequent competition with acetylcholine, choline, or nicotine) is unlikely to be a relevant aspect of this disease; and 4) The distance between the extracellular (ligand-binding) domain and the transmembrane-pore domain is critical for effective binding–gating coupling. Text eng https://hdl.handle.net/2142/122222 Copyright 2023 Nicole Godellas Ph.D. Dissertation Molecular & Integrative Physi University of Illinois at Urbana-Champaign Molecular & Integrative Physl