Development and application of quantitative ligand-binding studies to elucidate mechanistic aspects of pentameric ligand-gated ion channels

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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Molecular & Integrative Physi

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2023

Author dc:creator

• Godellas, Nicole E

Contributors dc:contributor

• Grosman, Claudio
• Anakk, Sayeepriyadarshini
• Llano, Daniel A
• Lingle, Christopher J

• Ligand-binding Assays
• Cys-loop Receptors
• Pentameric Ligand-gated Ion Channels
• Function
• Nicotinic Receptors
• Ion Channels
• Mechanisms

Statement dc:rights

• Copyright 2023 Nicole Godellas

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/122222

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F122222