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Probing function in ligand-gated ion channels without measuring ion transport
Although the functional properties of ion channels are most accurately assessed using electrophysiological approaches, a number of experimental situations call for alternative methods. Here, working on members of the pentameric ligand-gated ion channel (pLGIC) superfamily, we focused on the practica...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Rockefeller University Press
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9136306/ https://www.ncbi.nlm.nih.gov/pubmed/35612603 http://dx.doi.org/10.1085/jgp.202213082 |
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author | Godellas, Nicole E. Grosman, Claudio |
author_facet | Godellas, Nicole E. Grosman, Claudio |
author_sort | Godellas, Nicole E. |
collection | PubMed |
description | Although the functional properties of ion channels are most accurately assessed using electrophysiological approaches, a number of experimental situations call for alternative methods. Here, working on members of the pentameric ligand-gated ion channel (pLGIC) superfamily, we focused on the practical implementation of, and the interpretation of results from, equilibrium-type ligand-binding assays. Ligand-binding studies of pLGICs are by no means new, but the lack of uniformity in published protocols, large disparities between the results obtained for a given parameter by different groups, and a general disregard for constraints placed on the experimental observations by simple theoretical considerations suggested that a thorough analysis of this classic technique was in order. To this end, we present a detailed practical and theoretical study of this type of assay using radiolabeled α-bungarotoxin, unlabeled small-molecule cholinergic ligands, the human homomeric α7-AChR, and extensive calculations in the framework of a realistic five-binding-site reaction scheme. Furthermore, we show examples of the practical application of this method to tackle two longstanding questions in the field: our results suggest that ligand-binding affinities are insensitive to binding-site occupancy and that mutations to amino-acid residues in the transmembrane domain are unlikely to affect the channel’s affinities for ligands that bind to the extracellular domain. |
format | Online Article Text |
id | pubmed-9136306 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-91363062022-12-06 Probing function in ligand-gated ion channels without measuring ion transport Godellas, Nicole E. Grosman, Claudio J Gen Physiol Article Although the functional properties of ion channels are most accurately assessed using electrophysiological approaches, a number of experimental situations call for alternative methods. Here, working on members of the pentameric ligand-gated ion channel (pLGIC) superfamily, we focused on the practical implementation of, and the interpretation of results from, equilibrium-type ligand-binding assays. Ligand-binding studies of pLGICs are by no means new, but the lack of uniformity in published protocols, large disparities between the results obtained for a given parameter by different groups, and a general disregard for constraints placed on the experimental observations by simple theoretical considerations suggested that a thorough analysis of this classic technique was in order. To this end, we present a detailed practical and theoretical study of this type of assay using radiolabeled α-bungarotoxin, unlabeled small-molecule cholinergic ligands, the human homomeric α7-AChR, and extensive calculations in the framework of a realistic five-binding-site reaction scheme. Furthermore, we show examples of the practical application of this method to tackle two longstanding questions in the field: our results suggest that ligand-binding affinities are insensitive to binding-site occupancy and that mutations to amino-acid residues in the transmembrane domain are unlikely to affect the channel’s affinities for ligands that bind to the extracellular domain. Rockefeller University Press 2022-05-25 /pmc/articles/PMC9136306/ /pubmed/35612603 http://dx.doi.org/10.1085/jgp.202213082 Text en © 2022 Godellas and Grosman https://creativecommons.org/licenses/by-nc-sa/4.0/http://www.rupress.org/terms/This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/). |
spellingShingle | Article Godellas, Nicole E. Grosman, Claudio Probing function in ligand-gated ion channels without measuring ion transport |
title | Probing function in ligand-gated ion channels without measuring ion transport |
title_full | Probing function in ligand-gated ion channels without measuring ion transport |
title_fullStr | Probing function in ligand-gated ion channels without measuring ion transport |
title_full_unstemmed | Probing function in ligand-gated ion channels without measuring ion transport |
title_short | Probing function in ligand-gated ion channels without measuring ion transport |
title_sort | probing function in ligand-gated ion channels without measuring ion transport |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9136306/ https://www.ncbi.nlm.nih.gov/pubmed/35612603 http://dx.doi.org/10.1085/jgp.202213082 |
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