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.
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6 June 2022
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May 25 2022
Probing function in ligand-gated ion channels without measuring ion transport
Nicole E. Godellas
,
Nicole E. Godellas
1
Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL
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Claudio Grosman
1
Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL
2
Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, IL
3
Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, IL
Correspondence to Claudio Grosman: grosman@illinois.edu
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Nicole E. Godellas
1
Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL
1
Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL
2
Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, IL
3
Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, IL
Correspondence to Claudio Grosman: grosman@illinois.edu
Received:
January 07 2022
Accepted:
April 28 2022
Online Issn: 1540-7748
Print Issn: 0022-1295
Funding
Funder(s):
National Institutes of Health
- Award Id(s): R01-NS042169
© 2022 Godellas and Grosman
2022
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/).
J Gen Physiol (2022) 154 (6): e202213082.
Article history
Received:
January 07 2022
Accepted:
April 28 2022
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Citation
Nicole E. Godellas, Claudio Grosman; Probing function in ligand-gated ion channels without measuring ion transport. J Gen Physiol 6 June 2022; 154 (6): e202213082. doi: https://doi.org/10.1085/jgp.202213082
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