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The tethered agonist approach to mapping ion channel proteins – toward a structural model for the agonist binding site of the nicotinic acetylcholine receptor

Li, Lintong and Zhong, Wenge and Zacharias, Niki and Gibbs, Caroline and Lester, Henry A. and Dougherty, Dennis A. (2001) The tethered agonist approach to mapping ion channel proteins – toward a structural model for the agonist binding site of the nicotinic acetylcholine receptor. Chemistry and Biology, 8 (1). pp. 47-58. ISSN 1074-5521. doi:10.1016/s1074-5521(00)00055-7. https://resolver.caltech.edu/CaltechAUTHORS:20200422-150959147

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Abstract

Background: The integral membrane proteins of neurons and other excitable cells are generally resistant to high resolution structural tools. Structure–function studies, especially those enhanced by the nonsense suppression methodology for unnatural amino acid incorporation, constitute one of the most powerful probes of ion channels and related structures. The nonsense suppression methodology can also be used to incorporate functional side chains designed to deliver novel structural probes to membrane proteins. In this vein, we sought to generalize a potentially powerful tool – the tethered agonist approach – for mapping the agonist binding site of ligand-gated ion channels. Results: Using the in vivo nonsense suppression method for unnatural amino acid incorporation, a series of tethered quaternary ammonium derivatives of tyrosine have been incorporated into the nicotinic acetylcholine receptor. At three sites a constitutively active receptor results, but the pattern of activation as a function of chain length is different. At position α149, there is a clear preference for a three-carbon tether, while at position α93 tethers of 2–5 carbons are comparably effective. At position γ55/δ57 all tethers except the shortest one can activate the receptor. Based on these and other data, a model for the receptor binding site can be developed by analogy to the acetylcholine esterase crystal structure. Conclusion: Through the use of nonsense suppression techniques, the tethered agonist approach has been made into a general tool for probing receptor structures. When applied to the nicotinic receptor, the method places new restrictions on developing models for the agonist binding site.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/s1074-5521(00)00055-7DOIArticle
ORCID:
AuthorORCID
Lester, Henry A.0000-0002-5470-5255
Dougherty, Dennis A.0000-0003-1464-2461
Additional Information:© 2001 Elsevier. Under an Elsevier user license. Received 14 July 2000, Revised 27 October 2000, Accepted 30 October 2000, Available online 19 February 2001. ESI ionization, quadrupole mass spectrometry was carried out at the Caltech Protein Microanalytical Laboratory under the direction of Gary M. Hathaway. This work was supported by the NIH (NS 34407 and NS 11756).
Funders:
Funding AgencyGrant Number
NIHNS 34407
NIHNS 11756
Subject Keywords:Tethered agonist; Nicotinic acetylcholine receptor; Agonist binding site; Unnatural amino acid mutagenesis
Issue or Number:1
DOI:10.1016/s1074-5521(00)00055-7
Record Number:CaltechAUTHORS:20200422-150959147
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200422-150959147
Official Citation:Lintong Li, Wenge Zhong, Niki Zacharias, Caroline Gibbs, Henry A Lester, Dennis A Dougherty, The tethered agonist approach to mapping ion channel proteins – toward a structural model for the agonist binding site of the nicotinic acetylcholine receptor, Chemistry & Biology, Volume 8, Issue 1, 2001, Pages 47-58, ISSN 1074-5521, https://doi.org/10.1016/S1074-5521(00)00055-7.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:102731
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:23 Apr 2020 15:03
Last Modified:16 Nov 2021 18:14

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