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Replacing voltage sensor arginines with citrulline provides mechanistic insight into charge versus shape

Voltage-dependent activation of voltage-gated cation channels results from the outward movement of arginine-bearing helices within proteinaceous voltage sensors. The voltage-sensing residues in potassium channels have been extensively characterized, but current functional approaches do not allow a d...

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Autores principales: Infield, Daniel T., Lee, Elizabeth E.L., Galpin, Jason D., Galles, Grace D., Bezanilla, Francisco, Ahern, Christopher A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Rockefeller University Press 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028492/
https://www.ncbi.nlm.nih.gov/pubmed/29866793
http://dx.doi.org/10.1085/jgp.201812075
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author Infield, Daniel T.
Lee, Elizabeth E.L.
Galpin, Jason D.
Galles, Grace D.
Bezanilla, Francisco
Ahern, Christopher A.
author_facet Infield, Daniel T.
Lee, Elizabeth E.L.
Galpin, Jason D.
Galles, Grace D.
Bezanilla, Francisco
Ahern, Christopher A.
author_sort Infield, Daniel T.
collection PubMed
description Voltage-dependent activation of voltage-gated cation channels results from the outward movement of arginine-bearing helices within proteinaceous voltage sensors. The voltage-sensing residues in potassium channels have been extensively characterized, but current functional approaches do not allow a distinction between the electrostatic and steric contributions of the arginine side chain. Here we use chemical misacylation and in vivo nonsense suppression to encode citrulline, a neutral and nearly isosteric analogue of arginine, into the voltage sensor of the Shaker potassium channel. We functionally characterize the engineered channels and compare them with those bearing conventional mutations at the same positions. We observe effects on both voltage sensitivity and gating kinetics, enabling dissection of the roles of residue structure versus positive charge in channel function. In some positions, substitution with citrulline causes mild effects on channel activation compared with natural mutations. In contrast, substitution of the fourth S4 arginine with citrulline causes substantial changes in the conductance–voltage relationship and the kinetics of the channel, which suggests that a positive charge is required at this position for efficient voltage sensor deactivation and channel closure. The encoding of citrulline is expected to enable enhanced precision for the study of arginine residues located in crowded transmembrane environments in other membrane proteins. In addition, the method may facilitate the study of citrullination in vivo.
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spelling pubmed-60284922019-01-02 Replacing voltage sensor arginines with citrulline provides mechanistic insight into charge versus shape Infield, Daniel T. Lee, Elizabeth E.L. Galpin, Jason D. Galles, Grace D. Bezanilla, Francisco Ahern, Christopher A. J Gen Physiol Research Articles Voltage-dependent activation of voltage-gated cation channels results from the outward movement of arginine-bearing helices within proteinaceous voltage sensors. The voltage-sensing residues in potassium channels have been extensively characterized, but current functional approaches do not allow a distinction between the electrostatic and steric contributions of the arginine side chain. Here we use chemical misacylation and in vivo nonsense suppression to encode citrulline, a neutral and nearly isosteric analogue of arginine, into the voltage sensor of the Shaker potassium channel. We functionally characterize the engineered channels and compare them with those bearing conventional mutations at the same positions. We observe effects on both voltage sensitivity and gating kinetics, enabling dissection of the roles of residue structure versus positive charge in channel function. In some positions, substitution with citrulline causes mild effects on channel activation compared with natural mutations. In contrast, substitution of the fourth S4 arginine with citrulline causes substantial changes in the conductance–voltage relationship and the kinetics of the channel, which suggests that a positive charge is required at this position for efficient voltage sensor deactivation and channel closure. The encoding of citrulline is expected to enable enhanced precision for the study of arginine residues located in crowded transmembrane environments in other membrane proteins. In addition, the method may facilitate the study of citrullination in vivo. Rockefeller University Press 2018-07-02 /pmc/articles/PMC6028492/ /pubmed/29866793 http://dx.doi.org/10.1085/jgp.201812075 Text en © 2018 Infield et al. http://www.rupress.org/terms/https://creativecommons.org/licenses/by-nc-sa/4.0/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 Research Articles
Infield, Daniel T.
Lee, Elizabeth E.L.
Galpin, Jason D.
Galles, Grace D.
Bezanilla, Francisco
Ahern, Christopher A.
Replacing voltage sensor arginines with citrulline provides mechanistic insight into charge versus shape
title Replacing voltage sensor arginines with citrulline provides mechanistic insight into charge versus shape
title_full Replacing voltage sensor arginines with citrulline provides mechanistic insight into charge versus shape
title_fullStr Replacing voltage sensor arginines with citrulline provides mechanistic insight into charge versus shape
title_full_unstemmed Replacing voltage sensor arginines with citrulline provides mechanistic insight into charge versus shape
title_short Replacing voltage sensor arginines with citrulline provides mechanistic insight into charge versus shape
title_sort replacing voltage sensor arginines with citrulline provides mechanistic insight into charge versus shape
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028492/
https://www.ncbi.nlm.nih.gov/pubmed/29866793
http://dx.doi.org/10.1085/jgp.201812075
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