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Probing the Pore of ClC-0 by Substituted Cysteine Accessibility Method Using Methane Thiosulfonate Reagents
ClC channels are a family of protein molecules containing two ion-permeation pores. Although these transmembrane proteins are important for a variety of physiological functions, their molecular operations are only superficially understood. High-resolution X-ray crystallography techniques have recent...
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Formato: | Texto |
Lenguaje: | English |
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The Rockefeller University Press
2003
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2229544/ https://www.ncbi.nlm.nih.gov/pubmed/12885876 http://dx.doi.org/10.1085/jgp.200308845 |
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author | Lin, Chia-Wei Chen, Tsung-Yu |
author_facet | Lin, Chia-Wei Chen, Tsung-Yu |
author_sort | Lin, Chia-Wei |
collection | PubMed |
description | ClC channels are a family of protein molecules containing two ion-permeation pores. Although these transmembrane proteins are important for a variety of physiological functions, their molecular operations are only superficially understood. High-resolution X-ray crystallography techniques have recently revealed the structures of two bacterial ClC channels, but whether vertebrate ClC channel pores are similar to those of bacterial homologues is not clear. To study the pore architecture of the Torpedo ClC-0 channel, we employed the substituted-cysteine-accessibility method (SCAM) and used charged methane thiosulfonate (MTS) compounds to modify the introduced cysteine. Several conclusions were derived from this approach. First, the MTS modification pattern from Y512C to E526C in ClC-0, which corresponds to residues forming helix R in bacterial ClC channels, is indeed consistent with the suggested helical structure. Second, the ClC-0 pore is more accessible to the negatively charged than to the positively charged MTS compound, a pore property that is regulated by the intrinsic electrostatic potential in the pore. Finally, attempts to modify the introduced cysteine at positions intracellular to the selectivity filter did not result in larger MTS modification rates for the open-state channel, suggesting that the fast gate of ClC-0 cannot be located at a position intracellular to the Cl(−) selectivity filter. Thus, the proposal that the glutamate side chain is the fast gate of the channel is applicable to ClC-0, revealing a structural and functional conservation of ClC channels between bacterial and vertebrate species. |
format | Text |
id | pubmed-2229544 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2003 |
publisher | The Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-22295442008-04-16 Probing the Pore of ClC-0 by Substituted Cysteine Accessibility Method Using Methane Thiosulfonate Reagents Lin, Chia-Wei Chen, Tsung-Yu J Gen Physiol Article ClC channels are a family of protein molecules containing two ion-permeation pores. Although these transmembrane proteins are important for a variety of physiological functions, their molecular operations are only superficially understood. High-resolution X-ray crystallography techniques have recently revealed the structures of two bacterial ClC channels, but whether vertebrate ClC channel pores are similar to those of bacterial homologues is not clear. To study the pore architecture of the Torpedo ClC-0 channel, we employed the substituted-cysteine-accessibility method (SCAM) and used charged methane thiosulfonate (MTS) compounds to modify the introduced cysteine. Several conclusions were derived from this approach. First, the MTS modification pattern from Y512C to E526C in ClC-0, which corresponds to residues forming helix R in bacterial ClC channels, is indeed consistent with the suggested helical structure. Second, the ClC-0 pore is more accessible to the negatively charged than to the positively charged MTS compound, a pore property that is regulated by the intrinsic electrostatic potential in the pore. Finally, attempts to modify the introduced cysteine at positions intracellular to the selectivity filter did not result in larger MTS modification rates for the open-state channel, suggesting that the fast gate of ClC-0 cannot be located at a position intracellular to the Cl(−) selectivity filter. Thus, the proposal that the glutamate side chain is the fast gate of the channel is applicable to ClC-0, revealing a structural and functional conservation of ClC channels between bacterial and vertebrate species. The Rockefeller University Press 2003-08 /pmc/articles/PMC2229544/ /pubmed/12885876 http://dx.doi.org/10.1085/jgp.200308845 Text en Copyright © 2003, The Rockefeller University Press 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 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/). |
spellingShingle | Article Lin, Chia-Wei Chen, Tsung-Yu Probing the Pore of ClC-0 by Substituted Cysteine Accessibility Method Using Methane Thiosulfonate Reagents |
title | Probing the Pore of ClC-0 by Substituted Cysteine Accessibility Method Using Methane Thiosulfonate Reagents |
title_full | Probing the Pore of ClC-0 by Substituted Cysteine Accessibility Method Using Methane Thiosulfonate Reagents |
title_fullStr | Probing the Pore of ClC-0 by Substituted Cysteine Accessibility Method Using Methane Thiosulfonate Reagents |
title_full_unstemmed | Probing the Pore of ClC-0 by Substituted Cysteine Accessibility Method Using Methane Thiosulfonate Reagents |
title_short | Probing the Pore of ClC-0 by Substituted Cysteine Accessibility Method Using Methane Thiosulfonate Reagents |
title_sort | probing the pore of clc-0 by substituted cysteine accessibility method using methane thiosulfonate reagents |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2229544/ https://www.ncbi.nlm.nih.gov/pubmed/12885876 http://dx.doi.org/10.1085/jgp.200308845 |
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