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A Cysteine Scan of the Inner Vestibule of Cyclic Nucleotide–gated Channels Reveals Architecture and Rearrangement of the Pore
Cyclic nucleotide–gated (CNG) channels belong to the P-loop–containing family of ion channels that also includes KcsA, MthK, and Shaker channels. In this study, we investigated the structure and rearrangement of the CNGA1 channel pore using cysteine mutations and cysteine-specific modification. We c...
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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/PMC2217351/ https://www.ncbi.nlm.nih.gov/pubmed/12771192 http://dx.doi.org/10.1085/jgp.200308819 |
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author | Flynn, Galen E. Zagotta, William N. |
author_facet | Flynn, Galen E. Zagotta, William N. |
author_sort | Flynn, Galen E. |
collection | PubMed |
description | Cyclic nucleotide–gated (CNG) channels belong to the P-loop–containing family of ion channels that also includes KcsA, MthK, and Shaker channels. In this study, we investigated the structure and rearrangement of the CNGA1 channel pore using cysteine mutations and cysteine-specific modification. We constructed 16 mutant channels, each one containing a cysteine mutation at one of the positions between 384 and 399 in the S6 region of the pore. By measuring currents activated by saturating concentrations of the full agonist cGMP and the partial agonists cIMP and cAMP, we show that mutating S6 residues to cysteine caused both favorable and unfavorable changes in the free energy of channel opening. The time course of cysteine modification with 2-aminoethylmethane thiosulfonate hydrochloride (MTSEA) was complex. For many positions we observed decreases in current activated by cGMP and concomitant increases in current activated by cIMP and cAMP. A model where modification affected both gating and permeation successfully reproduced the complex time course of modification for most of the mutant channels. From the model fits to the time course of modification for each mutant channel, we quantified the following: (a) the bimolecular rate constant of modification in the open state, (b) the change in conductance, and (c) the change in the free energy of channel opening for modification of each cysteine. At many S6 cysteines, modification by MTSEA caused a decrease in conductance and a favorable change in the free energy of channel opening. Our results are interpreted within the structural framework of the known structures of KcsA and MthK. We conclude that: (a) MTSEA modification affects both gating and permeation, (b) the open configuration of the pore of CNGA1 channels is consistent with the structure of MthK, and (c) the modification of S6 residues disrupts the helical packing of the closed channel, making it easier for channels to open. |
format | Text |
id | pubmed-2217351 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2003 |
publisher | The Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-22173512008-04-16 A Cysteine Scan of the Inner Vestibule of Cyclic Nucleotide–gated Channels Reveals Architecture and Rearrangement of the Pore Flynn, Galen E. Zagotta, William N. J Gen Physiol Article Cyclic nucleotide–gated (CNG) channels belong to the P-loop–containing family of ion channels that also includes KcsA, MthK, and Shaker channels. In this study, we investigated the structure and rearrangement of the CNGA1 channel pore using cysteine mutations and cysteine-specific modification. We constructed 16 mutant channels, each one containing a cysteine mutation at one of the positions between 384 and 399 in the S6 region of the pore. By measuring currents activated by saturating concentrations of the full agonist cGMP and the partial agonists cIMP and cAMP, we show that mutating S6 residues to cysteine caused both favorable and unfavorable changes in the free energy of channel opening. The time course of cysteine modification with 2-aminoethylmethane thiosulfonate hydrochloride (MTSEA) was complex. For many positions we observed decreases in current activated by cGMP and concomitant increases in current activated by cIMP and cAMP. A model where modification affected both gating and permeation successfully reproduced the complex time course of modification for most of the mutant channels. From the model fits to the time course of modification for each mutant channel, we quantified the following: (a) the bimolecular rate constant of modification in the open state, (b) the change in conductance, and (c) the change in the free energy of channel opening for modification of each cysteine. At many S6 cysteines, modification by MTSEA caused a decrease in conductance and a favorable change in the free energy of channel opening. Our results are interpreted within the structural framework of the known structures of KcsA and MthK. We conclude that: (a) MTSEA modification affects both gating and permeation, (b) the open configuration of the pore of CNGA1 channels is consistent with the structure of MthK, and (c) the modification of S6 residues disrupts the helical packing of the closed channel, making it easier for channels to open. The Rockefeller University Press 2003-06 /pmc/articles/PMC2217351/ /pubmed/12771192 http://dx.doi.org/10.1085/jgp.200308819 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 Flynn, Galen E. Zagotta, William N. A Cysteine Scan of the Inner Vestibule of Cyclic Nucleotide–gated Channels Reveals Architecture and Rearrangement of the Pore |
title | A Cysteine Scan of the Inner Vestibule of Cyclic Nucleotide–gated Channels Reveals Architecture and Rearrangement of the Pore |
title_full | A Cysteine Scan of the Inner Vestibule of Cyclic Nucleotide–gated Channels Reveals Architecture and Rearrangement of the Pore |
title_fullStr | A Cysteine Scan of the Inner Vestibule of Cyclic Nucleotide–gated Channels Reveals Architecture and Rearrangement of the Pore |
title_full_unstemmed | A Cysteine Scan of the Inner Vestibule of Cyclic Nucleotide–gated Channels Reveals Architecture and Rearrangement of the Pore |
title_short | A Cysteine Scan of the Inner Vestibule of Cyclic Nucleotide–gated Channels Reveals Architecture and Rearrangement of the Pore |
title_sort | cysteine scan of the inner vestibule of cyclic nucleotide–gated channels reveals architecture and rearrangement of the pore |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2217351/ https://www.ncbi.nlm.nih.gov/pubmed/12771192 http://dx.doi.org/10.1085/jgp.200308819 |
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