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Ligand binding and activation properties of the purified bacterial cyclic nucleotide–gated channel SthK

Cyclic nucleotide–modulated ion channels play several essential physiological roles. They are involved in signal transduction in photoreceptors and olfactory sensory neurons as well as pacemaking activity in the heart and brain. Investigations of the molecular mechanism of their actions, including s...

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Autores principales: Schmidpeter, Philipp A.M., Gao, Xiaolong, Uphadyay, Vikrant, Rheinberger, Jan, Nimigean, Crina M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Rockefeller University Press 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5987880/
https://www.ncbi.nlm.nih.gov/pubmed/29752414
http://dx.doi.org/10.1085/jgp.201812023
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author Schmidpeter, Philipp A.M.
Gao, Xiaolong
Uphadyay, Vikrant
Rheinberger, Jan
Nimigean, Crina M.
author_facet Schmidpeter, Philipp A.M.
Gao, Xiaolong
Uphadyay, Vikrant
Rheinberger, Jan
Nimigean, Crina M.
author_sort Schmidpeter, Philipp A.M.
collection PubMed
description Cyclic nucleotide–modulated ion channels play several essential physiological roles. They are involved in signal transduction in photoreceptors and olfactory sensory neurons as well as pacemaking activity in the heart and brain. Investigations of the molecular mechanism of their actions, including structural and electrophysiological characterization, are restricted by the availability of stable, purified protein obtained from accessible systems. Here, we establish that SthK, a cyclic nucleotide–gated (CNG) channel from Spirochaeta thermophila, is an excellent model for investigating the gating of eukaryotic CNG channels at the molecular level. The channel has high sequence similarity with its eukaryotic counterparts and was previously reported to be activated by cyclic nucleotides in patch-clamp experiments with Xenopus laevis oocytes. We optimized protein expression and purification to obtain large quantities of pure, homogeneous, and active recombinant SthK protein from Escherichia coli. A negative-stain electron microscopy (EM) single-particle analysis indicated that this channel is a promising candidate for structural studies with cryo-EM. Using radioactivity and fluorescence flux assays, as well as single-channel recordings in lipid bilayers, we show that the protein is partially activated by micromolar concentrations of cyclic adenosine monophosphate (cAMP) and that channel activity is increased by depolarization. Unlike previous studies, we find that cyclic guanosine monophosphate (cGMP) is also able to activate SthK, but with much lower efficiency than cAMP. The distinct sensitivities to different ligands resemble eukaryotic CNG and hyperpolarization-activated and cyclic nucleotide–modulated channels. Using a fluorescence binding assay, we show that cGMP and cAMP bind to SthK with similar apparent affinities, suggesting that the large difference in channel activation by cAMP or cGMP is caused by the efficacy with which each ligand promotes the conformational changes toward the open state. We conclude that the functional characteristics of SthK reported here will permit future studies to analyze ligand gating and discrimination in CNG channels.
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spelling pubmed-59878802018-12-04 Ligand binding and activation properties of the purified bacterial cyclic nucleotide–gated channel SthK Schmidpeter, Philipp A.M. Gao, Xiaolong Uphadyay, Vikrant Rheinberger, Jan Nimigean, Crina M. J Gen Physiol Research Articles Cyclic nucleotide–modulated ion channels play several essential physiological roles. They are involved in signal transduction in photoreceptors and olfactory sensory neurons as well as pacemaking activity in the heart and brain. Investigations of the molecular mechanism of their actions, including structural and electrophysiological characterization, are restricted by the availability of stable, purified protein obtained from accessible systems. Here, we establish that SthK, a cyclic nucleotide–gated (CNG) channel from Spirochaeta thermophila, is an excellent model for investigating the gating of eukaryotic CNG channels at the molecular level. The channel has high sequence similarity with its eukaryotic counterparts and was previously reported to be activated by cyclic nucleotides in patch-clamp experiments with Xenopus laevis oocytes. We optimized protein expression and purification to obtain large quantities of pure, homogeneous, and active recombinant SthK protein from Escherichia coli. A negative-stain electron microscopy (EM) single-particle analysis indicated that this channel is a promising candidate for structural studies with cryo-EM. Using radioactivity and fluorescence flux assays, as well as single-channel recordings in lipid bilayers, we show that the protein is partially activated by micromolar concentrations of cyclic adenosine monophosphate (cAMP) and that channel activity is increased by depolarization. Unlike previous studies, we find that cyclic guanosine monophosphate (cGMP) is also able to activate SthK, but with much lower efficiency than cAMP. The distinct sensitivities to different ligands resemble eukaryotic CNG and hyperpolarization-activated and cyclic nucleotide–modulated channels. Using a fluorescence binding assay, we show that cGMP and cAMP bind to SthK with similar apparent affinities, suggesting that the large difference in channel activation by cAMP or cGMP is caused by the efficacy with which each ligand promotes the conformational changes toward the open state. We conclude that the functional characteristics of SthK reported here will permit future studies to analyze ligand gating and discrimination in CNG channels. Rockefeller University Press 2018-06-04 /pmc/articles/PMC5987880/ /pubmed/29752414 http://dx.doi.org/10.1085/jgp.201812023 Text en © 2018 Schmidpeter 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
Schmidpeter, Philipp A.M.
Gao, Xiaolong
Uphadyay, Vikrant
Rheinberger, Jan
Nimigean, Crina M.
Ligand binding and activation properties of the purified bacterial cyclic nucleotide–gated channel SthK
title Ligand binding and activation properties of the purified bacterial cyclic nucleotide–gated channel SthK
title_full Ligand binding and activation properties of the purified bacterial cyclic nucleotide–gated channel SthK
title_fullStr Ligand binding and activation properties of the purified bacterial cyclic nucleotide–gated channel SthK
title_full_unstemmed Ligand binding and activation properties of the purified bacterial cyclic nucleotide–gated channel SthK
title_short Ligand binding and activation properties of the purified bacterial cyclic nucleotide–gated channel SthK
title_sort ligand binding and activation properties of the purified bacterial cyclic nucleotide–gated channel sthk
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5987880/
https://www.ncbi.nlm.nih.gov/pubmed/29752414
http://dx.doi.org/10.1085/jgp.201812023
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