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

The Journal of General Physiology
Philipp Am SchmidpeterCrina M Nimigean

Abstract

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...Continue Reading

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Citations

Mar 20, 2019·The Journal of Biological Chemistry·Jacob L W MorganWilliam N Zagotta
Sep 5, 2019·The Journal of General Physiology·Leo C T NgEric A Accili
Nov 7, 2018·Nature Communications·Yinth Andrea Bernal SierraDietmar Schmitz
May 23, 2018·The Journal of General Physiology·Caitlin Sedwick
Nov 13, 2018·Bio-protocol·Philipp A M Schmidpeter, Crina M Nimigean
Sep 30, 2018·Nature Communications·Arin MarchesiSimon Scheuring
May 3, 2020·Proceedings of the National Academy of Sciences of the United States of America·Eric G B EvansStefan Stoll
Dec 18, 2020·Nature Communications·Philipp A M SchmidpeterCrina M Nimigean
Mar 19, 2021·British Journal of Pharmacology·Thilo HenßAlexander Gottschalk
Jul 18, 2021·Nature Communications·Raghavendar Reddy Sanganna GariSimon Scheuring
Aug 7, 2021·Pflügers Archiv : European journal of physiology·Luisa Maria Rosaria NapolitanoArin Marchesi

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Methods Mentioned

BETA
size-exclusion chromatography
x-ray
nuclear magnetic resonance
equilibrium titrations
gel filtration
gel-filtration
Fluorescence

Software Mentioned

Clampfit
PyMol
DoG Picker
Appion
RELION

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