Ligand binding and activation properties of the purified bacterial cyclic nucleotide-gated channel SthK
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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Binding and structural asymmetry governs ligand sensitivity in a cyclic nucleotide-gated ion channel
Fluorescence Titrations to Determine the Binding Affinity of Cyclic Nucleotides to SthK Ion Channels
Fluorescence Titrations to Determine the Binding Affinity of Cyclic Nucleotides to SthK Ion Channels
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