scholarly journals CryoEM structure of a prokaryotic cyclic nucleotide-gated ion channel

2017 ◽  
Vol 114 (17) ◽  
pp. 4430-4435 ◽  
Author(s):  
Zachary M. James ◽  
Andrew J. Borst ◽  
Yoni Haitin ◽  
Brandon Frenz ◽  
Frank DiMaio ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Cyclic Nucleotide ◽  
Sequence Similarity ◽  
Nucleotide Binding ◽  
Cardiac Pace ◽  
Hcn Channels ◽  
Cyclic Nucleotide Binding ◽  
Channel Opening ◽  
Cng Channel ◽  
Linker Domain

Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-regulated (HCN) ion channels play crucial physiological roles in phototransduction, olfaction, and cardiac pace making. These channels are characterized by the presence of a carboxyl-terminal cyclic nucleotide-binding domain (CNBD) that connects to the channel pore via a C-linker domain. Although cyclic nucleotide binding has been shown to promote CNG and HCN channel opening, the precise mechanism underlying gating remains poorly understood. Here we used cryoEM to determine the structure of the intact LliK CNG channel isolated from Leptospira licerasiae—which shares sequence similarity to eukaryotic CNG and HCN channels—in the presence of a saturating concentration of cAMP. A short S4–S5 linker connects nearby voltage-sensing and pore domains to produce a non–domain-swapped transmembrane architecture, which appears to be a hallmark of this channel family. We also observe major conformational changes of the LliK C-linkers and CNBDs relative to the crystal structures of isolated C-linker/CNBD fragments and the cryoEM structures of related CNG, HCN, and KCNH channels. The conformation of our LliK structure may represent a functional state of this channel family not captured in previous studies.

scholarly journals Discovery and Characterization of a Distinct Cyclic Nucleotide Binding Pocket in HCN Channels

2014 ◽  
Vol 106 (2) ◽  
pp. 627a
Author(s):  
Anna Moroni ◽  
Marco Lolicato ◽  
Annalisa Bucchi ◽  
Cristina Arrigoni ◽  
Stefano Zucca ◽  
...  
Keyword(s):  
Cyclic Nucleotide ◽  
Nucleotide Binding ◽  
Binding Pocket ◽  
Hcn Channels ◽  
Cyclic Nucleotide Binding

scholarly journals A Cyclic Nucleotide Modulated Prokaryotic K+ Channel

2004 ◽  
Vol 124 (3) ◽  
pp. 203-210 ◽  
Author(s):  
Crina M. Nimigean ◽  
Tania Shane ◽  
Christopher Miller
Keyword(s):  
Cyclic Nucleotide ◽  
Gel Filtration ◽  
Nucleotide Binding ◽  
K Channel ◽  
Ionic Flux ◽  
Heterogeneous Distribution ◽  
Homogeneous Population ◽  
Mesorhizobium Loti ◽  
Cyclic Nucleotide Binding ◽  
Channel Opening

A search of prokaryotic genomes uncovered a gene from Mesorhizobium loti homologous to eukaryotic K+ channels of the S4 superfamily that also carry a cyclic nucleotide binding domain at the COOH terminus. The gene was cloned from genomic DNA, and the protein, denoted MloK1, was overexpressed in Escherichia coli and purified. Gel filtration analysis revealed a heterogeneous distribution of protein sizes which, upon inclusion of cyclic nucleotide, coalesces into a homogeneous population, eluting at the size expected for a homotetramer. As followed by a radioactive 86Rb+ flux assay, the putative channel protein catalyzes ionic flux with a selectivity expected for a K+ channel. Ion transport is stimulated by cAMP and cGMP at submicromolar concentrations. Since this bacterial homologue does not have the “C-linker” sequence found in all eukaryotic S4-type cyclic nucleotide-modulated ion channels, these results show that this four-helix structure is not a general requirement for transducing the cyclic nucleotide-binding signal to channel opening.

scholarly journals Allosteric Regulation of the Cyclic Nucleotide-Binding Domain in HCN Channels

2015 ◽  
Vol 108 (2) ◽  
pp. 192a-193a
Author(s):  
Hannah A. DeBerg ◽  
Shahidul M. Islam ◽  
Michael C. Puljung ◽  
Benoit Roux ◽  
William N. Zagotta ◽  
...  
Keyword(s):  
Cyclic Nucleotide ◽  
Allosteric Regulation ◽  
Nucleotide Binding ◽  
Binding Domain ◽  
Hcn Channels ◽  
Nucleotide Binding Domain ◽  
Cyclic Nucleotide Binding ◽  
Cyclic Nucleotide Binding Domain

scholarly journals Salt Bridges and Gating in the COOH-terminal Region of HCN2 and CNGA1 Channels

2004 ◽  
Vol 124 (6) ◽  
pp. 663-677 ◽  
Author(s):  
Kimberley B. Craven ◽  
William N. Zagotta
Keyword(s):  
Crystal Structure ◽  
Cyclic Nucleotide ◽  
Sequence Similarity ◽  
Terminal Region ◽  
Hcn Channels ◽  
Salt Bridges ◽  
Linker Region ◽  
Channel Opening ◽  
Intersubunit Interactions ◽  
Cnga1 Channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels and cyclic nucleotide-gated (CNG) channels are activated by the direct binding of cyclic nucleotides. The intracellular COOH-terminal regions exhibit high sequence similarity in all HCN and CNG channels. This region contains the cyclic nucleotide-binding domain (CNBD) and the C-linker region, which connects the CNBD to the pore. Recently, the structure of the HCN2 COOH-terminal region was solved and shown to contain intersubunit interactions between C-linker regions. To explore the role of these intersubunit interactions in intact channels, we studied two salt bridges in the C-linker region: an intersubunit interaction between C-linkers of neighboring subunits, and an intrasubunit interaction between the C-linker and its CNBD. We show that breaking these salt bridges in both HCN2 and CNGA1 channels through mutation causes an increase in the favorability of channel opening. The wild-type behavior of both HCN2 and CNGA1 channels is rescued by switching the position of the positive and negative residues, thus restoring the salt bridges. These results suggest that the salt bridges seen in the HCN2 COOH-terminal crystal structure are also present in the intact HCN2 channel. Furthermore, the similar effects of the mutations on HCN2 and CNGA1 channels suggest that these salt bridge interactions are also present in the intact CNGA1 channel. As disrupting the interactions leads to channels with more favorable opening transitions, the salt bridges appear to stabilize a closed conformation in both the HCN2 and CNGA1 channels. These results suggest that the HCN2 COOH-terminal crystal structure contains the C-linker regions in the resting configuration even though the CNBD is ligand bound, and channel opening involves a rearrangement of the C-linkers and, thus, disruption of the salt bridges. Discovering that one portion of the COOH terminus, the CNBD, can be in the activated configuration while the other portion, the C-linker, is not activated has lead us to suggest a novel modular gating scheme for HCN and CNG channels.

scholarly journals Pathways of inter- and intrasubunit allosteric signaling in the C-linker disk and cyclic nucleotide-binding domain of HCN2 channels

2020 ◽  
Author(s):  
Christopher Pfleger ◽  
Jana Kusch ◽  
Mahesh Kondapuram ◽  
Tina Schwabe ◽  
Christian Sattler ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Cyclic Nucleotide ◽  
Signal Transmission ◽  
Nucleotide Binding ◽  
Binding Domain ◽  
Hcn Channels ◽  
Nucleotide Binding Domain ◽  
Membrane Hyperpolarization ◽  
Cyclic Nucleotide Binding ◽  
Cyclic Nucleotide Binding Domain

AbstractOpening of hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels is controlled by membrane hyperpolarization and binding of cyclic nucleotides to the tetrameric cyclic nucleotide-binding domain (CNBD), attached to the C-linker disk (CL). Confocal patch-clamp fluorometry revealed a pronounced cooperativity of ligand binding among protomers. However, by which pathways allosteric signal transmission occurs remained elusive. Here, we investigate how changes in the structural dynamics of the CL- CNBD of mouse HCN2 upon cAMP binding relate to inter- and intrasubunit signal transmission. Applying a rigidity theory-based approach, we identify two intersubunit and one intrasubunit pathways that differ in allosteric coupling strength between cAMP binding sites or towards the CL. These predictions agree with results from electrophysiological and patch-clamp fluorometry experiments. Our results map out distinct routes within the CL-CNBD that modulate different cAMP binding responses in HCN2 channels. They signify that functionally relevant submodules may exist within and across structurally discernable subunits in HCN channels.

Regulation of cell surface expression of functional pacemaker channels by a motif in the B-helix of the cyclic nucleotide-binding domain

2008 ◽  
Vol 295 (3) ◽  
pp. C642-C652 ◽  
Author(s):  
Hamed Nazzari ◽  
Damiano Angoli ◽  
Sarah S. Chow ◽  
Gina Whitaker ◽  
Leisha Leclair ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cyclic Nucleotide ◽  
Nucleotide Binding ◽  
Surface Expression ◽  
Binding Domain ◽  
Cell Surface Expression ◽  
Nucleotide Binding Domain ◽  
Cyclic Nucleotide Binding ◽  
Channel Opening ◽  
Cyclic Nucleotide Binding Domain

Previous studies have suggested that a portion of the cyclic nucleotide-binding domain (CNBD) of the hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) “pacemaker” channel, composed of the A- and B-helices and the interceding β-barrel, confers two functions: inhibition of channel opening in response to hyperpolarization and promotion of cell surface expression. The sequence determinants required for each of these functions are unknown. In addition, the mechanism underlying plasma membrane targeting by this subdomain has been limitedly explored. Here we identify a four-amino acid motif (EEYP) in the B-helix that strongly promotes channel export from the endoplasmic reticulum (ER) and cell surface expression but does not contribute to the inhibition of channel opening. This motif augments a step in the trafficking pathway and/or the efficiency of correct folding and assembly.

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