Structure of a eukaryotic cyclic-nucleotide-gated channel

Li, Minghui; Zhou, Xin; Wang, Shu; Michailidis, Ioannis E.; Gong, Ye; Su, Dai‐Shi; Li, Huan; Li, Xueming; Yang, Jian

doi:10.1038/nature20819

Cited by 130 publications

(180 citation statements)

References 57 publications

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“…However, recent cryoEM structures of apo and cAMP-bound HCN1 channel exhibit similar C-linker architectures, which resemble those observed in the crystal structures of isolated HCN C-linker/CNBD fragments (12)(13)(14). Conversely, the recently reported open-pore cryoEM structure of TAX4 (15), a CNG channel from Caenorhabditis elegans, shows a pronounced translation and rotation of the C-linker, relative to the pore domain (PD), compared with both HCN1 structures. Despite this new structural information on CNG and HCN channels, the nature of the structural rearrangements in the C-linker/CNBD associated with channel activation remain unclear.…”

mentioning

confidence: 91%

“…The intersubunit elbow-on-shoulder contacts form a ring directly beneath the TMDs, and this interaction has been proposed to be dynamic during the cyclic nucleotide-dependent gating of CNG and HCN channels (1). The C-linker of LliK has a different conformation and quaternary organization than that observed in the crystal structures of CNG and HCN carboxyl-terminal fragments (13,14,23), as well as the recent cryoEM structures of TAX4, HCN1, and CaM-inhibited Eag1 (12,15,28) (Fig. 3 G-J and Movies S1-S3).…”

Section: Identification and Characterization Of A Functional Prokaryomentioning

confidence: 99%

“…Crystal structures of all CNG and HCN channel C-linker/CNBD fragments (13,14,23,27), as well as the recent cryoEM structures of TAX4, HCN1, and CaM-inhibited Eag1 (12,15,28), demonstrate a compact architecture of the CNBDs and CNBHDs whereby each protomer forms (weak) intersubunit contacts with neighboring subunits (Fig. 3 C and D).…”

Section: Identification and Characterization Of A Functional Prokaryomentioning

confidence: 99%

“…Of the positively charged residues in the LliK S4 helix (R122, K125, R128, and R135), four in TAX4, three in Eag1, and three in HCN1 are conserved and similarly oriented (12,15,28) (Fig. 4 E and F and Fig.…”

Section: Identification and Characterization Of A Functional Prokaryomentioning

confidence: 99%

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CryoEM structure of a prokaryotic cyclic nucleotide-gated ion channel

James

Borst

Haitin

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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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 carboxylterminal 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.yclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels are cationpermeable ion channels regulated by the direct binding of cyclic nucleotides (cAMP or cGMP) (1). CNG channels are present in retinal photoreceptors and olfactory sensory neurons, where they perform chemoelectrical energy conversion in response to light or odor stimuli, respectively. Mutations in CNG channels have been associated with numerous inherited retinal degenerative disorders, achromatopsia, and anosmia (2). HCN channels are found in the cardiac sinoatrial node and throughout the nervous system, where they open in response to membrane hyperpolarization and generate a depolarizing current responsible for rhythmic firing (1). HCN channel mutations and mistrafficking have been associated with several disorders, including sinus bradycardia, epilepsy, and autism (3, 4).CNG and HCN channels possess a cyclic nucleotide-binding domain (CNBD) in their carboxyl-terminal region, and binding of cyclic nucleotide produces a large increase in the open probability of the channel pore. Cyclic nucleotide binding to HCN channels also shifts the voltage dependence of activation to more depolarized potentials, increasing the rate and extent of channel opening (5). CNG and HCN channels are part of a family that includes KCNH potassium channels (Fig. 1A and Fig. S1). KCNH channels, however, are distinct in that they possess a cyclic nucleotide-binding homology domain (CNBHD) occupied by an "intrinsic ligand" and are not directly regulated by cyclic nucleotides (6-8).CNG and HCN channels also harbor a C-linker domain situated between the pore and the CNBD. Based on its position, along with mutagenesis and cross-linking studies, this domain is thought t...

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mentioning

confidence: 91%

Section: Identification and Characterization Of A Functional Prokaryomentioning

confidence: 99%

Section: Identification and Characterization Of A Functional Prokaryomentioning

confidence: 99%

Section: Identification and Characterization Of A Functional Prokaryomentioning

confidence: 99%

See 2 more Smart Citations

CryoEM structure of a prokaryotic cyclic nucleotide-gated ion channel

James

Borst

Haitin

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Electrophysiological characterization of CNG channels expressed in Xenopus oocytes or mammalian cells (Biel et al , 1993 and 1994; Baumann et al , 1994; Weyand et al , 1994; Yu et al , 1996; Zagotta and Siegelbaum, 1996), shows that they are activated by micromolar concentrations of cyclic nucleotides (cNMP), but cAMP and cGMP can act differentially on different channel subtypes. High resolution structures of a few cyclic nucleotide-modulated channels have recently been solved (James et al , 2017; Lee and MacKinnon, 2017; Li et al , 2017; Rheinberger et al , 2018), highlighting the need for biophysical assays to characterize these channels in order to gain a better understanding of the molecular interactions during gating and regulation.…”

Section: [Background]mentioning

confidence: 99%

Fluorescence Titrations to Determine the Binding Affinity of Cyclic Nucleotides to SthK Ion Channels

Schmidpeter

Nimigean

2018

BIO-PROTOCOL

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The cyclic-nucleotide modulated ion channel family includes cyclic nucleotide-gated (CNG) and hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels, which play essential roles in visual and olfactory signaling and the heart pacemaking activity. Functionally, these channels have been extensively characterized by electrophysiological techniques from protein heterologously expressed in Xenopus oocytes and mammalian cells. On the other hand, expression and purification of these proteins for biophysical and structural analyses in vitro is problematic and expensive and, accordingly, only limited information on the purified channels is available in the literature. Here we describe a protocol for binding studies of fluorescently labeled cyclic nucleotides to a homologue of eukaryotic CNG channels. Furthermore, we describe how to directly probe binding of unlabeled cyclic nucleotides in a competition assay. The use of fluorescence as a sensitive probe for ligand binding reduces the amount of protein needed and enables fast and easy measurements using standard laboratory equipment.

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An Unorthodox Mechanism Underlying Voltage Sensitivity of TRPV1 Ion Channel

Yang

Lee

et al. 2020

Advanced Science

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While the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1) channel is a polymodal nociceptor for heat, capsaicin, and protons, the channel's responses to each of these stimuli are profoundly regulated by membrane potential, damping or even prohibiting its response at negative voltages and amplifying its response at positive voltages. Therefore, voltage sensitivity of TRPV1 is anticipated to play an important role in shaping pain responses. How voltage regulates TRPV1 activation remains unknown. Here, it is shown that voltage sensitivity does not originate from the S4 segment like classic voltage-gated ion channels; instead, outer pore acidic residues directly partake in voltage-sensitive activation, with their negative charges collectively constituting the observed gating charges. Outer pore gating-charge movement is titratable by extracellular pH and is allosterically coupled to channel activation, likely by influencing the upper gate in the ion selectivity filter. Elucidating this unorthodox voltage-gating process provides a mechanistic foundation for understanding TRPV1 polymodal gating and opens the door to novel approaches regulating channel activity for pain management.

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Structure of a eukaryotic cyclic-nucleotide-gated channel

Cited by 130 publications

References 57 publications

CryoEM structure of a prokaryotic cyclic nucleotide-gated ion channel

CryoEM structure of a prokaryotic cyclic nucleotide-gated ion channel

Fluorescence Titrations to Determine the Binding Affinity of Cyclic Nucleotides to SthK Ion Channels

An Unorthodox Mechanism Underlying Voltage Sensitivity of TRPV1 Ion Channel

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