Structure of the SthK Carboxy-Terminal Region Reveals a Gating Mechanism for Cyclic Nucleotide-Modulated Ion Channels

Kesters, Divya; Brams, Marijke; Nys, Mieke; Wijckmans, Eveline; Spurný, Radovan; Voets, Thomas; Tytgat, Jan; Kusch, Jana; Ulens, Chris

doi:10.1371/journal.pone.0116369

Cited by 30 publications

(49 citation statements)

References 33 publications

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“…Thus, our analysis suggests that the more potent facilitation by cAMP is due to stronger effects on the closed to open transitions as well as to its greater binding affinity and as compared with cGMP. A similar suggestion was made in a recent study on the SthK (Schmidpeter et al, 2018), a CNG channel from Spirochaeta thermophila that is structurally and evolutionarily related to the HCN channels (Brams et al, 2014;Kesters et al, 2015), which showed that cGMP binds with an affinity that is similar to cAMP but that it opens the channel less efficiently. Structures of the HCN2 channel show that cGMP binds in the syn configuration whereas cAMP binds in the anti configuration, and that each ligand makes unique interactions within the binding site (Zagotta et al, 2003).…”

Section: Discussionsupporting

confidence: 80%

Binding and structural asymmetry governs ligand sensitivity in a cyclic nucleotide–gated ion channel

Zhuang

Petegem

et al. 2019

Journal of General Physiology

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Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels open more easily when cAMP or cGMP bind to a domain in the intracellular C-terminus in each of four identical subunits. How sensitivity of the channels to these ligands is determined is not well understood. Here, we apply a mathematical model, which incorporates negative cooperativity, to gating and mutagenesis data available in the literature and combine the results with binding data collected using isothermal titration calorimetry. This model recapitulates the concentration–response data for the effects of cAMP and cGMP on wild-type HCN2 channel opening and, remarkably, predicts the concentration–response data for a subset of mutants with single-point amino acid substitutions in the binding site. Our results suggest that ligand sensitivity is determined by negative cooperativity and asymmetric effects on structure and channel opening, which are tuned by ligand-specific interactions and residues within the binding site.

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Section: Discussionsupporting

confidence: 80%

Binding and structural asymmetry governs ligand sensitivity in a cyclic nucleotide–gated ion channel

Zhuang

Petegem

et al. 2019

Journal of General Physiology

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“…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: 84%

“…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: 86%

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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“…Because cyclic nucleotide regulation is potentiated when the AЈ helix is coordinating metal ions with the membrane, it is plausible that upon cyclic nucleotide binding, the AЈ helix moves toward the membrane, and this movement makes the channel more likely to open. In a recent crystal structure of a C-terminal fragment of a bacterial CNG channel, SthK, the AЈ helix is moved upward, toward the presumed location of the membrane, when bound to an agonist (cAMP) but not when bound to an antagonist (cGMP) (39).…”

Section: Discussionmentioning

confidence: 99%

Regulation of CNGA1 Channel Gating by Interactions with the Membrane

Aman

Gordon

Zagotta

2016

Journal of Biological Chemistry

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Cyclic nucleotide-gated (CNG) channels are expressed in rod photoreceptors and open in response to direct binding of cyclic nucleotides. We have previously shown that potentiation of CNGA1 channels by transition metals requires a histidine in the A helix following the S6 transmembrane segment. Here, we used transition metal ion FRET and patch clamp fluorometry with a fluorescent, noncanonical amino acid (3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap)) to show that the potentiating transition metal Co 2؉ binds in or near the A helix. Adding high-affinity metal-binding sites to the membrane (stearoyl-nitrilotriacetic acid (C18-NTA)) increased potentiation for low Co 2؉ concentrations, indicating that the membrane can coordinate metal ions with the A helix. These results suggest that restraining the A helix to the plasma membrane potentiates CNGA1 channel opening. Similar interactions between the A helix and the plasma membrane may underlie regulation of structurally related hyperpolarizationactivated cyclic nucleotide-gated (HCN) and voltage-gated potassium subfamily H (KCNH) channels by plasma membrane components. Cyclic nucleotide-gated (CNG)2 channels are voltage-independent, nonselective cation channels that are activated by the direct binding of cGMP and cAMP (1, 2). They are expressed in photoreceptors and olfactory receptors, where they mediate visual and olfactory transduction, respectively (3). They are also expressed throughout the brain (4, 5), where they can modulate neuronal excitability (6) and long-term potentiation (7). CNG channels are structurally similar to hyperpolarization-activated cyclic nucleotide-gated (HCN) and voltage-gated potassium subfamily H (KCNH) channels (1, 8). They are composed of four subunits forming around the central ion-conducting pore (Fig. 1A). Each subunit has a C-terminal cyclic nucleotide-binding domain (CNBD), which is connected to the channel pore by a C-linker region (Fig. 1B) (9). In CNG and HCN channels, cyclic nucleotide binding to the CNBD causes a conformational change in the C-linker, which then favors opening of the channel pore.Although structural rearrangements of the CNBD have been extensively studied, rearrangements of the C-linker are less well understood. The C-linker is the site of virtually all of the intersubunit interactions in the C-terminal region. It is composed of several ␣ helices, the most N-terminal of which is the AЈ helix, directly following the S6 transmembrane segment (Fig. 1B) (9). Based on the structure of the isolated C-linker and CNBD of HCN2, the AЈ helix is thought to run nearly parallel to the membrane near the cytosolic surface.The first evidence that the AЈ helix plays a role in channel gating came from studies of transition metal ion effects on CNGA1 (rod) and CNGA2 (olfactory) channels. Micromolar concentrations of transition metals were found to potentiate activation of rod channels, increasing the currents in response to partial agonists and subsaturating concentrations of full agonists (10 -12). This effect wa...

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Structure of the SthK Carboxy-Terminal Region Reveals a Gating Mechanism for Cyclic Nucleotide-Modulated Ion Channels

Cited by 30 publications

References 33 publications

Binding and structural asymmetry governs ligand sensitivity in a cyclic nucleotide–gated ion channel

Binding and structural asymmetry governs ligand sensitivity in a cyclic nucleotide–gated ion channel

CryoEM structure of a prokaryotic cyclic nucleotide-gated ion channel

Regulation of CNGA1 Channel Gating by Interactions with the Membrane

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