Cyclic Nucleotide-modulated Channels Research Articles

Anisotropic Network Analysis of Open/Closed HCN4 Channel Advocates Asymmetric Subunit Cooperativity in cAMP Modulation of Gating.

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are opened in an allosteric manner by membrane hyperpolarization and cyclic nucleotides such as cAMP. Because of conflicting reports from experimental studies on whether cAMP binding to the four available binding sites in the channel tetramer operates cooperatively in gating, we employ here a computational approach as a promising route to examine ligand-induced conformational changes after binding to individual sites. By combining an elastic network model (ENM) with linear response theory (LRT) for modeling the apo-holo transition of the cyclic nucleotide-binding domain (CNBD) in HCN channels, we observe a distinct pattern of cooperativity matching the "positive-negative-positive" cooperativity reported from functional studies. This cooperativity pattern is highly conserved among HCN subtypes (HCN4, HCN1), but only to a lesser extent visible in structurally related channels, which are only gated by voltage (KAT1) or cyclic nucleotides (TAX4). This suggests an inherent cooperativity between subunits in HCN channels as part of a ligand-triggered gating mechanism in these channels.

PIP2 sterically inhibits pore opening of a CNG channel.

Cyclic nucleotide-gated (CNG), and hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels are key players in signaling cascades related to vision and olfaction, as well as pacemaking and neuropathic pain, respectively. Despite their structural homology, their functional regulation is quite different. For instance, the signaling lipid PIP2 inhibits CNG channels, but activates HCN channels. In both cases, the underlying mechanism is unknown.

Gating intermediates reveal inhibitory role of the voltage sensor in a cyclic nucleotide-modulated ion channel

Understanding how ion channels gate is important for elucidating their physiological roles and targeting them in pathophysiological states. Here, we used SthK, a cyclic nucleotide-modulated channel from Spirochaeta thermophila, to define a ligand-gating trajectory that includes multiple on-pathway intermediates. cAMP is a poor partial agonist for SthK and depolarization increases SthK activity. Tuning the energy landscape by gain-of-function mutations in the voltage sensor domain (VSD) allowed us to capture multiple intermediates along the ligand-activation pathway, highlighting the allosteric linkage between VSD, cyclic nucleotide-binding (CNBD) and pore domains. Small, lateral displacements of the VSD S4 segment were necessary to open the intracellular gate, pointing to an inhibitory VSD at rest. We propose that in wild-type SthK, depolarization leads to such VSD displacements resulting in release of inhibition. In summary, we report conformational transitions along the activation pathway that reveal allosteric couplings between key sites integrating to open the intracellular gate.

Uncoupling of Voltage- and Ligand-Induced Activation in HCN2 Channels by Glycine Inserts.

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetramers that generate electrical rhythmicity in special brain neurons and cardiomyocytes. The channels are activated by membrane hyperpolarization. The binding of cAMP to the four available cyclic nucleotide-binding domains (CNBD) enhances channel activation. We analyzed in the present study the mechanism of how the effect of cAMP binding is transmitted to the pore domain. Our strategy was to uncouple the C-linker (CL) from the channel core by inserting one to five glycine residues between the S6 gate and the A′-helix (constructs 1G to 5G). We quantified in full-length HCN2 channels the resulting functional effects of the inserted glycines by current activation as well as the structural dynamics and statics using molecular dynamics simulations and Constraint Network Analysis. We show functionally that already in 1G the cAMP effect on activation is lost and that with the exception of 3G and 5G the concentration-activation relationships are shifted to depolarized voltages with respect to HCN2. The strongest effect was found for 4G. Accordingly, the activation kinetics were accelerated by all constructs, again with the strongest effect in 4G. The simulations reveal that the average residue mobility of the CL and CNBD domains is increased in all constructs and that the junction between the S6 and A′-helix is turned into a flexible hinge, resulting in a destabilized gate in all constructs. Moreover, for 3G and 4G, there is a stronger downward displacement of the CL-CNBD than in HCN2 and the other constructs, resulting in an increased kink angle between S6 and A′-helix, which in turn loosens contacts between the S4-helix and the CL. This is suggested to promote a downward movement of the S4-helix, similar to the effect of hyperpolarization. In addition, exclusively in 4G, the selectivity filter in the upper pore region and parts of the S4-helix are destabilized. The results provide new insights into the intricate activation of HCN2 channels.

Anionic lipids unlock the gate of pacemaker channels

Hyperpolarization-activated and cyclic nucleotide-modulated channels (HCN) determine pacemaking activity in the heart and brain. Signaling lipids PIP and phosphatidic acid (PA) potentiate HCN activity, but the mechanisms are unknown, since functional studies were performed in cells, with limited control over membrane composition. Here we use SthK, a bacterial homolog of HCN, to investigate lipid-modulation in molecular detail. SthK activity depends on the charge of lipids as revealed by flux-assays and single-channel recordings in defined lipid environments.

Contribution of HCN1 variant to sinus bradycardia: A case report.

BackgroundMissense mutations in the hyperpolarization‐activated cyclic nucleotide‐modulated (HCN) channel 4 (HCN4) are one of the genetic causes of cardiac sinus bradycardia.ObjectiveTo investigate possible HCN4 channel mutation in a young patient with profound sinus bradycardia.MethodsDirect sequencing of HCN4 and whole‐exome sequencing were performed on DNA samples from the indexed patient (P), the patient's son (PS), and a family unrelated healthy long‐distance running volunteer (V). Resting heart rate was 31 bpm for P, 67 bpm for PS, and 50 bpm for V. Immunoblots, flow cytometry, and immunocytofluorescence confocal imaging were used to study cellular distribution of channel variants. Patch‐clamp electrophysiology was used to investigate the properties of mutant HCN1 channels.ResultsIn P no missense mutations were found in the HCN4 gene; instead, we found two heterozygous variants in the HCN1 gene: deletion of an N‐terminal glycine triplet (72GGG74, “N‐del”) and a novel missense variant, P851A, in the C‐terminal region. N‐del variant was found before and shared by PS. These two variations were not found in V. Compared to wild type, N‐del and P851A reduced cell surface expression and negatively shifted voltage‐activation with slower activation kinetics.ConclusionDecreased channel activity HCN1 mutant channel makes it unable to contribute to early depolarization of sinus node action potential, thus likely a main cause of the profound sinus bradycardia in this patient.

Identifying Novel Isoform-Specific HCN Channel Ligands using SPR

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are expressed abundantly in the heart and central nervous system where they control cardiac and neuronal rhythmicity. Four HCN channel isoforms exist in mammals, HCN1-4. While HCN2 is the most abundantly expressed isoform in the brain, HCN4 is the predominant isoform in the heart. Identification of isoform specific regulators of HCN channels is crucial for the development of drugs that can treat cardiovascular diseases without affecting the neuronal functions and vice versa. HCN channels consist of four subunits, each containing six transmembrane segments. In addition, HCN channel subunits contain a cyclic nucleotide-binding (CNB) domain in their C-terminal region, which is linked to the pore-forming transmembrane segment by a C-linker. Upon cyclic nucleotide binding to the CNB domain, the HCN channel undergoes a conformational change that facilitates channel pore opening. The role of CNB domain in regulating channel opening via ligand binding makes it an ideal target for isoform-specific small molecule regulators. To identify isoform specific binders of HCN channel CNB domains, we conducted a surface plasmon resonance (SPR)-based screen. For the SPR screen, the C-linker/CNB domains of HCN2 and HCN4 channels were expressed in BL21 cells and purified using Ni-affinity and size-exclusion chromatography. Then the isolated C-linker/CNB domains were immobilized on the SPR senor chip, and screened for binding to the small molecules in the Spectrum Library using SPR. Using this approach, we identified nine compounds that selectively bind to the CNB domain of HCN4, but not to HCN2 channels. These results indicate that CNB domains of HCN channels harbor binding sites for HCN isoform-specific small molecule ligands.

Molecular Insights into Lipid-Modulation of the Cyclic Nucleotide-Gated Channel SthK

Cyclic nucleotide-gated channels (CNG) and the closely related hyperpolarization activated and cyclic nucleotide-modulated (HCN) channels are key players in electrical signaling related to vision and olfaction, as well as pacemaking activity in the heart and brain, respectively. They are, however, hard to express and purify for biophysical studies under defined conditions leaving important questions about their regulation so far unanswered. The bacterial homolog SthK has been shown to share a high degree of structural and functional homology to eukaryotic CNG and HCN channels and is used as a bona fide model system to study their function and regulation in molecular detail.

Allosteric signaling in C-linker and cyclic nucleotide-binding domain of HCN2 channels

Opening 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 (CL) disk. Confocal patch-clamp fluorometry revealed 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 toward 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.

PDK1–AKT signaling pathway regulates the expression and function of cardiac hyperpolarization-activated cyclic nucleotide-modulated channels

AimThe enzyme 3-phosphoinositide-dependent protein kinase-1 (PDK1) is associated with cardiac and pathological remodeling and ion channel function regulation. However, whether it regulates hyperpolarization-activated cyclic nucleotide-modulated channels (HCNs) remains unclear. Main methodsIn the atrial myocytes of heart-specific PDK1 “knockout” mouse model and neonatal mice, protein kinase B (AKT)-related inhibitors or agonists as well as knockdown or overexpression plasmids were used to study the relationship between PDK1 and HCNs. Key findingsHCN1 expression and AKT phosphorylation at the Thr308 site were significantly decreased in atrial myocytes after PDK1 knockout or inhibition; in contrast, HCN2 and HCN4 levels were significantly increased. Also, a similar trend of HCNs expression has been observed in cultured atrial myocytes after PDK1 inhibition, as further demonstrated via immunofluorescence and patch-clamp experiments. Moreover, these results of PDK1 overexpression indicate an opposite trend compared with the previous experimental results. However, the results of PDK1 inhibition or overexpression could be reversed by activating or inhibiting AKT, respectively. SignificanceThese results indicate that the PDK1–AKT signaling pathway is involved in the regulation of HCN mRNA transcription, protein expression, HCN current density, and cell membrane location.

N6-modified cAMP derivatives that activate protein kinase A also act as full agonists of murine HCN2 channels

cAMP acts as a second messenger in many cellular processes. Three protein types mainly mediate cAMP-induced effects: PKA, exchange protein directly activated by cAMP (Epac), and cyclic nucleotide-modulated channels (cyclic nucleotide-gated or hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels). Discrimination among these cAMP signaling pathways requires specific targeting of only one protein. Previously, cAMP modifications at position N6 of the adenine ring (PKA) and position 2'-OH of the ribose (Epac) have been used to produce target-selective compounds. However, cyclic nucleotide-modulated ion channels were usually outside of the scope of these previous studies. These channels are widely distributed, so possible channel cross-activation by PKA- or Epac-selective agonists warrants serious consideration. Here we demonstrate the agonistic effects of three PKA-selective cAMP derivatives, N6-phenyladenosine-3',5'-cyclic monophosphate (N6-Phe-cAMP), N6-benzyladenosine-3',5'-cyclic monophosphate (N6-Bn-cAMP), and N6-benzoyl-adenosine-3',5'-cyclic monophosphate (N6-Bnz-cAMP), on murine HCN2 pacemaker channels. Electrophysiological characterization in Xenopus oocytes revealed that these derivatives differ in apparent affinities depending on the modification type but that their efficacy and effects on HCN2 activation kinetics are similar to those of cAMP. Docking experiments suggested a pivotal role of Arg-635 at the entrance of the binding pocket in HCN2, either causing stabilizing cation-π interactions with the aromatic ring in N6-Phe-cAMP or N6-Bn-cAMP or a steric clash with the aromatic ring in N6-Bnz-cAMP. A reduced apparent affinity of N6-Phe-cAMP toward the variants R635A and R635E strengthened that notion. We conclude that some PKA activators also effectively activate HCN2 channels. Hence, when studying PKA-mediated cAMP signaling with cAMP derivatives in a native environment, activation of HCN channels should be considered.

Lens Connexin Channels Have Differential Permeability to the Second Messenger cAMP.

PurposeGap junction channels exhibit connexin specific biophysical properties, including the selective intercellular passage of larger solutes, such as second messengers. Here, we have examined the cyclic nucleotide permeability of the lens connexins, which could influence events like epithelial cell division and differentiation.MethodsWe compared the cAMP permeability through channels composed of Cx43, Cx46, or Cx50 using simultaneous measurements of junctional conductance and intercellular transfer. For cAMP detection, the recipient cells were transfected with a cAMP sensor gene, the cyclic nucleotide-modulated channel from sea urchin sperm (SpIH). cAMP was introduced via patch pipette into the cell of the pair that did not express SpIH. SpIH-derived currents were recorded from the other cell of a pair that expressed SpIH. cAMP permeability was also directly visualized in transfected cells using a chemically modified fluorescent form of the molecule.ResultscAMP transfer was observed for homotypic Cx43 channels over a wide range of junctional conductance. Homotypic Cx46 channels also transferred cAMP, but permeability was reduced compared with Cx43. In contrast, homotypic Cx50 channels exhibited extremely low permeability to cAMP, when compared with either Cx43, or Cx46.ConclusionsThese data show that channels made from Cx43 and Cx46 result in the intercellular delivery of cAMP in sufficient quantity to activate cyclic nucleotide-modulated channels. The data also suggest that the greatly reduced cAMP permeability of Cx50 channels could play a role in the regulation of cell division in the lens.

Protein Redox State Monitoring Studies of Thiol Reactivity.

Protein cysteine thiol status is a major determinant of oxidative stress and oxidant signaling. The -SulfoBiotics- Protein Redox State Monitoring Kit provides a unique opportunity to investigate protein thiol states. This system adds a 15-kDa Protein-SHifter to reduced cysteine residues, and this molecular mass shift can be detected by gel electrophoresis. Even in biological samples, Protein-SHifter Plus allows the thiol states of specific proteins to be studied using Western blotting. Peroxiredoxin 6 (Prx6) is a unique one-cysteine peroxiredoxin that scavenges peroxides by utilizing conserved Cysteine-47. Human Prx6 also contains an additional non-conserved cysteine residue, while rat Prx6 only has the catalytic cysteine. In cultured cells, cysteine residues of Prx6 were found to be predominantly fully reduced. The treatment of human cells with hydrogen peroxide (H2O2) formed Prx6 with one cysteine reduced. Since catalytic cysteine becomes oxidized in rat cells by the same H2O2 treatment and treating denatured human Prx6 with H2O2 results in the oxidation of both cysteines, non-conserved cysteine may not be accessible to H2O2 in human cells. We also found that untreated cells contained Prx6 multimers bound through disulfide bonds. Surprisingly, treating cells with H2O2 eliminated these Prx6 multimers. In contrast, treating cell lysates with H2O2 promoted the formation of Prx6 multimers. Similarly, treating purified preparations of the recombinant cyclic nucleotide-binding domain of the human hyperpolarization-activated cyclic nucleotide-modulated channels with H2O2 promoted the formation of multimers. These studies revealed that the cellular environment defines the susceptibility of protein cysteines to H2O2 and determines whether H2O2 acts as a facilitator or a disrupter of disulfide bonds.

Novel Fluorescent Cyclic Nucleotide Derivatives to Study CNG and HCN Channel Function

A highly specific molecular interaction of diffusible ligands with their receptors belongs to the key processes in cellular signaling. Because an appropriate method to monitor the unitary binding events is still missing, most of our present knowledge is based on ensemble signals recorded from a big number of receptors, such as ion currents or fluorescence changes of suitably labeled receptors, and reasoning from these data to the ligand binding. To study the binding process itself, appropriately tagged ligands are required that fully activate the receptors and report the binding at the same time. Herein, we tailored a series of 18 novel fluorescent cyclic nucleotide derivatives by attaching 6 different dyes via different alkyl linkers to the 8-position of the purine ring of cGMP or cAMP. The biological activity was determined in inside-out macropatches containing either homotetrameric (CNGA2), heterotetrameric (CNGA2:CNGA4:CNGB1b), or hyperpolarization-activated cyclic nucleotide-modulated (HCN2) channels. All these novel fluorescent ligands are efficient to activate the channels, and the potency of most of them significantly exceeded that of the natural cyclic nucleotides cGMP or cAMP. Moreover, some of them showed an enhanced brightness when bound to the channels. The best of our derivatives bear great potential to systematically analyze the activation mechanism in CNG and HCN channels, at both the level of ensemble and single-molecule analyses.

Alpha 2 adrenoceptor agonist guanabenz directly inhibits hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channels in mesencephalic trigeminal nucleus neurons

Alpha 2 (α2-) adrenoceptor agonists, such as clonidine or dexmedetomidine, have been found to inhibit hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channels, not only by reducing intracellular cyclic AMP levels but also by directly blocking HCN channels. In this study, we examined the inhibitory effect of guanabenz, a centrally acting α2-adrenoceptor agonist with high specificity for α2A-subtype, on HCN channels in mesencephalic trigeminal nucleus (MTN) neurons which robustly express HCN channels and have been suggested to coexpress α2A-adrenoceptors. By performing whole-cell patch-clamp recording on MTN neurons in brainstem slices, hyperpolarization-activated inward current (Ih) was examined during guanabenz treatment. Guanabenz inhibited Ih in a dose-dependent manner, which was likely to be ZD7288-sensitive HCN current as it did not affect barium-sensitive inward rectifying potassium current. Guanabenz not only inhibited Ih but also shifted the voltage-dependent activation curve to hyperpolarizing potentials. Interestingly, Ih inhibition by guanabenz was not reversed by α2-adrenoceptor antagonist atipamezole treatment or by intracellular cyclic AMP perfusion, suggesting that the inhibition may not result from α2A-adrenoceptor signalling pathway but from direct inhibition of HCN channels. Coherent to our electrophysiological results, single-cell RT-PCR revealed that most MTN neurons lack α2A-adrenoceptor mRNA. Our study demonstrates that guanabenz can directly inhibit HCN channels in addition to its primary role of activating α2A-adrenoceptors.

The Role of HCN Domain in the Function of HCN Channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels generate funny currents (If) in the heart and hyperpolarization-activated currents (Ih) in the brain. HCN channels are assembled from four subunits, with each subunit containing six transmembrane segments (S1-S6) and a cyclic-nucleotide binding (CNB) domain in the C-terminal region linked to the pore-forming transmembrane segment with a C-linker. Recent cryo-EM structure of HCN1 channels (Lee et al., Cell, 2017, 168:111-120) identified a stretch of 45 amino acids directly preceding the S1 transmembrane segment as a 3-alpha-helical structural motif conserved in all four mammalian HCN channel isoforms. The 3-alpha-helical domain, dubbed HCN domain, forms direct interactions with the S4 transmembrane segment in the voltage sensor from the same subunit and the C-linker/CNB domain from the adjacent subunit. Here we explored the role of the HCN domain in the function of HCN channels. Deletion of the HCN domain abolished currents from HCN channels expressed in Xenopus laevis oocytes. Mutant HCN channels with alanines substituted for the interacting residues between the HCN domain and the C-linker/CNB domain gave rise to hyperpolarization-activated currents with the half-maximal activation voltage (V1/2) similar to the V1/2 of the wild-type HCN channels. Taken together our observations point towards the role of the HCN domain in the functional assembly of HCN channels.

Ligand discrimination and gating in cyclic nucleotide-gated ion channels from apo and partial agonist-bound cryo-EM structures.

Cyclic nucleotide-modulated channels have important roles in visual signal transduction and pacemaking. Binding of cyclic nucleotides (cAMP/cGMP) elicits diverse functional responses in different channels within the family despite their high sequence and structure homology. The molecular mechanisms responsible for ligand discrimination and gating are unknown due to lack of correspondence between structural information and functional states. Using single particle cryo-electron microscopy and single-channel recording, we assigned functional states to high-resolution structures of SthK, a prokaryotic cyclic nucleotide-gated channel. The structures for apo, cAMP-bound, and cGMP-bound SthK in lipid nanodiscs, correspond to no, moderate, and low single-channel activity, respectively, consistent with the observation that all structures are in resting, closed states. The similarity between apo and ligand-bound structures indicates that ligand-binding domains are strongly coupled to pore and SthK gates in an allosteric, concerted fashion. The different orientations of cAMP and cGMP in the 'resting' and 'activated' structures suggest a mechanism for ligand discrimination.

All four subunits of HCN2 channels contribute to the activation gating in an additive but intricate manner.

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetramers that elicit electrical rhythmicity in specialized brain neurons and cardiomyocytes. The channels are dually activated by voltage and binding of cyclic adenosine monophosphate (cAMP) to their four cyclic nucleotide-binding domains (CNBDs). Here we analyze the effects of cAMP binding to different concatemers of HCN2 channel subunits, each having a defined number of functional CNBDs. We show that each liganded CNBD promotes channel activation in an additive manner and that, in the special case of two functional CNBDs, functionality does not depend on the arrangement of the subunits. Correspondingly, the reverse process of deactivation is slowed by progressive liganding, but only if four and three ligands as well as two ligands in trans position (opposite to each other) are bound. In contrast, two ligands bound in cis positions (adjacent to each other) and a single bound ligand do not affect channel deactivation. These results support an activation mechanism in which each single liganded CNBD causes a turning momentum on the tetrameric ring-like structure formed by all four CNBDs and that at least two liganded subunits in trans positions are required to maintain activation.

Activation gating in HCN2 channels.

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels control electrical rhythmicity in specialized brain and heart cells. We quantitatively analysed voltage-dependent activation of homotetrameric HCN2 channels and its modulation by the second messenger cAMP using global fits of hidden Markovian models to complex experimental data. We show that voltage-dependent activation is essentially governed by two separable voltage-dependent steps followed by voltage-independent opening of the pore. According to this model analysis, the binding of cAMP to the channels exerts multiple effects on the voltage-dependent gating: It stabilizes the open pore, reduces the total gating charge from ~8 to ~5, makes an additional closed state outside the activation pathway accessible and strongly accelerates the ON-gating but not the OFF-gating. Furthermore, the open channel has a much slower computed OFF-gating current than the closed channel, in both the absence and presence of cAMP. Together, these results provide detailed new insight into the voltage- and cAMP-induced activation gating of HCN channels.

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

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.