Hyperpolarization-activated Cyclic Nucleotide-modulated 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.

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.

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.

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.

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.

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.

Role of Dimers in the cAMP-Dependent Activation of Hyperpolarization-Activated Cyclic-Nucleotide-Modulated (HCN) Ion Channels.

Hyperpolarization-activated cyclic-nucleotide-modulated (HCN) ion channels control rhythmicity in neurons and cardiomyocytes. Cyclic AMP (cAMP) modulates HCN activity through the cAMP-induced formation of a tetrameric gating ring spanning the intracellular region (IR) of HCN. Although evidence from confocal patch-clamp fluorometry indicates that the cAMP-dependent gating of HCN occurs through a dimer of dimers, the structural and dynamical basis of cAMP allostery in HCN dimers has so far remained elusive. Thus, here we examine how dimers influence IR structural dynamics, and the role that such structural dynamics play in HCN allostery. To this end, we performed molecular dynamics (MD) simulations of HCN4 IR dimers in their fully apo, fully holo, and partially cAMP-bound states, resulting in a total simulated time of 1.2 μs. Comparative analyses of these MD trajectories, as well as previous monomer and tetramer simulations utilized as benchmarks for comparison, reveal that dimers markedly sensitize the HCN IR to cAMP-modulated allostery. Our results indicate that dimerization fine-tunes the IR dynamics to enhance, relative to both monomers and tetramers, the allosteric intra- and interprotomer coupling between the cAMP-binding domain and tetramerization domain components of the IR. The resulting allosteric model provides a viable rationalization of electrophysiological data on the role of IR dimers in HCN activation.

Investigating cyclic nucleotide and cyclic dinucleotide binding to HCN channels by surface plasmon resonance.

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels control cardiac and neuronal rhythmicity. HCN channels contain cyclic nucleotide-binding domain (CNBD) in their C-terminal region linked to the pore-forming transmembrane segment with a C-linker. The C-linker couples the conformational changes caused by the direct binding of cyclic nucleotides to the HCN pore opening. Recently, cyclic dinucleotides were shown to antagonize the effect of cyclic nucleotides in HCN4 but not in HCN2 channels. Based on the structural analysis and mutational studies it has been proposed that cyclic dinucleotides affect HCN4 channels by binding to the C-linker pocket (CLP). Here, we first show that surface plasmon resonance (SPR) can be used to accurately measure cyclic nucleotide binding affinity to the C-linker/CNBD of HCN2 and HCN4 channels. We then used SPR to investigate cyclic dinucleotide binding in HCN channels. To our surprise, we detected no binding of cyclic dinucleotides to the isolated monomeric C-linker/CNBDs of HCN4 channels with SPR. The binding of cyclic dinucleotides was further examined with isothermal calorimetry (ITC), which indicated no binding of cyclic dinucleotides to both monomeric and tetrameric C-linker/CNBDs of HCN4 channels. Taken together, our results suggest that interaction of the C-linker/CNBD with other parts of the channel is necessary for cyclic-dinucleotide binding in HCN4 channels.

Investigating Ligand Binding to HCN Channels by Surface Plasmon Resonance

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels control cardiac and neuronal rhythmicity. HCN channels contain cyclic nucleotide-binding (CNB) domain in their C-terminal region linked to the pore-forming transmembrane segment with a C-linker. The C-linker couples the conformational changes caused by the direct binding of cyclic nucleotides in the CNB domain to the pore opening. Surface plasmon resonance (SPR) is a powerful biophysical tool for quantitatively investigating ligand-protein and protein-protein interactions. Here we used SPR to detect ligand binding to the isolated C-linker/CNB domain of HCN channels. The isolated C-linker/CNB domains of wild-type (WT) and L586W mutant HCN2 channels were immobilized on a NTA sensor chip and cAMP was injected over the protein coated surface. The mutant C-linker/CNB domain has been used before for ligand binding studies based on the changes in the fluorescence of the introduced tryptophan upon ligand binding. From the cyclic nucleotide concentration dependent SPR responses, we determined the binding affinity for cAMP to be 6.3 + 2.6 μM for the WT and 10.8 + 2.3μM for the mutant C-linker/CNB domains. The binding affinity determined for the mutant L586W C-linker-CNB domains determined with SPR is in agreement with the binding affinity of 13 + 2 μM determined with the fluorescence-based method. These results indicate that SPR is well suited for the detection of binding of known HCN channel ligands. Therefore, SPR can be used to identify novel HCN channel regulators for treatment of diseases associated with abnormal functions of these channels, such as epilepsy and cardiac arrhythmias.

Structural Basis of Tonic Inhibition by Dimers of Dimers in Hyperpolarization-Activated Cyclic-Nucleotide-Modulated (HCN) Ion Channels.

The hyperpolarization-activated cyclic-nucleotide-modulated (HCN) ion channels control rhythmicity in neurons and cardiomyocytes. Cyclic AMP (cAMP) modulates HCN activity through the cAMP-dependent formation of a tetrameric gating ring spanning the intracellular region (IR) of HCN. In the absence of cAMP, the IR cAMP-binding domain (CBD) mainly samples its inactive conformation, resulting in steric clashes that destabilize the IR tetramer. Although these clashes with the inactive CBD are released through tetramer dissociation into monomers, functional mutagenesis suggests that the apo IR is not fully monomeric. To investigate the inhibitory nonmonomeric IR species, we performed molecular dynamics simulations starting from "hybrid" structures that are tetrameric but contain inactive apo-state CBD conformations. The ensemble of simulated trajectories reveals that full dissociation of the tetramer into monomers is not necessary to release the steric hindrance with the inactive CBD. Specifically, we found that partial dissociation of the tetramer into dimers is sufficient to accommodate four inactive CBDs, while reduction of the quaternary symmetry of the nondissociated tetramer from 4- to 2-fold permits accommodation of two inactive CBDs. Our findings not only rationalize available electrophysiological, fluorometry, and sedimentation equilibrium data, but also provide unprecedented structural insight into previously elusive nonmonomeric autoinhibitory HCN species.

Same-Single-Cell Analysis of Pacemaker-Specific Markers in Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Subtypes Classified by Electrophysiology.

Insights into the expression of pacemaker-specific markers in human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte subtypes can facilitate the enrichment and track differentiation and maturation of hiPSC-derived pacemaker-like cardiomyocytes. To date, no study has directly assessed gene expression in each pacemaker-, atria-, and ventricular-like cardiomyocyte subtype derived from hiPSCs since currently the subtypes of these immature cardiomyocytes can only be identified by action potential profiles. Traditional acquisition of action potentials using patch-clamp recordings renders the cells unviable for subsequent analysis. We circumvented these issues by acquiring the action potential profile of a single cell optically followed by assessment of protein expression through immunostaining in that same cell. Our same-single-cell analysis for the first time revealed expression of proposed pacemaker-specific markers-hyperpolarization-activated cyclic nucleotide-modulated (HCN)4 channel and Islet (Isl)1-at the protein level in all three hiPSC-derived cardiomyocyte subtypes. HCN4 expression was found to be higher in pacemaker-like hiPSC-derived cardiomyocytes than atrial- and ventricular-like subtypes but its downregulation over time in all subtypes diminished the differences. Isl1 expression in pacemaker-like hiPSC-derived cardiomyocytes was initially not statistically different than the contractile subtypes but did become statistically higher than ventricular-like cells with time. Our observations suggest that although HCN4 and Isl1 are differentially expressed in hiPSC-derived pacemaker-like relative to ventricular-like cardiomyocytes, these markers alone are insufficient in identifying hiPSC-derived pacemaker-like cardiomyocytes. Stem Cells 2016;34:2670-2680.

Regulation of HCN Ion Channels by Non-canonical Cyclic Nucleotides.

The hyperpolarization-activated cyclic-nucleotide-modulated (HCN) proteins are cAMP-regulated ion channels that play a key role in nerve impulse transmission and heart rate modulation in neuronal and cardiac cells, respectively. Although they are regulated primarily by cAMP, other cyclic nucleotides such as cGMP, cCMP, and cUMP serve as partial agonists for the HCN2 and HCN4 isoforms. By competing with cAMP for binding, these non-canonical ligands alter ion channel gating, and in turn, modulate the cAMP-dependent activation profiles. The partial activation of non-canonical cyclic nucleotides can be rationalized by either a partial reversal of a two-state inactive/active conformational equilibrium, or by sampling of a third conformational state with partial activity. Furthermore, different mechanisms and degrees of activation have been observed upon binding of non-canonical cyclic nucleotides to HCN2 versus HCN4, suggesting that these ligands control HCN ion channels in an isoform-specific manner. While more work remains to be done to achieve a complete understanding of ion channel modulation by non-canonical cyclic nucleotides, it is already clear that such knowledge will ultimately prove invaluable in achieving a more complete understanding of ion channel signaling in vivo, as well as in the development of therapeutics designed to selectively modulate ion channel gating.

Conformational Flip of Nonactivated HCN2 Channel Subunits Evoked by Cyclic Nucleotides

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetrameric proteins that evoke electrical rhythmicity in specialized neurons and cardiomyocytes. The channels are activated by hyperpolarizing voltage but are also receptors for the intracellular ligand adenosine-3′,5′-cyclic monophosphate (cAMP) that enhances activation but is unable to activate the channels alone. Using fcAMP, a fluorescent derivative of cAMP, we analyzed the effect of ligand binding on HCN2 channels not preactivated by voltage. We identified a conformational flip of the channel as an intermediate state following the ligand binding and quantified it kinetically. Globally fitting the time courses of ligand binding and unbinding revealed modest cooperativity among the subunits in the conformational flip. The intensity of this cooperativity, however, was only moderate compared to channels preactivated by hyperpolarizing voltage. These data provide kinetic information about conformational changes proceeding in nonactivated HCN2 channels when cAMP binds. Moreover, our approach bears potential for analyzing the function of any other membrane receptor if a potent fluorescent ligand is available.

Role of Dynamics in the Autoinhibition and Activation of the Hyperpolarization-activated Cyclic Nucleotide-modulated (HCN) Ion Channels

The hyperpolarization-activated cyclic nucleotide-modulated (HCN) ion channels control rhythmicity in neurons and cardiomyocytes. Cyclic AMP allosterically modulates HCN through the cAMP-dependent formation of a tetrameric gating ring spanning the intracellular region (IR) of HCN, to which cAMP binds. Although the apo versus holo conformational changes of the cAMP-binding domain (CBD) have been previously mapped, only limited information is currently available on the HCN IR dynamics, which have been hypothesized to play a critical role in the cAMP-dependent gating of HCN. Here, using molecular dynamics simulations validated and complemented by experimental NMR and CD data, we comparatively analyze HCN IR dynamics in the four states of the thermodynamic cycle arising from the coupling between cAMP binding and tetramerization equilibria. This extensive set of molecular dynamics trajectories captures the active-to-inactive transition that had remained elusive for other CBDs, and it provides unprecedented insight on the role of IR dynamics in HCN autoinhibition and its release by cAMP. Specifically, the IR tetramerization domain becomes more flexible in the monomeric states, removing steric clashes that the apo-CDB structure would otherwise impose. Furthermore, the simulations reveal that the active/inactive structural transition for the apo-monomeric CBD occurs through a manifold of pathways that are more divergent than previously anticipated. Upon cAMP binding, these pathways become disallowed, pre-confining the CBD conformational ensemble to a tetramer-compatible state. This conformational confinement primes the IR for tetramerization and thus provides a model of how cAMP controls HCN channel gating.

Structure of the SthK carboxy-terminal region reveals a gating mechanism for cyclic nucleotide-modulated ion channels.

Cyclic nucleotide-sensitive ion channels are molecular pores that open in response to cAMP or cGMP, which are universal second messengers. Binding of a cyclic nucleotide to the carboxyterminal cyclic nucleotide binding domain (CNBD) of these channels is thought to cause a conformational change that promotes channel opening. The C-linker domain, which connects the channel pore to this CNBD, plays an important role in coupling ligand binding to channel opening. Current structural insight into this mechanism mainly derives from X-ray crystal structures of the C-linker/CNBD from hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels. However, these structures reveal little to no conformational changes upon comparison of the ligand-bound and unbound form. In this study, we take advantage of a recently identified prokaryote ion channel, SthK, which has functional properties that strongly resemble cyclic nucleotide-gated (CNG) channels and is activated by cAMP, but not by cGMP. We determined X-ray crystal structures of the C-linker/CNBD of SthK in the presence of cAMP or cGMP. We observe that the structure in complex with cGMP, which is an antagonist, is similar to previously determined HCN channel structures. In contrast, the structure in complex with cAMP, which is an agonist, is in a more open conformation. We observe that the CNBD makes an outward swinging movement, which is accompanied by an opening of the C-linker. This conformation mirrors the open gate structures of the Kv1.2 channel or MthK channel, which suggests that the cAMP-bound C-linker/CNBD from SthK represents an activated conformation. These results provide a structural framework for better understanding cyclic nucleotide modulation of ion channels, including HCN and CNG channels.

Family of prokaryote cyclic nucleotide-modulated ion channels.

Cyclic nucleotide-modulated ion channels are molecular pores that mediate the passage of ions across the cell membrane in response to cAMP or GMP. Structural insight into this class of ion channels currently comes from a related homolog, MloK1, that contains six transmembrane domains and a cytoplasmic cyclic nucleotide binding domain. However, unlike eukaryote hyperpolarization-activated cyclic nucleotide-modulated (HCN) and cyclic nucleotide-gated (CNG) channels, MloK1 lacks a C-linker region, which critically contributes to the molecular coupling between ligand binding and channel opening. In this study, we report the identification and characterization of five previously unidentified prokaryote homologs with high sequence similarity (24-32%) to eukaryote HCN and CNG channels and that contain a C-linker region. Biochemical characterization shows that two homologs, termed AmaK and SthK, can be expressed and purified as detergent-solubilized protein from Escherichia coli membranes. Expression of SthK channels in Xenopus laevis oocytes and functional characterization using the patch-clamp technique revealed that the channels are gated by cAMP, but not cGMP, are highly selective for K(+) ions over Na(+) ions, generate a large unitary conductance, and are only weakly voltage dependent. These properties resemble essential properties of various eukaryote HCN or CNG channels. Our results contribute to an understanding of the evolutionary origin of cyclic nucleotide-modulated ion channels and pave the way for future structural and functional studies.

Double Electron-Electron Resonance Studies of Ligand Induced Rearrangements of HCN Channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) ion channels are members of the superfamily of voltage-gated potassium channels. HCN channels play a role in controlling neuronal and cardiac pacemaking activity. They are regulated by the binding of cyclic nucleotides to a conserved, intracellular cyclic nucleotide-binding domain (CNBD). The CNBD is connected to the transmembrane domain through a C-linker. Crystallization studies of intracellular fragments of the C-linker and CNBD show little conformational difference in cAMP bound and unbound states. However, fluorescence and electrophysiology experiments suggest a significant conformational change in the C-linker and CNBD. Here, we use double electron-electron resonance (DEER) to study conformational changes of a soluble fragment of HCN channels in response to binding different cyclic nucleotide species. DEER is an electron paramagnetic resonance technique capable of measuring absolute distances and distance distributions between two spin-labeled protein residues separated by 2 to 8 nanometers. The ability to measure distance distributions allows us to probe whether binding of a partial agonist induces a conformational change smaller compared to the full agonist or results only in a shift of the conformational equilibrium. We find that in a soluble fragment of HCN2 consisting of the C-linker and CNBD, cAMP, the physiological agonist, leads to conformational changes of the CNBD that are much greater than suggested by crystallization. Binding of cCMP, known to be a partial agonist of the full channel, leads to conformational changes similar to cAMP. We also find that cGMP, known to be a full agonist on intact channels, acts as a partial agonist on the fragment, causing only a fraction of protein to undergo conformational change.