Gating movements and ion permeation in HCN4 pacemaker channels.

The HCN1-4 channel family is responsible for the hyperpolarization-activated cation current If/Ih that controls automaticity in cardiac and neuronal pacemaker cells. We present cryoelectron microscopy (cryo-EM) structures of HCN4 in the presence or absence of bound cAMP, displaying the pore domain in closed and open conformations. Analysis of cAMP-bound and -unbound structures sheds light on how ligand-induced transitions in the channel cytosolic portion mediate the effect of cAMP on channel gating and highlights the regulatory role of a Mg2+ coordination site formed between the C-linker and the S4-S5 linker. Comparison of open/closed pore states shows that the cytosolic gate opens through concerted movements of the S5 and S6 transmembrane helices. Furthermore, in combination with molecular dynamics analyses, the open pore structures provide insights into the mechanisms of K+/Na+ permeation. Our results contribute mechanistic understanding on HCN channel gating, cyclic nucleotide-dependent modulation, and ion permeation.

Structural determinants of ivabradine block of the open pore of HCN4.

HCN1-4 channels are the molecular determinants of the If/Ih current that crucially regulates cardiac and neuronal cell excitability. HCN dysfunctions lead to sinoatrial block (HCN4), epilepsy (HCN1), and chronic pain (HCN2), widespread medical conditions awaiting subtype-specific treatments. Here, we address the problem by solving the cryo-EM structure of HCN4 in complex with ivabradine, to date the only HCN-specific drug on the market. Our data show ivabradine bound inside the open pore at 3 Å resolution. The structure unambiguously proves that Y507 and I511 on S6 are the molecular determinants of ivabradine binding to the inner cavity, while F510, pointing outside the pore, indirectly contributes to the block by controlling Y507. Cysteine 479, unique to the HCN selectivity filter (SF), accelerates the kinetics of block. Molecular dynamics simulations further reveal that ivabradine blocks the permeating ion inside the SF by electrostatic repulsion, a mechanism previously proposed for quaternary ammonium ions.

A novel de novo HCN1 loss-of-function mutation in genetic generalized epilepsy causing increased neuronal excitability.

The causes of genetic epilepsies are unknown in the majority of patients. HCN ion channels have a widespread expression in neurons and increasing evidence demonstrates their functional involvement in human epilepsies. Among the four known isoforms, HCN1 is the most expressed in the neocortex and hippocampus and de novo HCN1 point mutations have been recently associated with early infantile epileptic encephalopathy. So far, HCN1 mutations have not been reported in patients with idiopathic epilepsy. Using a Next Generation Sequencing approach, we identified the de novo heterozygous p.Leu157Val (c.469C > G) novel mutation in HCN1 in an adult male patient affected by genetic generalized epilepsy (GGE), with normal cognitive development. Electrophysiological analysis in heterologous expression model (CHO cells) and in neurons revealed that L157V is a loss-of-function, dominant negative mutation causing reduced HCN1 contribution to net inward current and responsible for an increased neuronal firing rate and excitability, potentially predisposing to epilepsy. These data represent the first evidence that autosomal dominant missense mutations of HCN1 can also be involved in GGE, without the characteristics of epileptic encephalopathy reported previously. It will be important to include HCN1 screening in patients with GGE, in order to extend the knowledge of the genetic causes of idiopathic epilepsies, thus paving the way for the identification of innovative therapeutic strategies.

Mutation in S6 domain of HCN4 channel in patient with suspected Brugada syndrome modifies channel function.

Diseases such as the sick sinus and the Brugada syndrome are cardiac abnormalities, which can be caused by a number of genetic aberrances. Among them are mutations in HCN4, a gene, which encodes the hyperpolarization-activated, cyclic nucleotide-gated ion channel 4; this pacemaker channel is responsible for the spontaneous activity of the sinoatrial node. The present genetic screening of patients with suspected or diagnosed Brugada or sick sinus syndrome identified in 1 out of 62 samples the novel mutation V492F. It is located in a highly conserved site of hyperpolarization-activated cyclic nucleotide-gated (HCN)4 channel downstream of the filter at the start of the last transmembrane domain S6. Functional expression of mutant channels in HEK293 cells uncovered a profoundly reduced channel function but no appreciable impact on channel synthesis and trafficking compared to the wild type. The inward rectifying HCN4 current could be partially rescued by an expression of heteromeric channels comprising wt and mutant monomers. These heteromeric channels were responsive to cAMP but they required a more negative voltage for activation and they exhibited a lower current density than the wt channel. This suggests a dominant negative effect of the mutation in patients, which carry this heterozygous mutation. Such a modulation of HCN4 activity could be the cause of the diagnosed cardiac abnormality.

Cyclic dinucleotides bind the C-linker of HCN4 to control channel cAMP responsiveness.

cAMP mediates autonomic regulation of heart rate by means of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which underlie the pacemaker current If. cAMP binding to the C-terminal cyclic nucleotide binding domain enhances HCN open probability through a conformational change that reaches the pore via the C-linker. Using structural and functional analysis, we identified a binding pocket in the C-linker of HCN4. Cyclic dinucleotides, an emerging class of second messengers in mammals, bind the C-linker pocket (CLP) and antagonize cAMP regulation of the channel. Accordingly, cyclic dinucleotides prevent cAMP regulation of If in sinoatrial node myocytes, reducing heart rate by 30%. Occupancy of the CLP hence constitutes an efficient mechanism to hinder ß-adrenergic stimulation on If. Our results highlight the regulative role of the C-linker and identify a potential drug target in HCN4. Furthermore, these data extend the signaling scope of cyclic dinucleotides in mammals beyond their first reported role in innate immune system.

Embryonic stem cell-derived CD166+ precursors develop into fully functional sinoatrial-like cells.

RATIONALE: A cell-based biological pacemaker is based on the differentiation of stem cells and the selection of a population displaying the molecular and functional properties of native sinoatrial node (SAN) cardiomyocytes. So far, such selection has been hampered by the lack of proper markers. CD166 is specifically but transiently expressed in the mouse heart tube and sinus venosus, the prospective SAN. OBJECTIVE: We have explored the possibility of using CD166 expression for isolating SAN progenitors from differentiating embryonic stem cells. METHODS AND RESULTS: We found that in embryonic day 10.5 mouse hearts, CD166 and HCN4, markers of the pacemaker tissue, are coexpressed. Sorting embryonic stem cells for CD166 expression at differentiation day 8 selects a population of pacemaker precursors. CD166+ cells express high levels of genes involved in SAN development (Tbx18, Tbx3, Isl-1, Shox2) and function (Cx30.2, HCN4, HCN1, CaV1.3) and low levels of ventricular genes (Cx43, Kv4.2, HCN2, Nkx2.5). In culture, CD166+ cells form an autorhythmic syncytium composed of cells morphologically similar to and with the electrophysiological properties of murine SAN myocytes. Isoproterenol increases (+57%) and acetylcholine decreases (-23%) the beating rate of CD166-selected cells, which express the ß-adrenergic and muscarinic receptors. In cocultures, CD166-selected cells are able to pace neonatal ventricular myocytes at a rate faster than their own. Furthermore, CD166+ cells have lost pluripotency genes and do not form teratomas in vivo. CONCLUSIONS: We demonstrated for the first time the isolation of a nonteratogenic population of cardiac precursors able to mature and form a fully functional SAN-like tissue.

Deep bradycardia and heart block caused by inducible cardiac-specific knockout of the pacemaker channel gene Hcn4.

Cardiac pacemaking generation and modulation rely on the coordinated activity of several processes. Although a wealth of evidence indicates a relevant role of the I(f) ("funny," or pacemaker) current, whose molecular constituents are the hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels and particularly HCN4, work with mice where Hcn genes were knocked out, or functionally modified, has challenged this view. However, no previous studies used a cardiac-specific promoter to induce HCN4 ablation in adult mice. We report here that, in an inducible and cardiac-specific HCN4 knockout (ciHCN4-KO) mouse model, ablation of HCN4 consistently leads to progressive development of severe bradycardia (∼50% reduction of original rate) and AV block, eventually leading to heart arrest and death in about 5 d. In vitro analysis of sinoatrial node (SAN) myocytes isolated from ciHCN4-KO mice at the mean time of death revealed a strong reduction of both the I(f) current (by ∼70%) and of the spontaneous rate (by ∼60%). In agreement with functional results, immunofluorescence and Western blot analysis showed reduced expression of HCN4 protein in SAN tissue and cells. In ciHCN4-KO animals, the residual I(f) was normally sensitive to ß-adrenergic receptor (ß-AR) modulation, and the permanence of rate response to ß-AR stimulation was observed both in vivo and in vitro. Our data show that cardiac HCN4 channels are essential for normal heart impulse generation and conduction in adult mice and support the notion that dysfunctional HCN4 channels can be a direct cause of rhythm disorders. This work contributes to identifying the molecular mechanism responsible for cardiac pacemaking.

LEONARDO DA VINCI AND THE ORIGIN OF SEMEN.

It is well known that Leonardo da Vinci made several drawings of the human male anatomy. The early drawings (before 1500) were incorrect in identifying the origin of semen, where he followed accepted teaching of his time. It is widely thought that he did not correct this mistake, a view that is reflected in several biographies. In fact, he made a later drawing (after 1500) in which the description of the anatomy is remarkably accurate and must have been based on careful dissection. In addition to highlighting this fact, acknowledged previously in only one other source, this article reviews the background to Leonardo's knowledge of the relevant anatomy.

A high affinity switch for cAMP in the HCN pacemaker channels.

Binding of cAMP to Hyperpolarization activated cyclic nucleotide gated (HCN) channels facilitates pore opening. It is unclear why the isolated cyclic nucleotide binding domain (CNBD) displays in vitro lower affinity for cAMP than the full-length channel in patch experiments. Here we show that HCN are endowed with an affinity switch for cAMP. Alpha helices D and E, downstream of the cyclic nucleotide binding domain (CNBD), bind to and stabilize the holo CNBD in a high affinity state. These helices increase by 30-fold cAMP efficacy and affinity measured in patch clamp and ITC, respectively. We further show that helices D and E regulate affinity by interacting with helix C of the CNBD, similarly to the regulatory protein TRIP8b. Our results uncover an intramolecular mechanism whereby changes in binding affinity, rather than changes in cAMP concentration, can modulate HCN channels, adding another layer to the complex regulation of their activity.

Funny channel gene mutations associated with arrhythmias.

Many diverse data support a role of the funny current (If) in pacemaking and heart rate control. Among them, clinically relevant applications have special impact, since they translate the concept of funny channel-based pacemaking into practical developments of potential therapeutic value. For example, the If role in pacemaking is the basis for a pharmacological approach to heart rate control. Ivabradine, a specific f-channel blocker acting as a 'pure heart rate reducing' agent, is used today as a therapeutic tool in chronic stable angina and heart failure. Also, the pacemaking capability of funny channels has been the main rationale behind the development of 'biological' pacemakers, whose aim is to eventually replace the electronic pacemakers implanted today. Finally, the role of If in pacemaking implies that dysfunctional funny channels can contribute to rhythm disorders. This consideration has led to the search for inheritable forms of cardiac arrhythmias potentially linked to functional changes of HCN4 channels, the molecular correlates of funny channels. This review addresses recent reports where investigation of families with arrhythmias has allowed the identification of specific HCN4 channel mutations associated with different types of rhythm disorders, specifically with sinus bradycardia. The perspective of future research in this field is also addressed.

An LQTS6 MiRP1 mutation suppresses pacemaker current and is associated with sinus bradycardia.

BACKGROUND: Sinus node (SN) dysfunction is observed in some long-QT syndrome (LQTS) patients, but has not been studied as a function of LQTS genotype. LQTS6 involves mutations in the hERG ß-subunit MiRP1, which also interacts with hyperpolarization-activated, cyclic nucleotide gated (HCN) channels-the molecular correlate of SN pacemaker current (If ). An LQTS registry search identified a 55-year male with M54T MiRP1 mutation, history of sinus bradycardia (39-56 bpm), and prolonged QTc. OBJECTIVE: We tested if LQTS6 incorporates sinus bradycardia due to abnormal If . METHODS: We transiently co-transfected neonatal rat ventricular myocytes (to study currents in a myocyte background) with human HCN4 (hHCN4, primary SN isoform) or human HCN2 (hHCN2) and one of the following: empty vector, wild-type hMiRP1 (WT), M54T hMiRP1 (M54T). Current amplitude, voltage dependence, and kinetics were measured by whole cell patch clamp. RESULTS: M54T co-expression decreased HCN4 current density by 80% compared to hHCN4 alone or with WT, and also slowed HCN4 activation at physiologically relevant voltages. Neither WT nor M54T altered HCN4 voltage dependence. A computer simulation predicts that these changes in HCN4 current would decrease rate and be additive with published effects of M54T mutation on hERG kinetics on rate. CONCLUSIONS: We conclude that M54T LQTS6 mutation can cause sinus bradycardia through effects on both hERG and HCN currents. Patients with other LQTS6 mutations should be examined for SN dysfunction, and the effect on HCN current determined.

Silvio Weidmann: laying the foundations for unravelling the mechanism of heart rhythm.

Silvio Weidmann laid the basis of cardiac electrophysiology and was the forerunner in the search for mechanisms governing the electrical activity of the heart in his legendary first studies of Purkinje fibres in the 1950s. His work was the cornerstone of research in this field for many generations, and countless cardiologists and electrophysiologists have based their studies on the knowledge generated by Weidmann's pioneering data. This review summarizes his key contributions from the first intracellular recordings of cardiac membrane potentials in 1949 to the publication of his monograph in 1956. That summary is followed by an imagined dialogue between the authors and Silvio Weidmann himself, in the format of a one-act play. Both of us have such good recollections of our real-life conversations with Silvio Weidmann that we decided we could achieve a better feel for the history and issues by using a dialogue format. We hope that, in that way, we may transmit the character of Silvio Weidmann better for those readers who will not have known him personally. Silvio Weidmann was an extraordinarily sensitive and conversational person as well as a great scientist, and we feel it is worth the effort to convey that fact here. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Alkali metal cations modulate the geometry of different binding sites in HCN4 selectivity filter for permeation or block.

Hyperpolarization-activated cyclic-nucleotide gated (HCN) channels are important for timing biological processes like heartbeat and neuronal firing. Their weak cation selectivity is determined by a filter domain with only two binding sites for K+ and one for Na+. The latter acts as a weak blocker, which is released in combination with a dynamic widening of the filter by K+ ions, giving rise to a mixed K+/Na+ current. Here, we apply molecular dynamics simulations to systematically investigate the interactions of five alkali metal cations with the filter of the open HCN4 pore. Simulations recapitulate experimental data like a low Li+ permeability, considerable Rb+ conductance, a block by Cs+ as well as a punch through of Cs+ ions at high negative voltages. Differential binding of the cation species in specific filter sites is associated with structural adaptations of filter residues. This gives rise to ion coordination by a cation-characteristic number of oxygen atoms from the filter backbone and solvent. This ion/protein interplay prevents Li+, but not Na+, from entry into and further passage through the filter. The site equivalent to S3 in K+ channels emerges as a preferential binding and presumably blocking site for Cs+. Collectively, the data suggest that the weak cation selectivity of HCN channels and their block by Cs+ are determined by restrained cation-generated rearrangements of flexible filter residues.

Recessive loss-of-function mutation in the pacemaker HCN2 channel causing increased neuronal excitability in a patient with idiopathic generalized epilepsy.

The hyperpolarization-activated I(h) current, coded for by hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, controls synaptic integration and intrinsic excitability in many brain areas. Because of their role in pacemaker function, defective HCN channels are natural candidates for contributing to epileptogenesis. Indeed, I(h) is pathologically altered after experimentally induced seizures, and several independent data indicate a link between dysfunctional HCN channels and different forms of epilepsy. However, direct evidence for functional changes of defective HCN channels correlating with the disease in human patients is still elusive. By screening families with epilepsy for mutations in Hcn1 and Hcn2 genes, we found a recessive loss-of-function point mutation in the gene coding for the HCN2 channel in a patient with sporadic idiopathic generalized epilepsy. Of 17 screened members of the same family, the proband was the only one affected and homozygous for the mutation. The mutation (E515K) is located in the C-linker, a region known to affect channel gating. Functional analysis revealed that homomeric mutant, but not heteromeric wild-type/mutant channels, have a strongly inhibited function caused by a large negative shift of activation range and slowed activation kinetics, effectively abolishing the HCN2 contribution to activity. After transfection into acutely isolated newborn rat cortical neurons, homomeric mutant, but not heteromeric wild type/mutant channels, lowered the threshold of action potential firing and strongly increased cell excitability and firing frequency when compared with wild-type channels. This is the first evidence in humans for a single-point, homozygous loss-of-function mutation in HCN2 potentially associated with generalized epilepsy with recessive inheritance.

A caveolin-binding domain in the HCN4 channels mediates functional interaction with caveolin proteins.

Pacemaker (HCN) channels have a key role in the generation and modulation of spontaneous activity of sinoatrial node myocytes. Previous work has shown that compartmentation of HCN4 pacemaker channels within caveolae regulates important functions, but the molecular mechanism responsible is still unknown. HCN channels have a conserved caveolin-binding domain (CBD) composed of three aromatic amino acids at the N-terminus; we sought to evaluate the role of this CBD in channel-protein interaction by mutational analysis. We generated two HCN4 mutants with a disrupted CBD (Y259S, F262V) and two with conservative mutations (Y259F, F262Y). In CHO cells expressing endogenous caveolin-1 (cav-1), alteration of the CBD shifted channels activation to more positive potentials, slowed deactivation and made Y259S and F262V mutants insensitive to cholesterol depletion-induced caveolar disorganization. CBD alteration also caused a significant decrease of current density, due to a weaker HCN4-cav-1 interaction and accumulation of cytoplasmic channels. These effects were absent in mutants with a preserved CBD. In caveolin-1-free fibroblasts, HCN4 trafficking was impaired and current density reduced with all constructs; the activation curve of F262V was not altered relative to wt, and that of Y259S displayed only half the shift than in CHO cells. The conserved CBD present in all HCN isoforms mediates their functional interaction with caveolins. The elucidation of the molecular details of HCN4-cav-1 interaction can provide novel information to understand the basis of cardiac phenotypes associated with some forms of caveolinopathies.

An updated computational model of rabbit sinoatrial action potential to investigate the mechanisms of heart rate modulation.

The cellular basis of cardiac pacemaking is still debated. Reliable computational models of the sinoatrial node (SAN) action potential (AP) may help gain a deeper understanding of the phenomenon. Recently, novel models incorporating detailed Ca(2+)-handling dynamics have been proposed, but they fail to reproduce a number of experimental data, and more specifically effects of 'funny' (I(f)) current modifications. We therefore developed a SAN AP model, based on available experimental data, in an attempt to reproduce physiological and pharmacological heart rate modulation. Cell compartmentalization and intracellular Ca(2+)-handling mechanisms were formulated as in the Maltsev-Lakatta model, focusing on Ca(2+)-cycling processes. Membrane current equations were revised on the basis of published experimental data. Modifications of the formulation of currents/pumps/exchangers to simulate I(f) blockers, autonomic modulators and Ca(2+)-dependent mechanisms (ivabradine, caesium, acetylcholine, isoprenaline, BAPTA) were derived from experimental data. The model generates AP waveforms typical of rabbit SAN cells, whose parameters fall within the experimental ranges: 352 ms cycle length, 80 mV AP amplitude, -58 mV maximum diastolic potential (MDP), 108 ms APD(50), and 7.1 Vs(-1) maximum upstroke velocity. Rate modulation by I(f) -blocking drugs agrees with experimental findings: 20% and 22% caesium-induced (5mM) and ivabradine-induced (3 µM) rate reductions, respectively, due to changes in diastolic depolarization (DD) slope, with no changes in either MDP or take-off potential (TOP). The model consistently reproduces the effects of autonomic modulation: 20% rate decrease with 10 nM acetylcholine and 28%increase with 1 µM isoprenaline, again entirely due to increase in the DD slope,with no changes in either MDP or TOP. Model testing of BAPTA effects showed slowing of rate, -26%, without cessation of beating. Our up-to-date model describes satisfactorily experimental data concerning autonomic stimulation, funny-channel blockade and inhibition of the Ca(2+)-related system by BAPTA, making it a useful tool for further investigation. Simulation results suggest that a detailed description of the intracellular Ca(2+) fluxes is fully compatible with the observation that I(f) is a major component of pacemaking and rate modulation.

The role of the funny current in pacemaker activity.

Abstract: Pacemaking is a basic physiological process, and the cellular mechanisms involved in this function have always attracted the keen attention of investigators. The "funny" (I(f)) current, originally described in sinoatrial node myocytes as an inward current activated on hyperpolarization to the diastolic range of voltages, has properties suitable for generating repetitive activity and for modulating spontaneous rate. The degree of activation of the funny current determines, at the end of an action potential, the steepness of phase 4 depolarization; hence, the frequency of action potential firing. Because I(f) is controlled by intracellular cAMP and is thus activated and inhibited by beta-adrenergic and muscarinic M2 receptor stimulation, respectively, it represents a basic physiological mechanism mediating autonomic regulation of heart rate. Given the complexity of the cellular processes involved in rhythmic activity, an exact quantification of the extent to which I(f) and other mechanisms contribute to pacemaking is still a debated issue; nonetheless, a wealth of information collected since the current was first described more than 30 years ago clearly agrees to identify I(f) as a major player in both generation of spontaneous activity and rate control. I(f)- dependent pacemaking has recently advanced from a basic, physiologically relevant concept, as originally described, to a practical concept that has several potentially useful clinical applications and can be valuable in therapeutically relevant conditions. Typically, given their exclusive role in pacemaking, f-channels are ideal targets of drugs aiming to pharmacological control of cardiac rate. Molecules able to bind specifically to and block f-channels can thus be used as pharmacological tools for heart rate reduction with little or no adverse cardiovascular side effects. Indeed a selective f-channel inhibitor, ivabradine, is today commercially available as a tool in the treatment of stable chronic angina. Also, several loss-of-function mutations of HCN4 (hyperpolarization-activated, cyclic-nucleotide gated 4), the major constitutive subunit of f-channels in pacemaker cells, are known today to cause rhythm disturbances, such as for example inherited sinus bradycardia. Finally, gene- or cell-based methods for in situ delivery of f-channels to silent or defective cardiac muscle represent novel approaches for the development of biological pacemakers eventually able to replace electronic devices.

Discovery of Herbacetin as a Novel SGK1 Inhibitor to Alleviate Myocardial Hypertrophy.

Cardiac hypertrophy is a pivotal pathophysiological step of various cardiovascular diseases, which eventually leads to heart failure and death. Extracts of Rhodiola species (Ext.R), a class of commonly used medicinal herbs in Europe and East Asia, can attenuate cardiac hypertrophy both in vitro and in vivo. Serum/glucocorticoid regulated kinase 1 (SGK1) is identified as a potential target of Ext. R. By mass spectrometry-based kinase inhibitory assay, herbacetin (HBT) from Ext.R is identified as a novel SGK1 inhibitor with IC50 of 752 nmol. Thermal shift assay, KINOMEscan in vitro assay combined with molecular docking proves a direct binding between HBT and SGK1. Site-specific mutation of Asp177 in SGK1 completely ablates the inhibitory activity of HBT. The presence of OH groups at the C-3, C-8, C-4' positions of flavonoids is suggested to be favorable for the inhibition of SGK1 activity. Finally, HBT significantly suppresses cardiomyocyte hypertrophy in vitro and in vivo, reduces reactive oxygen species (ROS) synthesis and calcium accumulation. HBT decreases phosphorylation of SGK1 and regulates its downstream forkhead box protein O1 (FoxO1) signaling pathway. Taken together, the findings suggest that a panel of flavonoids structurally related to HBT may be novel leads for developing new therapeutics against cardiac hypertrophy.