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.

Combination of hydrophobicity and codon usage bias determines sorting of model K+ channel protein to either mitochondria or endoplasmic reticulum.

When the K+ channel-like protein Kesv from Ectocarpus siliculosus virus 1 is heterologously expressed in mammalian cells, it is sorted to the mitochondria. This targeting can be redirected to the endoplasmic reticulum (ER) by altering the codon usage in distinct regions of the gene or by inserting a triplet of hydrophobic amino acids (AAs) into the protein's C-terminal transmembrane domain (ct-TMD). Systematic variations in the flavor of the inserted AAs and/or its codon usage show that a positive charge in the inserted AA triplet alone serves as strong signal for mitochondria sorting. In cases of neutral AA triplets, mitochondria sorting are favored by a combination of hydrophilic AAs and rarely used codons; sorting to the ER exhibits the inverse dependency. This propensity for ER sorting is particularly high when a common codon follows a rarer one in the AA triplet; mitochondria sorting in contrast is supported by codon uniformity. Since parameters like positive charge, hydrophobic AAs, and common codons are known to facilitate elongation of nascent proteins in the ribosome the data suggest a mechanism in which local changes in elongation velocity and co-translational folding in the ct-TMD influence intracellular protein sorting.

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.

A light-gated potassium channel for sustained neuronal inhibition.

Currently available inhibitory optogenetic tools provide short and transient silencing of neurons, but they cannot provide long-lasting inhibition because of the requirement for high light intensities. Here we present an optimized blue-light-sensitive synthetic potassium channel, BLINK2, which showed good expression in neurons in three species. The channel is activated by illumination with low doses of blue light, and in our experiments it remained active over (tens of) minutes in the dark after the illumination was stopped. This activation caused long periods of inhibition of neuronal firing in ex vivo recordings of mouse neurons and impaired motor neuron response in zebrafish in vivo. As a proof-of-concept application, we demonstrated that in a freely moving rat model of neuropathic pain, the activation of a small number of BLINK2 channels caused a long-lasting (>30 min) reduction in pain sensation.

Structural and functional approaches to studying cAMP regulation of HCN channels.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are primarily activated by voltage and further modulated by cAMP. While cAMP binding alone does not open the channel, its presence facilitates the action of voltage, increasing channel open probability. Functional results indicate that the membrane-based voltage sensor domain (VSD) communicates with the cytosolic cyclic nucleotide-binding domain (CNBD), and vice-versa. Yet, a mechanistic explanation on how this could occur in structural terms is still lacking. In this review, we will discuss the recent advancement in understanding the molecular mechanisms connecting the VSD with the CNBD in the tetrameric organization of HCN channels unveiled by the 3D structures of HCN1 and HCN4. Data show that the HCN domain transmits cAMP signal to the VSD by bridging the cytosolic to the membrane domains. Furthermore, a metal ion coordination site connects the C-linker to the S4-S5 linker in HCN4, further facilitating cAMP signal transmission to the VSD in this isoform.

Inferring functional units in ion channel pores via relative entropy.

Coarse-grained protein models approximate the first-principle physical potentials. Among those modeling approaches, the relative entropy framework yields promising and physically sound results, in which a mapping from the target protein structure and dynamics to a model is defined and subsequently adjusted by an entropy minimization of the model parameters. Minimization of the relative entropy is equivalent to maximization of the likelihood of reproduction of (configurational ensemble) observations by the model. In this study, we extend the relative entropy minimization procedure beyond parameter fitting by a second optimization level, which identifies the optimal mapping to a (dimension-reduced) topology. We consider anisotropic network models of a diverse set of ion channels and assess our findings by comparison to experimental results.

Characterization of an N-terminal Nav1.5 channel variant - a potential risk factor for arrhythmias and sudden death?

BACKGROUND: Alterations in the SCN5A gene encoding the cardiac sodium channel Nav1.5 have been linked to a number of arrhythmia syndromes and diseases including long-QT syndrome (LQTS), Brugada syndrome (BrS) and dilative cardiomyopathy (DCM), which may predispose to fatal arrhythmias and sudden death. We identified the heterozygous variant c.316A > G, p.(Ser106Gly) in a 35-year-old patient with survived cardiac arrest. In the present study, we aimed to investigate the functional impact of the variant to clarify the medical relevance. METHODS: Mutant as well as wild type GFP tagged Nav1.5 channels were expressed in HEK293 cells. We performed functional characterization experiments using patch-clamp technique. RESULTS: Electrophysiological measurements indicated, that the detected missense variant alters Nav1.5 channel functionality leading to a gain-of-function effect. Cells expressing S106G channels show an increase in Nav1.5 current over the entire voltage window. CONCLUSION: The results support the assumption that the detected sequence aberration alters Nav1.5 channel function and may predispose to cardiac arrhythmias and sudden cardiac death.

Fusicoccin Activates KAT1 Channels by Stabilizing Their Interaction with 14-3-3 Proteins.

Plants acquire potassium (K+) ions for cell growth and movement via regulated diffusion through K+ channels. Here, we present crystallographic and functional data showing that the K+ inward rectifier KAT1 (K+Arabidopsis thaliana 1) channel is regulated by 14-3-3 proteins and further modulated by the phytotoxin fusicoccin, in analogy to the H+-ATPase. We identified a 14-3-3 mode III binding site at the very C terminus of KAT1 and cocrystallized it with tobacco (Nicotiana tabacum) 14-3-3 proteins to describe the protein complex at atomic detail. Validation of this interaction by electrophysiology shows that 14-3-3 binding augments KAT1 conductance by increasing the maximal current and by positively shifting the voltage dependency of gating. Fusicoccin potentiates the 14-3-3 effect on KAT1 activity by stabilizing their interaction. Crystal structure of the ternary complex reveals a noncanonical binding site for the toxin that adopts a novel conformation. The structural insights underscore the adaptability of fusicoccin, predicting more potential targets than so far anticipated. The data further advocate a common mechanism of regulation of the proton pump and a potassium channel, two essential elements in K+ uptake in plant cells.

Mechanical transduction of cytoplasmic-to-transmembrane-domain movements in a hyperpolarization-activated cyclic nucleotide-gated cation channel.

Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels play a critical role in the control of pacemaking in the heart and repetitive firing in neurons. In HCN channels, the intracellular cyclic nucleotide-binding domain (CNBD) is connected to the transmembrane portion of the channel (TMPC) through a helical domain, the C-linker. Although this domain is critical for mechanical signal transduction, the conformational dynamics in the C-linker that transmit the nucleotide-binding signal to the HCN channel pore are unknown. Here, we use linear response theory to analyze conformational changes in the C-linker of the human HCN1 protein, which couple cAMP binding in the CNBD with gating in the TMPC. By applying a force to the tip of the so-called "elbow" of the C-linker, the coarse-grained calculations recapitulate the same conformational changes triggered by cAMP binding in experimental studies. Furthermore, in our simulations, a displacement of the C-linker parallel to the membrane plane (i.e. horizontally) induced a rotational movement resulting in a distinct tilting of the transmembrane helices. This movement, in turn, increased the distance between the voltage-sensing S4 domain and the surrounding transmembrane domains and led to a widening of the intracellular channel gate. In conclusion, our computational approach, combined with experimental data, thus provides a more detailed understanding of how cAMP binding is mechanically coupled over long distances to promote voltage-dependent opening of HCN channels.

Photolithographic Fabrication of Micro Apertures in Dry Film Polymer Sheets for Channel Recordings in Planar Lipid Bilayers.

Planar lipid bilayers constitute a versatile method for measuring the activity of protein channels and pores on a single molecule level. Ongoing efforts attempt to tailor this method for detecting biomedically relevant target analytes or for high-throughput screening of drugs. To improve the mechanical stability of bilayer recordings, we use a thin-film epoxy resist ADEX as septum in free-standing vertical bilayers. Defined apertures with diameters between 30 µm and 100 µm were micro-fabricated by photolithography. The performance of these septa was tested by functional reconstitution of the K+ channel KcvNTS in lipid bilayers spanned over apertures in ADEX or Teflon films; the latter is conventionally used in bilayer recordings and serves as reference. We observe that the functional properties of the K+ channel are identical in both materials while ADEX provides no advantage in terms of capacitance and signal-to-noise ratio. In contrast to Teflon, however, ADEX enables long-term experimental recordings while the stability of the lipid bilayer is not compromised by pipetting solutions in and out of the recording chamber. Combined with the fact that the ADEX films can be cleaned with acetone, our results suggest that ADEX carries great potential for multiplexing bilayer chambers in robust and reusable sensing devices.

Influence of genetic modifiers on sudden cardiac death cases.

Sequence variants in the ion channel genes KCNH2 and SCN5A may cause the cardiac disorder long QT syndrome (LQTS). This disorder is associated with incomplete penetrance and variable expression in KCNH2- or SCN5A-mutation carriers. Common genetic variants, if associated with a mutation, may affect the severity of this cardiac disorder. This study identified rare mutations in the cardiac ion channel genes KCNH2 and SCN5A in a SCD case, as well as in a LQTS-affected family with a history of SCD. Moreover, common variants were found to occur together within the same genes. These findings support the concept that common single-nucleotide polymorphisms (SNPs) in genes encoding cardiac ion channels can directly modulate the functional effect of mutations and therefore enhance or weaken the risk of cardiac events.

High-resolution membrane capacitance measurements for studying endocytosis and exocytosis in yeast.

Fusion of exocytotic vesicles with the plasma membrane gives rise to an increase in membrane surface area, whereas the surface area is decreased when vesicles are internalized during endocytosis. Changes in membrane surface area, resulting from fusion and fission of membrane vesicles, can be followed by monitoring the corresponding proportional changes in membrane capacitance. Using the cell-attached configuration of the patch-clamp techniques we were able to resolve the elementary processes of endo- and exocytosis in yeast protoplasts at high temporal and spatial resolution. Spontaneous capacitance changes were predominantly in the range of 0.2-1 fF which translates to vesicle diameters of 90-200 nm. The size distribution revealed that endocytotic vesicles with a median at about 132 nm were smaller than exocytotic vesicles with a median at 155 nm. In energized and metabolizing protoplasts, endo- and exocytotic events occurred at frequencies of 1.6 and 2.7 events per minute, respectively. Even though these numbers appear very low, they are in good agreement with the observed growth rate of yeast cells and protoplasts.

Identification of Intrahelical Bifurcated H-Bonds as a New Type of Gate in K+ Channels.

Gating of ion channels is based on structural transitions between open and closed states. To uncover the chemical basis of individual gates, we performed a comparative experimental and computational analysis between two K+ channels, KcvS and KcvNTS. These small viral encoded K+ channel proteins, with a monomer size of only 82 amino acids, resemble the pore module of all complex K+ channels in terms of structure and function. Even though both proteins share about 90% amino acid sequence identity, they exhibit different open probabilities with ca. 90% in KcvNTS and 40% in KcvS. Single channel analysis, mutational studies and molecular dynamics simulations show that the difference in open probability is caused by one long closed state in KcvS. This state is structurally created in the tetrameric channel by a transient, Ser mediated, intrahelical hydrogen bond. The resulting kink in the inner transmembrane domain swings the aromatic rings from downstream Phes in the cavity of the channel, which blocks ion flux. The frequent occurrence of Ser or Thr based helical kinks in membrane proteins suggests that a similar mechanism could also occur in the gating of other ion channels.

Characterization of a novel KCNJ2 sequence variant detected in Andersen-Tawil syndrome patients.

BACKGROUND: Mutations in the KCNJ2 gene encoding the ion channel Kir2.1 have been linked to the Andersen-Tawil syndrome (ATS). Molecular genetic screening performed in a family exhibiting clinical ATS phenotypes unmasked a novel sequence variant (c.434A > G, p.Y145C) in this gene. The aim of this study was to investigate the effect of this variant on Kir2.1 ion channel functionality. METHODS: Mutant as well as wild type GFP tagged Kir2.1 channels were expressed in HEK293 cells. In order to examine the effect of the new variant, electrophysiological measurements were performed using patch clamp technique. Cellular localization of the mutant in comparison to the wild type ion channel was analyzed by confocal laser scanning microscopy. RESULTS: The currents of cells expressing only mutant channels or a mixture of wild type and mutant were significantly reduced compared to those expressing wild type (WT) channels (p < 0.01). Whereas WT expressing cells exhibited at -120 mV an averaged current of -4.5 ± 1.9 nA, the mutant generates only a current of -0.17 ± 0.07 nA. A co-expression of mutant and WT channel generates only a partial rescue of the WT current. Confocal laser scanning microscopy indicated that the novel variant is not interfering with synthesis and/or protein trafficking. CONCLUSIONS: The detected sequence variant causes loss-of-function of the Kir2.1 channel and explains the clinical phenotypes observed in Andersen-Tawil syndrome patients.

The small neurotoxin apamin blocks not only small conductance Ca2+ activated K+ channels (SK type) but also the voltage dependent Kv1.3 channel.

Apamin is frequently used as a specific blocker of small-conductance Ca2+-activated (SK type) K+ channels. Here we show that the small neurotoxin is not as specific as anticipated. It is also a high-affinity inhibitor with an IC50 of 13 nM of the Kv1.3 channel; it blocks the latter with potency similar to the Kv1.3 blocker PAP-1. Since SK type channels and Kv1.3 channels are frequently coexpressed in different tissues such as cells of the immune system, apamin must be used with caution as a pharmacological tool.

Structural basis for the mutual antagonism of cAMP and TRIP8b in regulating HCN channel function.

cAMP signaling in the brain mediates several higher order neural processes. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels directly bind cAMP through their cytoplasmic cyclic nucleotide binding domain (CNBD), thus playing a unique role in brain function. Neuronal HCN channels are also regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit that antagonizes the effects of cAMP by interacting with the channel CNBD. To unravel the molecular mechanisms underlying the dual regulation of HCN channel activity by cAMP/TRIP8b, we determined the NMR solution structure of the HCN2 channel CNBD in the cAMP-free form and mapped on it the TRIP8b interaction site. We reconstruct here the full conformational changes induced by cAMP binding to the HCN channel CNBD. Our results show that TRIP8b does not compete with cAMP for the same binding region; rather, it exerts its inhibitory action through an allosteric mechanism, preventing the cAMP-induced conformational changes in the HCN channel CNBD.

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.

Viral potassium channels as a robust model system for studies of membrane-protein interaction.

The viral channel KcvNTS belongs to the smallest K(+) channels known so far. A monomer of a functional homotetramer contains only 82 amino acids. As a consequence of the small size the protein is almost fully submerged into the membrane. This suggests that the channel is presumably sensitive to its lipid environment. Here we perform a comparative analysis for the function of the channel protein embedded in three different membrane environments. 1. Single-channel currents of KcvNTS were recorded with the patch clamp method on the plasma membrane of HEK293 cells. 2. They were also measured after reconstitution of recombinant channel protein into classical planar lipid bilayers and 3. into horizontal bilayers derived from giant unilamellar vesicles (GUVs). The recombinant channel protein was either expressed and purified from Pichia pastoris or from a cell-free expression system; for the latter a new approach with nanolipoprotein particles was used. The data show that single-channel activity can be recorded under all experimental conditions. The main functional features of the channel like a large single-channel conductance (80pS), high open-probability (>50%) and the approximate duration of open and closed dwell times are maintained in all experimental systems. An apparent difference between the approaches was only observed with respect to the unitary conductance, which was ca. 35% lower in HEK293 cells than in the other systems. The reason for this might be explained by the fact that the channel is tagged by GFP when expressed in HEK293 cells. Collectively the data demonstrate that the small viral channel exhibits a robust function in different experimental systems. This justifies an extrapolation of functional data from these systems to the potential performance of the channel in the virus/host interaction. This article is part of a Special Issue entitled: Viral Membrane Proteins-Channels for Cellular Networking.