HCN4 provides a 'depolarization reserve' and is not required for heart rate acceleration in mice

Cardiac pacemaking involves a variety of ion channels, but their relative importance is controversial and remains to be determined. Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, which underlie the I(f) current of sinoatrial cells, are thought to be key players in cardiac automaticity. In addition, the increase in heart rate following beta-adrenergic stimulation has been attributed to the cAMP-mediated enhancement of HCN channel activity. We have now studied mice in which the predominant sinoatrial HCN channel isoform HCN4 was deleted in a temporally controlled manner. Here, we show that deletion of HCN4 in adult mice eliminates most of sinoatrial I(f) and results in a cardiac arrhythmia characterized by recurrent sinus pauses. However, the mutants show no impairment in heart rate acceleration during sympathetic stimulation. Our results reveal that unexpectedly the channel does not play a role for the increase of the heart rate; however, HCN4 is necessary for maintaining a stable cardiac rhythm, especially during the transition from stimulated to basal cardiac states.     

Identification of the Molecular Site of Ivabradine Binding to HCN4 Channels

Ivabradine is a specific heart rate-reducing agent approved as a treatment of chronic stable angina. Its mode of action involves a selective and specific block of HCN channels, the molecular components of sinoatrial "funny" (f)-channels. Different studies suggest that the binding site of ivabradine is located in the inner vestibule of HCN channels, but the molecular details of ivabradine binding are unknown. We thus sought to investigate by mutagenesis and in silico analysis which residues of the HCN4 channel, the HCN isoform expressed in the sinoatrial node, are involved in the binding of ivabradine. Using homology modeling, we verified the presence of an inner cavity below the channel pore and identified residues lining the cavity; these residues were replaced with alanine (or valine) either alone or in combination, and WT and mutant channels were expressed in HEK293 cells. Comparison of the block efficiency of mutant vs WT channels, measured by patch-clamp, revealed that residues Y506, F509 and I510 are involved in ivabradine binding. For each mutant channel, docking simulations correctly explain the reduced block efficiency in terms of proportionally reduced affinity for ivabradine binding. In summary our study shows that ivabradine occupies a cavity below the channel pore, and identifies specific residues facing this cavity that interact and stabilize the ivabradine molecule. This study provides an interpretation of known properties of f/HCN4 channel block by ivabradine such as the “open channel block”, the current-dependence of block and the property of "trapping" of drug molecules in the closed configuration.     

Functional expression of the human HCN3 channel

Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels underlie the inward pacemaker current, termed I(f)/I(h), in a variety of tissues. Many details are known for the HCN subtypes 1, 2, and 4. We now successfully cloned the cDNA for HCN3 from human brain and compared the electrophysiological properties of hHCN3 to the other three HCN subtypes. Overexpression of human HCN3 channels in HEK293 cells resulted in a functional channel protein. Similar to hHCN2 channels, hHCN3 channels are activated with a rather slow time constant of 1244 +/- 526 ms at -100 mV, which is a greater time constant than that of HCN1 but a smaller one than that of HCN4 channels. The membrane potential for half-maximal activation V((1/2)) was -77 +/- 5.4 mV, and the reversal potential E(rev) was -20.5 +/- 4 mV, resulting in a permeability ratio P(Na)/P(K) of 0.3. Like all other HCNs, hHCN3 was inhibited rapidly and reversibly by extracellular cesium and slowly and irreversibly by extracellular applied ZD7288. Surprisingly, the human HCN3 channel was not modulated by intracellular cAMP, a hallmark of the other known HCN channels. Sequence comparison revealed >80% homology of the transmembrane segments, the pore region, and the cyclic nucleotide binding domain of hHCN3 with the other HCN channels. The missing response to cAMP distinguishes human HCN3 from both the well cAMP responding HCN subtypes 2 and 4 and the weak responding subtype 1.     

Pore topology of the hyperpolarization-activated cyclic nucleotide-gated channel from sea urchin sperm

The current flow through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, referred to as I(h), plays a major role in several fundamental biological processes. The sequence of the presumed pore region of HCN channels is reminiscent of that of most known K(+)-selective channels. In the present work, the pore topology of an HCN channel from sea urchin sperm, called SpHCN, was investigated by means of the substituted-cysteine accessibility method (SCAM). The I(h) current in the wild-type (w.t.) SpHCN channel was irreversibly blocked by intracellular Cd(2+). This blockage was not observed in mutant C428S. Extracellular Cd(2+) did not cause any inhibition of the I(h) current in the w.t. SpHCN channel, but blocked the current in mutant channels K433C and F434C. Large extracellular anions blocked the current both in the w.t. and K433Q mutant channel. These results suggest that 1) cysteine in position 428 faces the intracellular medium; 2) lysine and phenylalanine in position 433 and 434, respectively, face the extracellular side of the membrane; and 3) lysine 433 does not mediate the anion blockade. Additionally, our study confirms that the K(+) channel signature sequence GYG also forms the inner pore in HCN channels.     

Hyperpolarization-activated cyclic nucleotide-gated channel 1 is a molecular determinant of the cardiac pacemaker current I(f)

The pacemaker current I(f) of the sinoatrial node (SAN) is a major determinant of cardiac diastolic depolarization and plays a key role in controlling heart rate and its modulation by neurotransmitters. Substantial expression of two different mRNAs (HCN4, HCN1) of the family of pacemaker channels (HCN) is found in rabbit SAN, suggesting that the native channels may be formed by different isoforms. Here we report the cloning and heterologous expression of HCN1 from rabbit SAN and its specific localization in pacemaker myocytes. rbHCN1 is an 822-amino acid protein that, in human embryonic kidney 293 cells, displayed electrophysiological properties similar to those of I(f), suggesting that HCN1 can form a pacemaker channel. The presence of HCN1 in the SAN myocytes but not in nearby heart regions, and the electrophysiological properties of the channels formed by it, suggest that HCN1 plays a central and specific role in the formation of SAN pacemaker currents.     

Functional roles of charged residues in the putative voltage sensor of the HCN2 pacemaker channel

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels contribute to pacemaking activity in specialized neurons and cardiac myocytes. HCN channels have a structure similar to voltage-gated K(+) channels but have a much larger putative S4 transmembrane domain and open in response to membrane hyperpolarization instead of depolarization. As an initial attempt to define the structural basis of HCN channel gating, we have characterized the functional roles of the charged residues in the S2, S3, and S4 transmembrane domains. The nine basic residues and a single Ser in S4 were mutated individually to Gln, and the function of mutant channels was analyzed in Xenopus oocytes using two-microelectrode voltage clamp techniques. Surface membrane expression of hemagglutinin-epitope-tagged channel proteins was examined by chemiluminescence. Our results suggest that 1) Lys-291, Arg-294, Arg-297, and Arg-300 contribute to the voltage dependence of gating but not to channel folding or trafficking to the surface membrane; 2) Lys-303 and Ser-306 are essential for gating, but not for channel folding/trafficking; 3) Arg-312 is important for folding but not gating; and 4) Arg-309, Arg-315, and Arg-318 are crucial for normal protein folding/trafficking and may charge-pair with Asp residues located in the S2 and S3 domains.     

Myristoylated peptides potentiate the funny current (I(f)) in sinoatrial myocytes

The funny current, I(f), in sinoatrial myocytes is thought to contribute to the sympathetic fight-or-flight increase in heart rate. I(f) is produced by hyperpolarization-activated cyclic nucleotide sensitive-4 (HCN4) channels, and it is widely believed that sympathetic regulation of I(f) occurs via direct binding of cAMP to HCN4, independent of phosphorylation. However, we have recently shown that Protein Kinase A (PKA) activity is required for sympathetic regulation of I(f) and that PKA can directly phosphorylate HCN4. In the present study, we examined the effects of a myristoylated PKA inhibitory peptide (myr-PKI) on I(f) in mouse sinoatrial myocytes. We found that myr-PKI and another myristoylated peptide potently and specifically potentiated I(f) via a mechanism that did not involve PKA inhibition and that was independent of the peptide sequence, Protein Kinase C, or phosphatidylinositol-4,5-bisphosphate. The off-target activation of I(f) by myristoylated peptides limits their usefulness for studies of pacemaker mechanisms in sinoatrial myocytes.     

The human gene coding for HCN2, a pacemaker channel of the heart.

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, underlying 'pacemaker' currents (I(f)/Ih), are involved in pacemaker activity of cardiac sinoatrial node myocytes and central neurons. Several cDNAs deriving from four different genes were recently identified which code for channels characterized by six transmembrane domains and a cyclic nucleotide binding domain. We report here the identification of the human HCN2 gene and show that its functional expression in a human kidney cell line generates a current with properties similar to the native pacemaker f-channel of the heart. The hHCN2 gene maps to the telomeric region of chromosome 19, band p13.3. This is the first identification of a genetic locus coding for an HCN channel.     

mu-conotoxin GIIIA interactions with the voltage-gated Na(+) channel predict a clockwise arrangement of the domains

Voltage-gated Na(+) channels underlie the electrical activity of most excitable cells, and these channels are the targets of many antiarrhythmic, anticonvulsant, and local anesthetic drugs. The channel pore is formed by a single polypeptide chain, containing four different, but homologous domains that are thought to arrange themselves circumferentially to form the ion permeation pathway. Although several structural models have been proposed, there has been no agreement concerning whether the four domains are arranged in a clockwise or a counterclockwise pattern around the pore, which is a fundamental question about the tertiary structure of the channel. We have probed the local architecture of the rat adult skeletal muscle Na(+) channel (mu1) outer vestibule and selectivity filter using mu-conotoxin GIIIA (mu-CTX), a neurotoxin of known structure that binds in this region. Interactions between the pore-forming loops from three different domains and four toxin residues were distinguished by mutant cycle analysis. Three of these residues, Gln-14, Hydroxyproline-17 (Hyp-17), and Lys-16 are arranged approximately at right angles to each other in a plane above the critical Arg-13 that binds directly in the ion permeation pathway. Interaction points were identified between Hyp-17 and channel residue Met-1240 of domain III and between Lys-16 and Glu-403 of domain I and Asp-1532 of domain IV. These interactions were estimated to contribute -1.0+/-0.1, -0.9+/-0.3, and -1.4+/-0.1 kcal/mol of coupling energy to the native toxin-channel complex, respectively. mu-CTX residues Gln-14 and Arg-1, both on the same side of the toxin molecule, interacted with Thr-759 of domain II. Three analytical approaches to the pattern of interactions predict that the channel domains most probably are arranged in a clockwise configuration around the pore as viewed from the extracellular surface.     

Aldosterone modulates I(f) current through gene expression in cultured neonatal rat ventricular myocytes

Mineralocorticoid receptor (MR) antagonists decrease the incidence of sudden cardiac death in patients with heart failure, as has been reported in two clinical trials (Randomized Aldactone Evaluation Study and Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study). Aldosterone has been shown to increase the propensity to arrhythmias by changing the expression or function of various ion channels. In this study, we investigate the effect of aldosterone on the expression of hyperpolarization-activated current (I(f)) channels in cultured neonatal rat ventricular myocytes, using the whole cell patch-clamp technique, real-time PCR, and Western blotting. Incubation with 10 nM aldosterone for 17-24 h significantly accelerates the rate of spontaneous beating by increasing diastolic depolarization. I(f) current elicited by hyperpolarization from -50 to -130 mV significantly increases aldosterone by 10 nM (by 1.9-fold). Exposure to aldosterone for 1.5 h increases hyperpolarization-activated cyclic nucleotide-gated (HCN) 2 mRNA by 26.3% and HCN4 mRNA by 47.2%, whereas HCN1 mRNA expression remains unaffected. Aldosterone (24-h incubation) increases the expression of HCN2 protein (by 60.0%) and HCN4 protein (by 84.8%), but not HCN1 protein. MR antagonists (1 microM eplerenone or 0.1 microM spironolactone) abolish the increase of I(f) channel expression (currents, mRNA, and protein levels) by 10 nM aldosterone. In contrast, 1 microM aldosterone downregulated I(f) channel gene expression. Glucocorticoid receptor antagonist (100 nM RU-38486) did not affect the increase of I(f) current by 10 nM aldosterone. These findings suggest that aldosterone in physiological concentrations upregulates I(f) channel gene expression by MR activation in cardiac myocytes and may increase excitability, which may have a potential proarrhythmic bearing under pathophysiological conditions.     

Hysteresis in the voltage dependence of HCN channels: conversion between two modes affects pacemaker properties

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channels are important for rhythmic activity in the brain and in the heart. In this study, using ionic and gating current measurements, we show that cloned spHCN channels undergo a hysteresis in their voltage dependence during normal gating. For example, both the gating charge versus voltage curve, Q(V), and the conductance versus voltage curve, G(V), are shifted by about +60 mV when measured from a hyperpolarized holding potential compared with a depolarized holding potential. In addition, the kinetics of the tail current and the activation current change in parallel to the voltage shifts of the Q(V) and G(V) curves. Mammalian HCN1 channels display similar effects in their ionic currents, suggesting that the mammalian HCN channels also undergo voltage hysteresis. We propose a model in which HCN channels transit between two modes. The voltage dependence in the two modes is shifted relative to each other, and the occupancy of the two modes depends on the previous activation of the channel. The shifts in the voltage dependence are fast (tau approximately 100 ms) and are not accompanied by any apparent inactivation. In HCN1 channels, the shift in voltage dependence is slower in a 100 mM K extracellular solution compared with a 1 mM K solution. Based on these findings, we suggest that molecular conformations similar to slow (C-type) inactivation of K channels underlie voltage hysteresis in HCN channels. The voltage hysteresis results in HCN channels displaying different voltage dependences during different phases in the pacemaker cycle. Computer simulations suggest that voltage hysteresis in HCN channels decreases the risk of arrhythmia in pacemaker cells.     

MiRP1 modulates HCN2 channel expression and gating in cardiac myocytes

MinK-related protein (MiRP1 or KCNE2) interacts with the hyperpolarization-activated, cyclic nucleotide-gated (HCN) family of pacemaker channels to alter channel gating in heterologous expression systems. Given the high expression levels of MiRP1 and HCN subunits in the cardiac sinoatrial node and the contribution of pacemaker channel function to impulse initiation in that tissue, such an interaction could be of considerable physiological significance. However, the functional evidence for MiRP1/HCN interactions in heterologous expression studies has been accompanied by inconsistencies between studies in terms of the specific effects on channel function. To evaluate the effect of MiRP1 on HCN expression and function in a physiological context, we used an adenovirus approach to overexpress a hemagglutinin (HA)-tagged MiRP1 (HAMiRP1) and HCN2 in neonatal rat ventricular myocytes, a cell type that expresses both MiRP1 and HCN2 message at low levels. HA-MiRP1 co-expression with HCN2 resulted in a 4-fold increase in maximal conductance of pacemaker currents compared with HCN2 expression alone. HCN2 activation and deactivation kinetics also changed, being significantly more rapid for voltages between -60 and -95 mV when HA-MiRP1 was co-expressed with HCN2. However, the voltage dependence of activation was not affected. Co-immunoprecipitation experiments demonstrated that expressed HA-MiRP1 and HCN2, as well as endogenous MiRP1 and HCN2, co-assemble in ventricular myocytes. The results indicate that MiRP1 acts as a beta subunit for HCN2 pacemaker channel subunits and alters channel gating at physiologically relevant voltages in cardiac cells.     

Separable gating mechanisms in a Mammalian pacemaker channel

Despite permeability to both K(+) and Na(+), hyperpolarization-activated cyclic nucleotide-gated (HCN) pacemaker channels contain the K(+) channel signature sequence, GYG, within the selectivity filter of the pore. Here, we show that this region is involved in regulating gating in a mouse isoform of the pacemaker channel (mHCN2). A mutation in the GYG sequence of the selectivity filter (G404S) had different effects on the two components of the wild-type current; it eliminated the slowly activating current (I(f)) but, surprisingly, did not affect the instantaneous current (I(inst)). Confocal imaging and immunocytochemistry showed G404S protein on the periphery of the cells, consistent with the presence of channels on the plasma membrane. Experiments with the wild-type channel showed that the rate of I(f) deactivation and I(f) amplitude had a parallel dependence on the ratio of K(+)/Na(+) driving forces. In addition, the amplitude of fully activated I(f), unlike I(inst), was not well predicted by equal and independent flow of K(+) and Na(+). The data are consistent with two separable gating mechanisms associated with pacemaker channels: one (I(f)) that is sensitive to voltage, to a mutation in the selectivity filter, and to driving forces for permeating cations and another (I(inst)) that is insensitive to these influences.     

Inhibition of matrix metalloproteinase MMP-2 activates chloride current in human airway epithelial cells.

Matrix metalloproteinases (MMPs) are involved in the remodeling and degradation of the extracellular matrix. Recently, it has been found that MMPs also contribute to processes not directly related to tissue remodeling, such as platelet aggregation or degranulation of airway gland cells. Since mucus secretion is closely related to ion channel function, we investigated whether MMPs could also be involved in the regulation of ion channels. We used human airway submucosal cell line Calu-3 to study the effects of MMPs on whole-cell current and transepithelial short-circuit current (I(sc)). Phenanthroline, a specific inhibitor of MMPs, increased whole-cell current with the half-maximally effective dose of 5.2 microM, and reversibly activated I(sc) in transepithelial measurements. Current stimulated by phenanthroline displayed linear current-voltage relationships and had inhibitor pharmacology and ion selectivity consistent with cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel activity. Zymography and Western blot showed significant expression of MMP-2 in Calu-3 cells. Moreover, anti-MMP-2 antibodies (1 microg/mL) increased whole-cell current and I(sc), whereas human recombinant MMP-2 (10 ng/mL) reduced it. We also studied the expression of MMPs and the effects of phenanthroline on whole-cell current in A549 cells, which are derived from airway surface epithelium and do not express CFTR Cl- channels. While these cells also showed significant expression of MMP-2, inhibition of this enzyme with phenanthroline exerted no significant effect on whole-cell current. It is concluded that MMP-2 is involved in the regulation of CFTR Cl- channels in human airways.     

S4 movement in a mammalian HCN channel

Hyperpolarization-activated, cyclic nucleotide-gated ion channels (HCN) mediate an inward cation current that contributes to spontaneous rhythmic firing activity in the heart and the brain. HCN channels share sequence homology with depolarization-activated Kv channels, including six transmembrane domains and a positively charged S4 segment. S4 has been shown to function as the voltage sensor and to undergo a voltage-dependent movement in the Shaker K+ channel (a Kv channel) and in the spHCN channel (an HCN channel from sea urchin). However, it is still unknown whether S4 undergoes a similar movement in mammalian HCN channels. In this study, we used cysteine accessibility to determine whether there is voltage-dependent S4 movement in a mammalian HCN1 channel. Six cysteine mutations (R247C, T249C, I251C, S253C, L254C, and S261C) were used to assess S4 movement of the heterologously expressed HCN1 channel in Xenopus oocytes. We found a state-dependent accessibility for four S4 residues: T249C and S253C from the extracellular solution, and L254C and S261C from the internal solution. We conclude that S4 moves in a voltage-dependent manner in HCN1 channels, similar to its movement in the spHCN channel. This S4 movement suggests that the role of S4 as a voltage sensor is conserved in HCN channels. In addition, to determine the reason for the different cAMP modulation and the different voltage range of activation in spHCN channels compared with HCN1 channels, we constructed a COOH-terminal-deleted spHCN. This channel appeared to be similar to a COOH-terminal-deleted HCN1 channel, suggesting that the main functional differences between spHCN and HCN1 channels are due to differences in their COOH termini or in the interaction between the COOH terminus and the rest of the channel protein in spHCN channels compared with HCN1 channels.     

Regulation of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) Channel Activity by cCMP

Activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is facilitated in vivo by direct binding of the second messenger cAMP. This process plays a fundamental role in the fine-tuning of HCN channel activity and is critical for the modulation of cardiac and neuronal rhythmicity. Here, we identify the pyrimidine cyclic nucleotide cCMP as another regulator of HCN channels. We demonstrate that cCMP shifts the activation curves of two members of the HCN channel family, HCN2 and HCN4, to more depolarized voltages. Moreover, cCMP speeds up activation and slows down deactivation kinetics of these channels. The two other members of the HCN channel family, HCN1 and HCN3, are not sensitive to cCMP. The modulatory effect of cCMP is reversible and requires the presence of a functional cyclic nucleotide-binding domain. We determined an EC(50) value of ～30 μm for cCMP compared with 1 μm for cAMP. Notably, cCMP is a partial agonist of HCN channels, displaying an efficacy of ～0.6. cCMP increases the frequency of pacemaker potentials from isolated sinoatrial pacemaker cells in the presence of endogenous cAMP concentrations. Electrophysiological recordings indicated that this increase is caused by a depolarizing shift in the activation curve of the native HCN current, which in turn leads to an enhancement of the slope of the diastolic depolarization of sinoatrial node cells. In conclusion, our findings establish cCMP as a gating regulator of HCN channels and indicate that this cyclic nucleotide has to be considered in HCN channel-regulated processes.     

A new hyperpolarization-activated, cyclic nucleotide-gated channel from sea urchin sperm flagella

A sea urchin sperm flagellar hyperpolarization-activated, cyclic nucleotide-gated (HCN) channel is known (SpHCN1) that is modulated by cAMP. Here, we describe a second flagellar HCN channel (SpHCN2) cloned from the same sea urchin species. SpHCN2 is 638 amino acids compared to 767 for SpHCN1. SpHCN2 has all the domains of an HCN channel, including six transmembrane segments (S1-S6), the ion pore, and the cyclic nucleotide-binding domain. The two full-length proteins are 33% identical and 51% similar. The six transmembrane segments vary from 46-79% identity. S4, which is the voltage sensor, is 79% identical between the two proteins. The ion selectivity filter sequence is GYG in the ion pore of SpHCN1 and GFG in SpHCN2. By sequence, SpHCN2 is 73.5kDa, but it migrates on SDS-PAGE at 64kDa. Western immunoblots show localization to flagella, which is confirmed by immunofluorescence. A neighbor-joining tree shows that SpHCN2 is basal to all known HCN channels. SpHCN2 might be the simplest pacemaker channel yet discovered.     

K+ channel regulator KCR1 suppresses heart rhythm by modulating the pacemaker current If

Hyperpolarization-activated, cyclic nucleotide sensitive (HCN) channels underlie the pacemaker current I(f), which plays an essential role in spontaneous cardiac activity. HCN channel subunits (HCN1-4) are believed to be modulated by additional regulatory proteins, which still have to be identified. Using biochemistry, molecularbiology and electrophysiology methods we demonstrate a protein-protein interaction between HCN2 and the K(+) channel regulator protein 1, named KCR1. In coimmunoprecipitation experiments we show that KCR1 and HCN2 proteins are able to associate. Heterologously expressed HCN2 whole-cell current density was significantly decreased by KCR1. KCR1 profoundly suppressed I(HCN2) single-channel activity, indicating a functional interaction between KCR1 and the HCN2 channel subunit. Endogenous KCR1 expression could be detected in adult and neonatal rat ventriculocytes. Adenoviral-mediated overexpression of KCR1 in rat cardiomyocytes (i) reduced I(f) whole-cell currents, (ii) suppressed most single-channel gating parameters, (iii) altered the activation kinetics, (iv) suppressed spontaneous action potential activity, and (v) the beating rate. More importantly, siRNA-based knock-down of endogenous KCR1 increased the native I(f) current size and single-channel activity and accelerated spontaneous beating rate, supporting an inhibitory action of endogenous KCR1 on native I(f). Our observations demonstrate for the first time that KCR1 modulates I(HCN2)/I(f) channel gating and indicate that KCR1 serves as a regulator of cardiac automaticity.     

Voltage-dependent gating of hyperpolarization-activated, cyclic nucleotide-gated pacemaker channels: molecular coupling between the S4-S5 and C-linkers

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels have a transmembrane topology that is highly similar to voltage-gated K(+) channels, yet HCN channels open in response to membrane hyperpolarization instead of depolarization. The structural basis for the "inverted" voltage dependence of HCN gating and how voltage sensing by the S1-S4 domains is coupled to the opening of the intracellular gate formed by the S6 domain are unknown. Coupling could arise from interaction between specific residues or entire transmembrane domains. We previously reported that the mutation of specific residues in the S4-S5 linker of HCN2 (i.e. Tyr-331 and Arg-339) prevented normal channel closure presumably by disruption of a crucial interaction with the activation gate. Here we hypothesized that the C-linker, a carboxyl terminus segment that connects S6 to the cyclic nucleotide binding domain, interacts with specific residues of the S4-S5 linker to mediate coupling. The recently solved structure of the C-linker of HCN2 indicates that an alpha-helix (the A'-helix) is located near the end of each S6 domain, the presumed location of the activation gate. Ala-scanning mutagenesis of the end of S6 and the A'-helix identified five residues that were important for normal gating as mutations disrupted channel closure. However, partial deletion of the C-linker indicated that the presence of only two of these residues was required for normal coupling. Further mutation analyses suggested that a specific electrostatic interaction between Arg-339 of the S4-S5 linker and Asp-443 of the C-linker stabilizes the closed state and thus participates in the coupling of voltage sensing and activation gating in HCN channels.