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.     

Identification of a surface charged residue in the S3-S4 linker of the pacemaker (HCN) channel that influences activation gating

I(f), encoded by the hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel family, is a key player in cardiac and neuronal pacing. Although HCN channels structurally resemble voltage-gated K(+) (Kv) channels, their structure-function correlation is much less clear. Here we probed the functional importance of the HCN1 S3-S4 linker by multiple substitutions of its residues. Neutralizing Glu(235), an acidic S3-S4 linker residue conserved in all hyperpolarization-activated channels, by Ala substitution produced a depolarizing activation shift (V(12) = -65.0 +/- 0.7 versus -70.6 +/- 0.7 mV for wild-type HCN1); the charge-reversed mutation E235R shifted activation even more positively (-56.2 +/- 0.5 mV). Increasing external Mg(2+) mimicked the progressive rightward shifts of E235A and E235R by gradually shifting activation (V(12) = 1 < 3 < 10 < 30 mm); Delta V(12) induced by 30 mm Mg(2+) was significantly attenuated for E235A (+7.9 +/- 1.2 versus +11.3 +/- 0.9 mV for wild-type HCN1) and E235R (+3.3 +/- 1.4 mV) channels, as if surface charges were already shielded. Consistent with an electrostatic role, the energetic changes associated with Delta V(12) resulting from various Glu(235) substitutions (i.e. Asp, Ala, Pro, His, Lys, and Arg) displayed a strong correlation with their charges (Delta Delta G = -2.1 +/- 0.3 kcal/mol/charge; r = 0.94). In contrast, D233E, D233A, D233G, and D233R did not alter activation gating. D233C (in C318S background) was also not externally accessible when probed with methanethiosulfonate ethylammonium (MTSEA). We conclude that the S3-S4 linker residue Glu(235) influences activation gating, probably by acting as a surface charge.     

Helical secondary structure of the external S3-S4 linker of pacemaker (HCN) channels revealed by site-dependent perturbations of activation phenotype

If, encoded by the hyperpolarization-activated cyclic nucleotide-modulated channel family (HCN1-4), contributes significantly to neuronal and cardiac pacing. Recently, we reported that the S3-S4 residue Glu-235 of HCN1 influences activation by acting as a surface charge. However, it is uncertain whether other residues of the external S3-S4 linker are also involved in gating. Furthermore, the secondary conformation of the linker is not known. Here we probed the structural and functional role of the HCN1 S3-S4 linker by introducing systematic mutations into the entire linker (defined as 229-237) and studying their effects. We found that the mutations K230A (-62.2 +/- 3.4 mV versus -72.2 +/- 1.7 mV of wild type (WT)), G231A (-64.4 +/- 1.3 mV), M232A (V(1/2) = -63.1 +/- 1.1 mV), and E235G (-65.4 +/- 1.5 mV) produced depolarizing activation shifts. Although E229A and M232A decelerated gating kinetics (<13- and 3-fold, respectively), K230A and G231A accelerated both activation and deactivation (< approximately 2-3-fold). D233A, S234A, V236A, and Y237A channels exhibited WT properties (p > 0.05). Shortening the linker (EVY235-237deltadeltadelta) caused depolarizing activation shift and slowed kinetics that could not be explained by removing the charge at position 235 alone. Secondary structural predictions by the modeling algorithms SSpro2 and PROF, along with refinements by our experimental data, suggest that part of the S3-S4 linker conforms a helical structure with the functionally important residues Met-232, Glu-235, and Gly-231 (|deltadeltaG|>1 kcal/mol) clustered on one side.     

Endogenous channels in HEK cells and potential roles in HCN ionic current measurements

A transformed line of human embryonic kidney epithelial cells (HEK 293) is commonly used as an expression system for exogenous ion channel genes. Previously, it has been shown that these cells contain mRNAs for a variety of ion channels. Expression of some of these genes has been confirmed at the protein level. Patch-clamp electrophysiology experiments confirm the presence of multiple ion channels and molecular data agree with pharmacological profiles of identified channels. In this work, we show that endogenous voltage-gated potassium channels in HEK cells are a significant source of outward current at positive potentials. We show that both non-transfected HEK cells and HEK cells transfected with hyperpolarization-activated cyclic-nucleotide gated (HCN) channels have a significant amount of voltage-gated potassium (K(V)) current when certain tail current voltage-clamp protocols are used to assay HCN current activation. Specifically, tail current protocols that use a depolarized holding potential of -40 mV followed by hyperpolarizing pulses (-80 to -140 mV) and then a tail pulse potential of +20 mV indicate K(V) channels undergo closed-state inactivation at the more depolarized holding potential of -40 mV, followed by recovery from inactivation (but no activation) at hyperpolarizing potentials and high amount of activation at the positive tail potential. Our results indicate that pulse protocols with positive tail pulses are inaccurate assays for HCN current in certain HEK cells. Surprisingly, HEK-293 cells were found to contain mRNA for HCN2 and HCN3 although we have not detected a significant and consistent endogenous I(f)-like current in these cells.     

Mutations in the S4 domain of a pacemaker channel alter its voltage dependence

In an attempt to study the functional role of the positively charged amino acids present in the S4 segment of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels, we have introduced single and sequential amino acid replacements throughout this domain in the mouse type 2 HCN channel (mHCN2). Sequential neutralization of the first three positively charged amino acids resulted in cumulative shifts of the midpoint voltage activation constant towards more hyperpolarizing potentials. The contribution of each amino acid substitution was approximately -20 mV. Amino acid replacements to neutralize either the first (K291Q) or fourth (R300Q) positively charged amino acid resulted in the same shift (about 20 mV) towards more hyperpolarized potentials. Replacing the first positively charged amino acid with the negatively charged glutamic acid (K291E) produced a shift of approximately -50 mV in the same direction. None of the above amino acid substitutions had any measurable effect on the time course of channel activation. This suggests that the S4 domain of HCN channels critically controls the voltage dependence of channel opening but is not involved in regulating activation kinetics. No channel activity was detected in mutants with neutralization of the last six positively charged amino acids from the S4 domain, suggesting that these amino acids cannot be altered without impairing channel function.     

Structural changes during HCN channel gating defined by high affinity metal bridges

Hyperpolarization-activated cyclic nucleotide-sensitive nonselective cation (HCN) channels are activated by membrane hyperpolarization, in contrast to the vast majority of other voltage-gated channels that are activated by depolarization. The structural basis for this unique characteristic of HCN channels is unknown. Interactions between the S4-S5 linker and post-S6/C-linker region have been implicated previously in the gating mechanism of HCN channels. We therefore introduced pairs of cysteines into these regions within the sea urchin HCN channel and performed a Cd(2+)-bridging scan to resolve their spatial relationship. We show that high affinity metal bridges between the S4-S5 linker and post-S6/C-linker region can induce either a lock-open or lock-closed phenotype, depending on the position of the bridged cysteine pair. This suggests that interactions between these regions can occur in both the open and closed states, and that these regions move relative to each other during gating. Concatenated constructs reveal that interactions of the S4-S5 linker and post-S6/C-linker can occur between neighboring subunits. A structural model based on these interactions suggests a mechanism for HCN channel gating. We propose that during voltage-dependent activation the voltage sensors, together with the S4-S5 linkers, drive movement of the lower ends of the S5 helices around the central axis of the channel. This facilitates a movement of the pore-lining S6 helices, which results in opening of the channel. This mechanism may underlie the unique voltage dependence of HCN channel gating.     

Structural elements of instantaneous and slow gating in hyperpolarization-activated cyclic nucleotide-gated channels

Hyperpolarization-activated cyclic nucleotide-gated (HCN) subunits produce a slowly activating current in response to hyperpolarization (If) and an instantaneous voltage-independent current (Iinst) when expressed in Chinese hamster ovary (CHO) cells. Here we found that a mutation in the S4-S5 linker of HCN2 (Y331D) produced an additional mixed cationic instantaneous current. However, this current was inhibited by external Cs+ like If and unlike Iinst. Together with a concomitant reduction in If, the data suggest that the Y331D mutation disrupted channel closing placing the channel in a "If-like," and not an "Iinst-like," state. The "If-like" instantaneous current represented approximately 70% of total If over voltages ranging from +20 to -150 mV in high K+ solutions. If activated at more depolarized potentials and the activation curve was less steep, whereas deactivation was significantly slowed, consistent with the idea that the mutation inhibited channel closing. The data suggest that the mutation produced allosteric effects on the activation gate (S6 segment) and/or on voltage-sensing elements. We also found that decreases in the ratio of external K+/Na+ further disrupted channel closing in the mutant channel. Finally, our data suggest that the structures involved in producing Iinst are similar between the HCN1 and HCN2 isoforms and that excess HCN protein on the plasma membrane of CHO cells relative to native cells is not responsible for Iinst. The data are consistent with Iinst flowing through a "leaky" closed state but do not rule out flow through a second configuration of recombinant HCN channels or up-regulated endogenous channels/subunits.     

Dissecting the structural and functional roles of the S3-S4 linker of pacemaker (hyperpolarization-activated cyclic nucleotide-modulated) channels by systematic length alterations

If or Ih, a key player in neuronal and cardiac pacing, is encoded by the hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel gene family. We have recently reported that the S3-S4 linker (i.e. residues 229EKGMDSEVY237 of HCN1) prominently influences the activation phenotypes of HCN channels and that part of the linker may conform a secondary helical structure. Here we further dissected the structural and functional roles of this linker by systematic alterations of its length. In contrast to voltage-gated K+ channels, complete deletion of the S3-S4 linker (Delta229-237) did not produce functional channels. Similarly, the deletions Delta229-234, Delta232-234, and Delta232-237 also abolished normal current activity. Interestingly, Delta229-231, Delta233-237, Delta234-237, Delta235-237, Delta229-231/Delta233-237, Delta229-231/Delta234-237, and Delta229-231/Delta235-237 all yielded robust hyperpolarization-activated inward currents, indicating that loss-of-function caused by deletion could be rescued by keeping the single functionally important residue Met232 alone. Whereas shortening the linker by deletion generally shifted steady-state activation in the depolarizing direction (e.g. DeltaV1/2 of Delta229-231, Delta233-237, Delta235-237 > +10 mV relative to wild type), linker prolongation by duplicating the entire linker (Dup229-237) or by glutamine insertion (InsQ233Q, InsQQ233QQ and InsQQQ233QQQ, or Ins237QQQ) produced length-dependent progressive hyperpolarizing activation shifts (-35 mV < DeltaV1/2 < -4 mV). Based on these results, we conclude that only Met232 is prerequisite for channels to function, but the length and other constituents of the S3-S4 linker shape the ultimate activation phenotype. Our results also highlight several evolutionary similarities and differences between HCN and voltage-gated K+ channels. Manipulations of the S3-S4 linker length may provide a flexible approach to customize HCN gating for engineering electrically active cells (such as stem cell-derived neuronal and cardiac pacemakers) for gene- and cell-based therapies.     

A single histidine residue determines the pH sensitivity of the pacemaker channel HCN2

Hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels control the rhythmic activity of heart and neuronal networks. The activation of these channels is regulated in a complex manner by hormones and neurotransmitters. In addition it was suggested that the channels may be controlled by the pH of the cytosol. Here we demonstrate that HCN2, a member of the HCN channel family, is directly modulated by the intracellular pH in the physiological range. Protons inhibit HCN2 channels by shifting the voltage dependence of channel activation to more negative voltages. By using site-directed mutagenesis, we have identified a single histidine residue (His-321) localized at the boundary between the voltage-sensing S4 helix and the cytoplasmic S4-S5 linker of the channel that is a major determinant of pH sensitivity. Replacement of His-321 by either arginine, glutamine, or glutamate results in channels that are no longer sensitive to shifts in intracellular pH. In contrast, cAMP-mediated modulation is completely intact in mutant channels indicating that His-321 is not involved in the molecular mechanism that controls modulation of HCN channel activity by cyclic nucleotides. Because His-321 is conserved in all four HCN channels known so far, regulation by intracellular pH is likely to constitute a general feature of both cardiac and neuronal pacemaker channels.     

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.     

Properties of hyperpolarization-activated pacemaker current defined by coassembly of HCN1 and HCN2 subunits and basal modulation by cyclic nucleotide

Members of the HCN channel family generate hyperpolarization-activated cation currents (Ih) that are directly regulated by cAMP and contribute to pacemaker activity in heart and brain. The four HCN isoforms show distinct but overlapping patterns of expression in different tissues. Here, we report that HCN1 and HCN2, isoforms coexpressed in neocortex and hippocampus that differ markedly in their biophysical properties, coassemble to generate heteromultimeric channels with novel properties. When expressed in Xenopus oocytes, HCN1 channels activate 5-10-fold more rapidly than HCN2 channels. HCN1 channels also activate at voltages that are 10-20 mV more positive than those required to activate HCN2. In cell-free patches, the steady-state activation curve of HCN1 channels shows a minimal shift in response to cAMP (+4 mV), whereas that of HCN2 channels shows a pronounced shift (+17 mV). Coexpression of HCN1 and HCN2 yields Ih currents that activate with kinetics and a voltage dependence that tend to be intermediate between those of HCN1 and HCN2 homomers, although the coexpressed channels do show a relatively large shift by cAMP (+14 mV). Neither the kinetics, steady-state voltage dependence, nor cAMP dose-response curve for the coexpressed Ih can be reproduced by the linear sum of independent populations of HCN1 and HCN2 homomers. These results are most simply explained by the formation of heteromeric channels with novel properties. The properties of these heteromeric channels closely resemble the properties of I(h) in hippocampal CA1 pyramidal neurons, cells that coexpress HCN1 and HCN2. Finally, differences in Ih channel properties recorded in cell-free patches versus intact oocytes are shown to be due, in part, to modulation of Ih by basal levels of cAMP in intact cells.     

The cGMP-dependent protein kinase II Is an inhibitory modulator of the hyperpolarization-activated HCN2 channel

Opening of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is facilitated by direct binding of cyclic nucleotides to a cyclic nucleotide-binding domain (CNBD) in the C-terminus. Here, we show for the first time that in the HCN2 channel cGMP can also exert an inhibitory effect on gating via cGMP-dependent protein kinase II (cGKII)-mediated phosphorylation. Using coimmunoprecipitation and immunohistochemistry we demonstrate that cGKII and HCN2 interact and colocalize with each other upon heterologous expression as well as in native mouse brain. We identify the proximal C-terminus of HCN2 as binding region of cGKII and show that cGKII phosphorylates HCN2 at a specific serine residue (S641) in the C-terminal end of the CNBD. The cGKII shifts the voltage-dependence of HCN2 activation to 2-5 mV more negative voltages and, hence, counteracts the stimulatory effect of cGMP on gating. The inhibitory cGMP effect can be either abolished by mutation of the phosphorylation site in HCN2 or by impairing the catalytic domain of cGKII. By contrast, the inhibitory effect is preserved in a HCN2 mutant carrying a CNBD deficient for cGMP binding. Our data suggest that bidirectional regulation of HCN2 gating by cGMP contributes to cellular fine-tuning of HCN channel activity.     

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.     

Src tyrosine kinase alters gating of hyperpolarization-activated HCN4 pacemaker channel through Tyr531

We recently discovered that the constitutively active Src tyrosine kinase can enhance hyperpolarization-activated, cyclic nucleotide-gated (HCN) 4 channel activity by binding to the channel protein. To investigate the mechanism of modulation by Src of HCN channels, we studied the effects of a selective inhibitor of Src tyrosine kinase, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), on HCN4 and its mutant channels expressed in HEK 293 cells by using a whole cell patch-clamp technique. We found that PP2 can inhibit HCN4 currents by negatively shifting the voltage dependence of channel activation, decreasing the whole cell channel conductance, and slowing activation and deactivation kinetics. Screening putative tyrosine residues subject to phosphorylation yielded two candidates: Tyr(531) and Tyr(554). Substituting HCN4-Tyr(531) with phenylalanine largely abolished the effects of PP2 on HCN4 channels. Replacing HCN4-Tyr(554) with phenylalanine did not abolish the effects of PP2 on voltage-dependent activation but did eliminate PP2-induced slowing of channel kinetics. The inhibitory effects of HCN channels associated with reduced Src tyrosine activity is confirmed in HL-1 cardiomyocytes. Finally, we found that PP2 can decrease the heart rate in a mouse model. These results demonstrate that Src tyrosine kinase enhances HCN4 currents by shifting their activation to more positive potentials and increasing the whole cell channel conductance as well as speeding the channel kinetics. The tyrosine residue that mediates most of Src's actions on HCN4 channels is Tyr(531).     

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.     

Structural Basis for the cAMP-dependent Gating in the Human HCN4 Channel

Hyperpolarization-activated cAMP-regulated (HCN) channels play important physiological roles in both cardiovascular and central nervous systems. Among the four HCN isoforms, HCN2 and HCN4 show high expression levels in the human heart, with HCN4 being the major cardiac isoform. The previously published crystal structure of the mouse HCN2 (mHCN2) C-terminal fragment, including the C-linker and the cyclic-nucleotide binding domain (CNBD), has provided many insights into cAMP-dependent gating in HCN channels. However, structures of other mammalian HCN channel isoforms have been lacking. Here we used a combination of approaches including structural biology, biochemistry, and electrophysiology to study cAMP-dependent gating in HCN4 channel. First we solved the crystal structure of the C-terminal fragment of human HCN4 (hHCN4) channel at 2.4 Å. Overall we observed a high similarity between mHCN2 and hHCN4 crystal structures. Functional comparison between two isoforms revealed that compared with mHCN2, the hHCN4 protein exhibited marked different contributions to channel function, such as a ∼3-fold reduction in the response to cAMP. Guided by structural differences in the loop region between β4 and β5 strands, we identified residues that could partially account for the differences in response to cAMP between mHCN2 and hHCN4 proteins. Moreover, upon cAMP binding, the hHCN4 C-terminal protein exerts a much prolonged effect in channel deactivation that could have significant physiological contributions.     

Functional heteromerization of HCN1 and HCN2 pacemaker channels

An important step toward understanding the molecular basis of the functional diversity of pacemaker currents in spontaneously active cells has been the identification of a gene family encoding hyperpolarization-activated cyclic nucleotide-sensitive cation nonselective (HCN) channels. Three of the four gene products that have been expressed so far give rise to pacemaker channels with distinct activation kinetics and are differentially distributed among the brain, with considerable overlap between some isoforms. This raises the possibility that HCN channels may coassemble to form heteromeric channels in some areas, similar to other K(+) channels. In this study, we have provided evidence for functional heteromerization of HCN1 and HCN2 channels using a concatenated cDNA construct encoding two connected subunits. We have observed that heteromeric channels activate several-fold faster than HCN2 and only a little slower than HCN1. Furthermore, the voltage dependence of activation is more similar to HCN2, whereas the cAMP sensitivity is intermediate between HCN1 and HCN2. This phenotype shows marked similarity to the current arising from coexpressed HCN1 and HCN2 subunits in oocytes and the native pacemaker current in CA1 pyramidal neurons. We suggest that heteromerization may increase the functional diversity beyond the levels expected from the number of HCN channel genes and their differential distribution.     

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.     

State-Dependent Accessibility of the P-S6 Linker of Pacemaker (HCN) Channels Supports a Dynamic Pore-to-Gate Coupling Model

The hyperpolarization-activated cyclic nucleotide-modulated channel gene family (HCN1-4) encodes the membrane depolarizing current that underlies pacemaking. Although the topology of HCN resembles K_v channels, much less is known about their structure-function correlation. Previously, we identified several pore residues in the S5-P linker and P-loop that are externally accessible and/or influence HCN gating, and proposed an evolutionarily conserved pore-to-gate mechanism. Here we sought dynamic evidence by assessing the functional consequences of Cys-scanning substitutions in the unexplored P-S6 linker (residues 352–359), the HCN1-R background (that is, resistant to sulfhydryl-reactive agents). None of A352C, Q353C, A354C, P355C, V356C, S357C, M358C, or S359C produced functional currents; the loss-of-function of Q353C, A354C, S357C, and M358C could be rescued by the reducing agent dithiothreitol. Q353C, A354C, and S357C, but not M358C and HCN1-R, were sensitive to Cd²⁺ blockade (IC₅₀ = 3–12 μM vs. >1 mM). External application of the positively charged covalent sulfhydryl modifier MTSET irreversibly reduced I _−140mV of Q353C and A354C to 27.9 ± 3.4% and 58.2 ± 13.1% of the control, respectively, and caused significant steady-state activation shifts (∆V _1/2 = –21.1 ± 1.6 for Q353C and −10.0 ± 2.9 mV for A354C). Interestingly, MTSET reactivity was also state dependent. MTSET, however, affected neither S357C nor M358C, indicating site specificity. Collectively, we have identified novel P-S6 residues whose extracellular accessibility was sterically and state dependent and have provided the first functional evidence consistent with a dynamic HCN pore-to-gate model.     

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.