Regulation of hyperpolarization-activated HCN channels by cAMP through a gating switch in binding domain symmetry

Recent X-ray structures show that the binding domains of tetrameric ligand-gated channels form either a 4-fold symmetric gating ring or a 2-fold symmetric dimer of dimers. To determine how such structures function to coordinate the binding of multiple ligands during channel activation, we examined the action of cAMP to enhance the opening of the hyperpolarization-activated HCN2 channels, whose cytoplasmic C terminus forms a gating ring in the presence of cAMP. Using tandem dimers and tetramers in which cAMP binding to selected HCN2 subunits was prevented by a point mutation or deletion, we provide the first direct determination of the energetic effects on gating of each of four ligand binding events and demonstrate the importance of the gating ring for cAMP regulation. We suggest that cAMP binding enhances channel opening by promoting assembly of the gating ring from an unliganded state in which the four subunits interact as a 2-fold symmetric dimer of dimers.     

Structural dynamics in the gating ring of cyclic nucleotide-gated ion channels

For ligand-gated ion channels, the binding of a ligand to an intracellular or extracellular domain generates changes in transmembrane pore-forming helices, which alters ion flow. The molecular mechanism for this allostery, however, remains unknown. Here we explore the structure and conformational rearrangements of the C-terminal gating ring of the cyclic nucleotide-gated channel CNGA1 during activation by cyclic nucleotides with patch-clamp fluorometry. By monitoring fluorescent resonance energy transfer (FRET) between membrane-resident quenchers and fluorophores attached to the channel, we detected no movement orthogonal to the membrane during channel activation. By monitoring FRET between fluorophores within the C-terminal region, we determined that the C-terminal end of the C-linker and the end of the C-helix move apart when channels open. We conclude that during channel activation, a portion of the gating ring moves parallel to the plasma membrane, hinging toward the central axis of the channel.     

Probability Fluxes and Transition Paths in a Markovian Model Describing Complex Subunit Cooperativity in HCN2 Channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are voltage-gated tetrameric cation channels that generate electrical rhythmicity in neurons and cardiomyocytes. Activation can be enhanced by the binding of adenosine-3′,5′-cyclic monophosphate (cAMP) to an intracellular cyclic nucleotide binding domain. Based on previously determined rate constants for a complex Markovian model describing the gating of homotetrameric HCN2 channels, we analyzed probability fluxes within this model, including unidirectional probability fluxes and the probability flux along transition paths. The time-dependent probability fluxes quantify the contributions of all 13 transitions of the model to channel activation. The binding of the first, third and fourth ligand evoked robust channel opening whereas the binding of the second ligand obstructed channel opening similar to the empty channel. Analysis of the net probability fluxes in terms of the transition path theory revealed pronounced hysteresis for channel activation and deactivation. These results provide quantitative insight into the complex interaction of the four structurally equal subunits, leading to non-equality in their function.     

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.     

Inner activation gate in S6 contributes to the state-dependent binding of cAMP in full-length HCN2 channel

Recently, applications of the patch-clamp fluorometry (PCF) technique in studies of cyclic nucleotide-gated (CNG) and hyperpolarization-activated, cyclic nucleotide-regulated (HCN) channels have provided direct evidence for the long-held notion that ligands preferably bind to and stabilize these channels in an open state. This state-dependent ligand-channel interaction involves contributions from not only the ligand-binding domain but also other discrete structural elements within the channel protein. This insight led us to investigate whether the pore of the HCN channel plays a role in the ligand-whole channel interaction. We used three well-characterized HCN channel blockers to probe the ion-conducting passage. The PCF technique was used to simultaneously monitor channel activity and cAMP binding. Two ionic blockers, Cs(+) and Mg(2+), effectively block channel conductance but have no obvious effect on cAMP binding. Surprisingly, ZD7288, an open channel blocker specific for HCN channels, significantly reduces the activity-dependent increase in cAMP binding. Independent biochemical assays exclude any nonspecific interaction between ZD7288 and isolated cAMP-binding domain. Because ZD7228 interacts with the inner pore region, where the activation gate is presumably located, we did an alanine scanning of the intracellular end of S6, from T426 to A435. Mutations of three residues, T426, M430, and H434, which are located at regular intervals on the S6 α-helix, enhance cAMP binding. In contrast, mutations of two residues in close proximity, F431A and I432A, dampen the response. Our results demonstrate that movements of the structural elements near the activation gate directly affect ligand binding affinity, which is a simple mechanistic explanation that could be applied to the interpretation of ligand gating in general.     

Flavonoid Regulation of HCN2 Channels

The hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are pacemaker channels whose currents contribute to rhythmic activity in the heart and brain. HCN channels open in response to hyperpolarizing voltages, and the binding of cAMP to their cyclic nucleotide-binding domain (CNBD) facilitates channel opening. Here, we report that, like cAMP, the flavonoid fisetin potentiates HCN2 channel gating. Fisetin sped HCN2 activation and shifted the conductance-voltage relationship to more depolarizing potentials with a half-maximal effective concentration (EC₅₀) of 1.8 μm. When applied together, fisetin and cAMP regulated HCN2 gating in a nonadditive fashion. Fisetin did not potentiate HCN2 channels lacking their CNBD, and two independent fluorescence-based binding assays reported that fisetin bound to the purified CNBD. These data suggest that the CNBD mediates the fisetin potentiation of HCN2 channels. Moreover, binding assays suggest that fisetin and cAMP partially compete for binding to the CNBD. NMR experiments demonstrated that fisetin binds within the cAMP-binding pocket, interacting with some of the same residues as cAMP. Together, these data indicate that fisetin is a partial agonist for HCN2 channels.     

Trafficking and gating of hyperpolarization-activated cyclic nucleotide-gated channels are regulated by interaction with tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) and cyclic AMP at distinct sites

Ion channel trafficking and gating are often influenced by interactions with auxiliary subunits. Tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) is an auxiliary subunit for neuronal hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. TRIP8b interacts directly with two distinct sites of HCN channel pore-forming subunits to control channel trafficking and gating. Here we use mutagenesis combined with electrophysiological studies to define and distinguish the functional importance of the HCN/TRIP8b interaction sites. Interaction with the last three amino acids of the HCN1 C terminus governed the effect of TRIP8b on channel trafficking, whereas TRIP8b interaction with the HCN1 cyclic nucleotide binding domain (CNBD) affected trafficking and gating. Biochemical studies revealed that direct interaction between TRIP8b and the HCN1 CNBD was disrupted by cAMP and that TRIP8b binding to the CNBD required an arginine residue also necessary for cAMP binding. In accord, increasing cAMP levels in cells antagonized the up-regulation of HCN1 channels mediated by a TRIP8b construct binding the CNBD exclusively. These data illustrate the distinct roles of the two TRIP8b-HCN interaction domains and suggest that TRIP8b and cAMP may directly compete for binding the HCN CNBD to control HCN channel gating, kinetics, and trafficking.     

cAMP Modulation of the cytoplasmic domain in the HCN2 channel investigated by molecular simulations

The hyperpolarization-activated cyclic nucleotide-modulated (HCN) cation channels are opened by membrane hyperpolarization, while their activation is modulated by the binding of cyclic adenosine monophosphate (cAMP) in the cytoplasm. Here we investigate the molecular basis of cAMP channel modulation by performing molecular dynamics simulations of a segment comprising the C-linker and the cyclic nucleotide binding domain (CNBD) in the presence and absence of cAMP, based on the available crystal structure of HCN2 from mouse. In presence of cAMP, the protein undergoes an oscillation of the quaternary structure on the order of 10 ns, not observed in the apoprotein. In contrast, the absence of ligand causes conformational rearrangements within the CNBDs, driving these domains to a more flexible state, similar to that described in CNBDs of other proteins. This increased flexibility causes a rather disordered movement of the CNBDs, resulting in an inhibitory effect on the channel. We propose that the cAMP-triggered large-scale oscillation plays an important role for the channel's function, being coupled to a motion of the C-linker which, in turn, modulates the gating of the channel.     

A novel mechanism of modulation of hyperpolarization-activated cyclic nucleotide-gated channels by Src kinase

Hyperpolarization-activated cyclic nucleotide-gated channels (HCN1-4) play a crucial role in the regulation of cell excitability. Importantly, they contribute to spontaneous rhythmic activity in brain and heart. HCN channels are principally activated by membrane hyperpolarization and binding of cAMP. Here, we identify tyrosine phosphorylation by Src kinase as another mechanism affecting channel gating. Inhibition of Src by specific blockers slowed down activation kinetics of native and heterologously expressed HCN channels. The same effect on HCN channel activation was observed in cells cotransfected with a dominant-negative Src mutant. Immunoprecipitation demonstrated that Src binds to and phosphorylates native and heterologously expressed HCN2. Src interacts via its SH3 domain with a sequence of HCN2 encompassing part of the C-linker and the cyclic nucleotide binding domain. We identified a highly conserved tyrosine residue in the C-linker of HCN channels (Tyr476 in HCN2) that confers modulation by Src. Replacement of this tyrosine by phenylalanine in HCN2 or HCN4 abolished sensitivity to Src inhibitors. Mass spectrometry confirmed that Tyr476 is phosphorylated by Src. Our results have functional implications for HCN channel gating. Furthermore, they indicate that tyrosine phosphorylation contributes in vivo to the fine tuning of HCN channel activity.     

A conserved tripeptide in CNG and HCN channels regulates ligand gating by controlling C-terminal oligomerization

Cyclic nucleotides directly enhance the opening of the tetrameric CNG and HCN channels, although the mechanism remains unclear. We examined why HCN and certain CNG subunits form functional homomeric channels, whereas other CNG subunits only function in heteromeric channels. The "defect" in the CNGA4 subunit that prevents its homomeric expression was localized to its C-linker, which connects the transmembrane domain to the binding domain and contains a tripeptide that decreases the efficacy of ligand gating. Remarkably, replacement of the homologous HCN tripeptide with the CNGA4 sequence transformed cAMP into an inverse agonist that inhibits HCN channel opening. Using analytical ultracentrifugation, we identified the structural basis for this gating switch: whereas cAMP normally enhances the assembly of HCN C-terminal domains into a tetrameric gating ring, inclusion of the CNGA4 tripeptide reversed this action so that cAMP now causes gating ring disassembly. Thus, ligand gating depends on the dynamic oligomerization of C-terminal binding domains.     

Regulation of hyperpolarization-activated HCN channel gating and cAMP modulation due to interactions of COOH terminus and core transmembrane regions

Members of the hyperpolarization-activated cation (HCN) channel family generate HCN currents (I(h)) that are directly regulated by cAMP and contribute to pacemaking activity in heart and brain. The four different HCN isoforms show distinct biophysical properties. In cell-free patches from Xenopus oocytes, the steady-state activation curve of HCN2 channels is 20 mV more hyperpolarized compared with HCN1. Whereas the binding of cAMP to a COOH-terminal cyclic nucleotide binding domain (CNBD) markedly shifts the activation curve of HCN2 by 17 mV to more positive potentials, the response of HCN1 is much less pronounced (4 mV shift). A previous deletion mutant study suggested that the CNBD inhibits hyperpolarization-gating in the absence of cAMP; the binding of cAMP shifts gating to more positive voltages by relieving this inhibition. The differences in basal gating and cAMP responsiveness between HCN1 and HCN2 were proposed to result from a greater inhibitory effect of the CNBD in HCN2 compared with HCN1. Here, we use a series of chimeras between HCN1 and HCN2, in which we exchange the NH(2) terminus, the transmembrane domain, or distinct domains of the COOH terminus, to investigate further the molecular bases for the modulatory action of cAMP and for the differences in the functional properties of the two channels. Differences in cAMP regulation between HCN1 and HCN2 are localized to sequence differences within the COOH terminus of the two channels. Surprisingly, exchange of the CNBDs between HCN1 and HCN2 has little effect on basal gating and has only a modest one on cAMP modulation. Rather, differences in cAMP modulation depend on the interaction between the CNBD and the C-linker, a conserved 80-amino acid region that connects the last (S6) transmembrane segment to the CNBD. Differences in basal gating depend on both the core transmembrane domain and the COOH terminus. These data, taken in the context of the previous data on deletion mutants, suggest that the inhibitory effect of the CNBD on basal gating depends on its interactions with both the C-linker and core transmembrane domain of the channel. The extent to which cAMP binding is able to relieve this inhibition is dependent on the interaction between the C-linker and the CNBD.     

Binding of the auxiliary subunit TRIP8b to HCN channels shifts the mode of action of cAMP

Hyperpolarization-activated cyclic nucleotide–regulated cation (HCN) channels generate the hyperpolarization-activated cation current Ih present in many neurons. These channels are directly regulated by the binding of cAMP, which both shifts the voltage dependence of HCN channel opening to more positive potentials and increases maximal Ih at extreme negative voltages where voltage gating is complete. Here we report that the HCN channel brain-specific auxiliary subunit TRIP8b produces opposing actions on these two effects of cAMP. In the first action, TRIP8b inhibits the effect of cAMP to shift voltage gating, decreasing both the sensitivity of the channel to cAMP (K_1/2) and the efficacy of cAMP (maximal voltage shift); conversely, cAMP binding inhibits these actions of TRIP8b. These mutually antagonistic actions are well described by a cyclic allosteric mechanism in which TRIP8b binding reduces the affinity of the channel for cAMP, with the affinity of the open state for cAMP being reduced to a greater extent than the cAMP affinity of the closed state. In a second apparently independent action, TRIP8b enhances the action of cAMP to increase maximal Ih. This latter effect cannot be explained by the cyclic allosteric model but results from a previously uncharacterized action of TRIP8b to reduce maximal current through the channel in the absence of cAMP. Because the binding of cAMP also antagonizes this second effect of TRIP8b, application of cAMP produces a larger increase in maximal Ih in the presence of TRIP8b than in its absence. These findings may provide a mechanistic explanation for the wide variability in the effects of modulatory transmitters on the voltage gating and maximal amplitude of Ih reported for different neurons in the brain.     

Activity-dependent regulation of HCN pacemaker channels by cyclic AMP: signaling through dynamic allosteric coupling

Signal transduction in neurons is a dynamic process, generally thought to be driven by transient changes in the concentration of second messengers. Here we describe a novel regulatory mechanism in which the dynamics of signaling through cyclic AMP are mediated by activity-dependent changes in the affinity of the hyperpolarization-activated, cation nonselective (HCN) channels for cAMP, rather than by changes in cAMP concentration. Due to the allosteric coupling of channel opening and ligand binding, changes in cellular electrical activity that alter the opening of the HCN channels modify the binding of static, basal levels of cAMP. These changes in ligand binding produce long-lasting changes in channel function which can contribute to the regulation of rhythmic firing patterns.     

Cellular context and multiple channel domains determine cAMP sensitivity of HCN4 channels: Ligand-independent relief of autoinhibition in HCN4

Hyperpolarization-activated, cyclic nucleotide–sensitive (HCN) channels produce the I_f and I_h currents, which are critical for cardiac pacemaking and neuronal excitability, respectively. HCN channels are modulated by cyclic AMP (cAMP), which binds to a conserved cyclic nucleotide–binding domain (CNBD) in the C terminus. The unliganded CNBD has been shown to inhibit voltage-dependent gating of HCNs, and cAMP binding relieves this “autoinhibition,” causing a depolarizing shift in the voltage dependence of activation. Here we report that relief of autoinhibition can occur in the absence of cAMP in a cellular context- and isoform-dependent manner: when the HCN4 isoform was expressed in Chinese hamster ovary (CHO) cells, the basal voltage dependence was already shifted to more depolarized potentials and cAMP had no further effect on channel activation. This “pre-relief” of autoinhibition was specific both to HCN4 and to CHO cells; cAMP shifted the voltage dependence of HCN2 in CHO cells and of HCN4 in human embryonic kidney (HEK) cells. The pre-relief phenotype did not result from different concentrations of soluble intracellular factors in CHO and HEK cells, as it persisted in excised cell-free patches. Likewise, it did not arise from a failure of cAMP to bind to the CNBD of HCN4 in CHOs, as indicated by cAMP-dependent slowing of deactivation. Instead, a unique ∼300–amino acid region of the distal C terminus of HCN4 (residues 719–1012, downstream of the CNBD) was found to be necessary, but not sufficient, for the depolarized basal voltage dependence and cAMP insensitivity of HCN4 in CHO cells. Collectively, these data suggest a model in which multiple HCN4 channel domains conspire with membrane-associated intracellular factors in CHO cells to relieve autoinhibition in HCN4 channels in the absence of cAMP. These findings raise the possibility that such ligand-independent regulation could tune the activity of HCN channels and other CNBD-containing proteins in many physiological systems.     

cAMP Control of HCN2 Channel Mg2+ Block Reveals Loose Coupling between the Cyclic Nucleotide-Gating Ring and the Pore

Hyperpolarization-activated cyclic nucleotide-regulated HCN channels underlie the Na⁺-K⁺ permeable I_H pacemaker current. As with other voltage-gated members of the 6-transmembrane K_V channel superfamily, opening of HCN channels involves dilation of a helical bundle formed by the intracellular ends of S6 albeit this is promoted by inward, not outward, displacement of S4. Direct agonist binding to a ring of cyclic nucleotide-binding sites, one of which lies immediately distal to each S6 helix, imparts cAMP sensitivity to HCN channel opening. At depolarized potentials, HCN channels are further modulated by intracellular Mg²⁺ which blocks the open channel pore and blunts the inhibitory effect of outward K⁺ flux. Here, we show that cAMP binding to the gating ring enhances not only channel opening but also the kinetics of Mg²⁺ block. A combination of experimental and simulation studies demonstrates that agonist acceleration of block is mediated via acceleration of the blocking reaction itself rather than as a secondary consequence of the cAMP enhancement of channel opening. These results suggest that the activation status of the gating ring and the open state of the pore are not coupled in an obligate manner (as required by the often invoked Monod-Wyman-Changeux allosteric model) but couple more loosely (as envisioned in a modular model of protein activation). Importantly, the emergence of second messenger sensitivity of open channel rectification suggests that loose coupling may have an unexpected consequence: it may endow these erstwhile “slow” channels with an ability to exert voltage and ligand-modulated control over cellular excitability on the fastest of physiologically relevant time scales.     

Conformational Flip of Nonactivated HCN2 Channel Subunits Evoked by Cyclic Nucleotides

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

Subunits act independently in a cyclic nucleotide-activated K(+) channel

Ion channels gated by cyclic nucleotides have crucial roles in neuronal excitability and signal transduction of sensory neurons. Here, we studied ligand binding of a cyclic nucleotide-activated K(+) channel from Mesorhizobium loti and its isolated cyclic nucleotide-binding domain. The channel and the binding domain alone bind cyclic AMP with similar affinity in a non-cooperative manner. The cAMP sensitivities of binding and activation coincide. Thus, each subunit in the tetrameric channel acts independently of the others. The binding and gating properties of the bacterial channel are distinctively different from those of eukaryotic cyclic nucleotide-gated channels.     

PKA-independent activation of If by cAMP in mouse sinoatrial myocytes

Hyperpolarization-activated, cyclic nucleotide-sensitive (HCN4) channels produce the “funny current,” I_f, which contributes to spontaneous pacemaking in sinoatrial myocytes (SAMs). The C-terminus of HCN channels inhibits voltage-dependent gating, and cAMP binding relieves this “autoinhibition.” We previously showed 1) that autoinhibition in HCN4 can be relieved in the absence of cAMP in some cellular contexts and 2) that PKA is required for β adrenergic receptor (βAR) signaling to HCN4 in SAMs. Together, these results raise the possibility that native HCN channels in SAMs may be insensitive to direct activation by cAMP. Here, we examined PKA-independent activation of I_f by cAMP in SAMs. We observed similar robust activation of I_f by exogenous cAMP and Rp-cAMP (an analog than cannot activate PKA). Thus PKA-dependent βAR-to-HCN signaling does not result from cAMP insensitivity of sinoatrial HCN channels and might instead arise via PKA-dependent limitation of cAMP production and/or cAMP access to HCN channels in SAMs.     

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.     

Voltage sensor movement and cAMP binding allosterically regulate an inherently voltage-independent closed-open transition in HCN channels

The hyperpolarization-activated cyclic nucleotide-modulated cation (HCN) channels are regulated by both membrane voltage and the binding of cyclic nucleotides to a cytoplasmic, C-terminal cyclic nucleotide-binding domain (CNBD). Here we have addressed the mechanism of this dual regulation for HCN2 channels, which activate with slow kinetics that are strongly accelerated by cAMP, and HCN1 channels, which activate with rapid kinetics that are weakly enhanced by cAMP. Surprisingly, we find that the rate of opening of HCN2 approaches a maximal value with extreme hyperpolarization, indicating the presence of a voltage-independent kinetic step in the opening process that becomes rate limiting at very negative potentials. cAMP binding enhances the rate of this voltage-independent opening step. In contrast, the rate of opening of HCN1 is much greater than that of HCN2 and does not saturate with increasing hyperpolarization over the voltage range examined. Domain-swapping chimeras between HCN1 and HCN2 reveal that the S4-S6 transmembrane region largely determines the limiting rate in opening kinetics at negative voltages. Measurements of HCN2 tail current kinetics also reveal a voltage-independent closing step that becomes rate limiting at positive voltages; the rate of this closing step is decreased by cAMP. These results are consistent with a cyclic allosteric model in which a closed-open transition that is inherently voltage independent is subject to dual allosteric regulation by voltage sensor movement and cAMP binding. This mechanism accounts for several properties of HCN channel gating and has potentially important physiological implications.