A conserved domain in the NH2 terminus important for assembly and functional expression of pacemaker channels

Pacemaker channels are formed by co-assembly of hyperpolarization-activated cyclic nucleotide-gated (HCN) subunits. Previously, we suggested that the NH(2) termini of the mouse HCN2 isoform were important for subunit co-assembly and functional channel expression. Using an alignment strategy together with yeast two-hybrid assays, patch clamp electrophysiology, and confocal imaging, we have now identified a domain within the NH(2) terminus of the HCN2 subunit that is responsible for interactions between NH(2) termini and promoting the trafficking of functional channels to the plasma membrane. This domain is composed of 52 amino acids, is located adjacent to the putative first transmembrane segment, and is highly conserved among the mammalian HCN isoforms. This conserved domain, but not the remaining unconserved NH(2)-terminal regions of HCN2, specifically interacted with itself in yeast two-hybrid assays. Moreover, the conserved domain was important for expression of currents. Whereas relatively normal whole cell HCN2 currents were produced by channels containing only the conserved domain, further deletion of this region, leaving only a more polar and putative coiled-coil segment, eliminated HCN2 currents and resulted in proteins that localized predominantly in perinuclear compartments. Thus, we suggest that this conserved domain is the critical NH(2)-terminal determinant of subunit co-assembly and trafficking of pacemaker channels.     

Tetramerization dynamics of C-terminal domain underlies isoform-specific cAMP gating in hyperpolarization-activated cyclic nucleotide-gated channels

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are dually activated by hyperpolarization and binding of cAMP to their cyclic nucleotide binding domain (CNBD). HCN isoforms respond differently to cAMP; binding of cAMP shifts activation of HCN2 and HCN4 by 17 mV but shifts that of HCN1 by only 2-4 mV. To explain the peculiarity of HCN1, we solved the crystal structures and performed a biochemical-biophysical characterization of the C-terminal domain (C-linker plus CNBD) of the three isoforms. Our main finding is that tetramerization of the C-terminal domain of HCN1 occurs at basal cAMP concentrations, whereas those of HCN2 and HCN4 require cAMP saturating levels. Therefore, HCN1 responds less markedly than HCN2 and HCN4 to cAMP increase because its CNBD is already partly tetrameric. This is confirmed by voltage clamp experiments showing that the right-shifted position of V(½) in HCN1 is correlated with its propensity to tetramerize in vitro. These data underscore that ligand-induced CNBD tetramerization removes tonic inhibition from the pore of HCN 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.     

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.     

Role of subunit heteromerization and N-linked glycosylation in the formation of functional hyperpolarization-activated cyclic nucleotide-gated channels

The coassembly of homologous subunits to heteromeric complexes serves as an important mechanism in generating ion channel diversity. Here, we have studied heteromerization in the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel family. Using a combination of fluorescence confocal microscopy, coimmunoprecipitation, and electrophysiology we found that upon coexpression in HEK293 cells almost all dimeric combinations of HCN channel subunits give rise to the formation of stable channel complexes in the plasma membrane. We also identified HCN1/HCN2 heteromers in mouse brain indicating that heteromeric channels exist in vivo. Surprisingly, HCN2 and HCN3 did not coassemble to heteromeric channels. This finding indicates that heteromerization requires specific structural determinants that are not present in all HCN channel combinations. Using N-glycosidase F we show that native as well as recombinant HCN channels are glycosylated resulting in a 10-20-kDa shift in the molecular weight. Tunicamycin, an inhibitor of N-linked glycosylation, blocked surface membrane expression of HCN2. Similarly, a mutant HCN2 channel in which the putative N-glycosylation site in the loop between S5 and the pore helix was replaced by glutamine (HCN2N380Q) was not inserted into the plasma membrane and did not yield detectable whole-cell currents. These results indicate that N-linked glycosylation is required for cell surface trafficking of HCN channels. Cotransfection of HCN2N380Q with HCN4, but not with HCN3, rescued cell surface expression of HCN2N380Q. Immunoprecipitation revealed that this rescue was due to the formation of a HCN2N380Q/HCN4 heteromeric channel. Taken together our results indicate that subunit heteromerization and glycosylation are important determinants of the formation of native HCN channels.     

Hyperpolarization-activated and cyclic nucleotide-gated channels are differentially expressed in juxtaglomerular cells in the olfactory bulb of mice

In the olfactory bulb, input from olfactory receptor neurons is processed by neuronal networks before it is relayed to higher brain regions. In many neurons, hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels generate and control oscillations of the membrane potential. Oscillations also appear crucial for information processing in the olfactory bulb. Four channel isoforms exist (HCN1–HCN4) that can form homo- or heteromers. Here, we describe the expression pattern of HCN isoforms in the olfactory bulb of mice by using a novel and comprehensive set of antibodies against all four isoforms. HCN isoforms are abundantly expressed in the olfactory bulb. HCN channels can be detected in most cell populations identified by commonly used marker antibodies. The combination of staining with marker and HCN antibodies has revealed at least 17 different staining patterns in juxtaglomerular cells. Furthermore, HCN isoforms give rise to an unexpected wealth of co-expression patterns but are rarely expressed in the same combination and at the same level in two given cell populations. Therefore, heteromeric HCN channels may exist in several cell populations in vivo. Our results suggest that HCN channels play an important role in olfactory information processing. The staining patterns are consistent with the possibility that both homomeric and heteromeric HCN channels are involved in oscillations of the membrane potential of juxtaglomerular cells.     

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.     

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.     

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.     

Activity-dependent heteromerization of the hyperpolarization-activated, cyclic-nucleotide gated (HCN) channels: role of N-linked glycosylation

Formation of heteromeric complexes of ion channels via co-assembly of different subunit isoforms provides an important mechanism for enhanced channel diversity. We have previously demonstrated co-association of the hyperpolarization activated cyclic-nucleotide gated (HCN1/HCN2) channel isoforms that was regulated by network (seizure) activity in developing hippocampus. However, the mechanisms that underlie this augmented expression of heteromeric complexes have remained unknown. Glycosylation of the HCN channels has been implicated in the stabilization and membrane expression of heteromeric HCN1/HCN2 constructs in heterologous systems. Therefore, we used in vivo and in vitro systems to test the hypothesis that activity modifies HCN1/HCN2 heteromerization in neurons by modulating the glycosylation state of the channel molecules. Seizure-like activity (SA) increased HCN1/HCN2 heteromerization in hippocampus in vivo as well as in hippocampal organotypic slice cultures. This activity increased the abundance of glycosylated HCN1 but not HCN2-channel molecules. In addition, glycosylated HCN1 channels were preferentially co-immunoprecipitated with the HCN2 isoforms. Provoking SA in vitro in the presence of the N-linked glycosylation blocker tunicamycin abrogated the activity-dependent increase of HCN1/HCN2 heteromerization. Thus, hippocampal HCN1 molecules have a significantly higher probability of being glycosylated after SA, and this might promote a stable heteromerization with HCN2.     

Allosteric signaling in C-linker and cyclic nucleotide-binding domain of HCN2 channels

Opening of hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels is controlled by membrane hyperpolarization and binding of cyclic nucleotides to the tetrameric cyclic nucleotide-binding domain (CNBD), attached to the C-linker (CL) disk. Confocal patch-clamp fluorometry revealed pronounced cooperativity of ligand binding among protomers. However, by which pathways allosteric signal transmission occurs remained elusive. Here, we investigate how changes in the structural dynamics of the CL-CNBD of mouse HCN2 upon cAMP binding relate to inter- and intrasubunit signal transmission. Applying a rigidity-theory-based approach, we identify two intersubunit and one intrasubunit pathways that differ in allosteric coupling strength between cAMP-binding sites or toward the CL. These predictions agree with results from electrophysiological and patch-clamp fluorometry experiments. Our results map out distinct routes within the CL-CNBD that modulate different cAMP-binding responses in HCN2 channels. They signify that functionally relevant submodules may exist within and across structurally discernable subunits in HCN channels.     

Salt bridges and gating in the COOH-terminal region of HCN2 and CNGA1 channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels and cyclic nucleotide-gated (CNG) channels are activated by the direct binding of cyclic nucleotides. The intracellular COOH-terminal regions exhibit high sequence similarity in all HCN and CNG channels. This region contains the cyclic nucleotide-binding domain (CNBD) and the C-linker region, which connects the CNBD to the pore. Recently, the structure of the HCN2 COOH-terminal region was solved and shown to contain intersubunit interactions between C-linker regions. To explore the role of these intersubunit interactions in intact channels, we studied two salt bridges in the C-linker region: an intersubunit interaction between C-linkers of neighboring subunits, and an intrasubunit interaction between the C-linker and its CNBD. We show that breaking these salt bridges in both HCN2 and CNGA1 channels through mutation causes an increase in the favorability of channel opening. The wild-type behavior of both HCN2 and CNGA1 channels is rescued by switching the position of the positive and negative residues, thus restoring the salt bridges. These results suggest that the salt bridges seen in the HCN2 COOH-terminal crystal structure are also present in the intact HCN2 channel. Furthermore, the similar effects of the mutations on HCN2 and CNGA1 channels suggest that these salt bridge interactions are also present in the intact CNGA1 channel. As disrupting the interactions leads to channels with more favorable opening transitions, the salt bridges appear to stabilize a closed conformation in both the HCN2 and CNGA1 channels. These results suggest that the HCN2 COOH-terminal crystal structure contains the C-linker regions in the resting configuration even though the CNBD is ligand bound, and channel opening involves a rearrangement of the C-linkers and, thus, disruption of the salt bridges. Discovering that one portion of the COOH terminus, the CNBD, can be in the activated configuration while the other portion, the C-linker, is not activated has lead us to suggest a novel modular gating scheme for HCN and CNG channels.     

MAGI-1a functions as a scaffolding protein for the distal renal tubular basolateral K+ channels

As the K(+) recycling pathway for renal Na(+) reabsorption, renal tubular K(+) channels participate in the fluid and electrolyte homeostasis. Previously, we showed that the Kir5.1/Kir4.1 heteromer, which is a heteromeric assembly of two inwardly rectifying K(+) channels, composes the principal basolateral K(+) channels in distal renal tubules and that two motifs in the carboxyl-terminal portion of the Kir4.1 subunit regulate its functional expression. In this study, by using yeast two-hybrid screening, we identified a new isoform of membrane-associated guanylate kinase with inverted domain structure 1 (MAGI-1a-long) as a scaffolding protein for the basolateral K(+) channels. MAGI-1a-long interacted with the PSD-95/Dlg/ZO-1 (PDZ)-binding motif of Kir4.1 by its fifth PDZ domain, and a high salt diet, which could suppress mineralocorticoid secretion, facilitated the interaction. The phosphorylation of serine 377 in the PDZ-binding motif disrupted the interaction, and the disruption of the interaction altered the intracellular localization of the channels from the basolateral side to perinuclear components. These results demonstrate that the phosphorylation-dependent scaffolding of the basolateral K(+) channels by MAGI-1a-long participates in the renal regulation of the fluid and electrolyte homeostasis.     

Resonance assignments of the nucleotide-free wildtype MloK1 cyclic nucleotide-binding domain

Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels play crucial roles in neuronal excitability and signal transduction of sensory cells. These channels are activated by binding of cyclic nucleotides to their intracellular cyclic nucleotide-binding domain (CNBD). A comparison of the structures of wildtype ligand-free and ligand-bound CNBD is essential to elucidate the mechanism underlying nucleotide-dependent activation of CNBDs. We recently reported the solution structure of the Mesorhizobium loti K1 (MloK1) channel CNBD in complex with cAMP. We have now extended these studies and achieved nearly complete assignments of ¹H, ¹³C and ¹⁵N resonances of the nucleotide-free CNBD. A completely new assignment of the nucleotide-free wildtype CNBD was necessary due to the sizable chemical shift differences as compared to the cAMP bound CNBD and the slow exchange behaviour between both forms. Scattering of these chemical shift differences over the complete CNBD suggests that nucleotide binding induces significant overall conformational changes.     

Analysis of the cyclic nucleotide binding domain of the HERG potassium channel and interactions with KCNE2

Mutations in the cyclic nucleotide binding domain (CNBD) of the human ether-a-go-go-related gene (HERG) K+ channel are associated with LQT2, a form of hereditary Long QT syndrome (LQTS). Elevation of cAMP can modulate HERG K+ channels both by direct binding and indirect regulation through protein kinase A. To assess the physiological significance of cAMP binding to HERG, we introduced mutations to disrupt the cyclic nucleotide binding domain. Eight mutants including two naturally occurring LQT2 mutants V822M and R823W were constructed. Relative cAMP binding capacity was reduced or absent in CNBD mutants. Mutant homotetramers carry little or no K+ current despite normal protein abundance and surface expression. Co-expression of mutant and wild-type HERG resulted in currents with altered voltage dependence but without dominant current suppression. The data from co-expression of V822M and wild-type HERG best fit a model where one normal subunit within a tetramer allows nearly normal current expression. The presence of KCNE2, an accessory protein that associates with HERG, however, conferred a partially dominant current suppression by CNBD mutants. Thus KCNE2 plays a pivotal role in determining the phenotypic severity of some forms of LQT2, which suggests that the CNBD of HERG may be involved in its interaction with KCNE2.     

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.     

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.     

A novel dimerization interface of cyclic nucleotide binding domain, which is disrupted in presence of cAMP: implications for CNG channels gating

Cyclic nucleotide binding domain (CNBD) is a ubiquitous domain of effector proteins involved in signalling cascades of prokaryota and eukaryota. CNBD activation by cyclic nucleotide monophosphate (cNMP) is studied well in the case of several proteins. However, this knowledge is hardly applicable to cNMP-modulated cation channels. Despite the availability of CNBD crystal structures of bacterial cyclic nucleotide-gated (CNG) and mammalian hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels in presence and absence of the cNMP, the full understanding of CNBD conformational changes during activation is lacking. Here, we describe a novel CNBD dimerization interface found in crystal structures of bacterial CNG channel MlotiK1 and mammalian cAMP-activated guanine nucleotide-exchange factor Epac2. Molecular dynamics simulations show that the found interface is stable on the studied timescale of 100?ns, in contrast to the dimerization interface, reported previously. Comparisons with cN-bound structures of CNBD show that the dimerization is incompatible with cAMP binding. Thus, the cAMP-dependent monomerization of CNBD may be an alternative mechanism of the cAMP sensing. Based on these findings, we propose a model of the bacterial CNG channel modulation by cAMP.     

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.     

Characterisation of a novel gene family of putative cyclic nucleotide- and calmodulin-regulated ion channels in Arabidopsis thaliana

In plants, cyclic GMP is involved in signal transduction in response to light and gibberellic acid. For cyclic AMP, a potential role during the plant cell cycle was recently reported. However, cellular targets for cyclic nucleotides in plants are largely unknown. Here we report on the identification and characterisation of a new gene family in Arabidopsis, which share features with cyclic nucleotide-gated channels from animals and inward-rectifying K⁺ channels from plants. The identified gene family comprises six members (Arabidopsis thaliana cyclic nucleotide-gated channels, AtCNGC1–6) with significant homology among the deduced proteins. Hydrophobicity analysis predicted six membrane-spanning domains flanked by hydrophilic amino and carboxy termini. A putative cyclic nucleotide binding domain (CNBD) which contains several residues that are invariant in other CNBDs was located in the carboxy terminus. This domain overlaps with a predicted calmodulin (CaM) binding site, suggesting interaction between cyclic nucleotide and CaM regulation. We demonstrated interaction of the carboxy termini of AtCNGC1 and AtCNGC2 with CaM in yeast, indicating that the CaM binding sites are functional. Furthermore, it was shown that both AtCNGC1 and AtCNGC2 can partly complement the K⁺-uptake-deficient yeast mutant CY162. Therefore, we propose that the identified genes constitute a family of plant cyclic nucleotide- and CaM-regulated ion channels.