Hyperpolarization-activated Cyclic Nucleotide-regulated Channels Research Articles

CryoEM structure of a prokaryotic cyclic nucleotide-gated ion channel

Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-regulated (HCN) ion channels play crucial physiological roles in phototransduction, olfaction, and cardiac pace making. These channels are characterized by the presence of a carboxyl-terminal cyclic nucleotide-binding domain (CNBD) that connects to the channel pore via a C-linker domain. Although cyclic nucleotide binding has been shown to promote CNG and HCN channel opening, the precise mechanism underlying gating remains poorly understood. Here we used cryoEM to determine the structure of the intact LliK CNG channel isolated from Leptospira licerasiae-which shares sequence similarity to eukaryotic CNG and HCN channels-in the presence of a saturating concentration of cAMP. A short S4-S5 linker connects nearby voltage-sensing and pore domains to produce a non-domain-swapped transmembrane architecture, which appears to be a hallmark of this channel family. We also observe major conformational changes of the LliK C-linkers and CNBDs relative to the crystal structures of isolated C-linker/CNBD fragments and the cryoEM structures of related CNG, HCN, and KCNH channels. The conformation of our LliK structure may represent a functional state of this channel family not captured in previous studies.

Crystal Structure of the Unliganded form of Cyclic Nucleotide Binding Domain (CNBD) from HCN2 Channel

Hyperpolarization-activated cyclic nucleotide-regulated channels (HCN) are allosterically regulated by cAMP via their cytoplasmic cyclic nucleotide binding domain (cNBD). Although the crystal structures of cAMP-bound form of cNBDs from several HCN channels have been solved, the exact mechanism of the allosteric regulation remains elusive and the differences in biochemical properties between cNBDs from different channels cannot be readily explained by the available structure. In part, this is because a unique conformation of the unliganded cNBD has not been previously crystallized. We now report a crystal structure of the apo form of cNBD from mouse HCN2 (residues 494-640) fused to maltose binding protein. The structure, obtained at 2.1 A resolution, clearly shows the protein in the open conformation of the cAMP binding pocket and suggests a plausible pathway for a conformational change upon ligand binding. A P-helix in the binding site retains its helical conformation in the crystal. All-atom molecular dynamics simulations over the course of 1 μs on the holo and apo crystal structures in the absence of cAMP support the notion that P-helix does not unwind in solution.

Dysfunction of Cortical Dendritic Integration in Neuropathic Pain Reversed by Serotoninergic Neuromodulation

Neuropathic pain is caused by long-term modifications of neuronal function in the peripheral nervous system, the spinal cord, and supraspinal areas. Although functional changes in the forebrain are thought to contribute to the development of persistent pain, their significance and precise subcellular nature remain unexplored. Using somatic and dendritic whole-cell patch-clamp recordings from neurons in the anterior cingulate cortex, we discovered that sciatic nerve injury caused an activity-dependent dysfunction of hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels in the dendrites of layer 5 pyramidal neurons resulting in enhanced integration of excitatory postsynaptic inputs and increased neuronal firing. Specific activation of the serotonin receptor type 7 (5-HT7R) alleviated the lesion-induced pathology by increasing HCN channel function, restoring normal dendritic integration, and reducing mechanical pain hypersensitivity in nerve-injured animals in vivo. Thus, serotoninergic neuromodulation at the forebrain level can reverse the dendritic dysfunction induced by neuropathic pain and may represent a potential therapeutical target.

HCN1 Channels as Targets for Anesthetic and Nonanesthetic Propofol Analogs in the Amelioration of Mechanical and Thermal Hyperalgesia in a Mouse Model of Neuropathic Pain

Chronic pain after peripheral nerve injury is associated with afferent hyperexcitability and upregulation of hyperpolarization-activated, cyclic nucleotide-regulated (HCN)-mediated IH pacemaker currents in sensory neurons. HCN channels thus constitute an attractive target for treating chronic pain. HCN channels are ubiquitously expressed; analgesics targeting HCN1-rich cells in the peripheral nervous system must spare the cardiac pacemaker current (carried mostly by HCN2 and HCN4) and the central nervous system (where all four isoforms are expressed). The alkylphenol general anesthetic propofol (2,6-di-iso-propylphenol) selectively inhibits HCN1 channels versus HCN2-HCN4 and exhibits a modest pharmacokinetic preference for the periphery. Consequently, we hypothesized that propofol, and congeners, should be antihyperalgesic. Alkyl-substituted propofol analogs have different rank-order potencies with respect to HCN1 inhibition, GABA(A) receptor (GABA(A)-R) potentiation, and general anesthesia. Thus, 2,6- and 2,4-di-tertbutylphenol (2,6- and 2,4-DTBP, respectively) are more potent HCN1 antagonists than propofol, whereas 2,6- and 2,4-di-sec-butylphenol (2,6- and 2,4-DSBP, respectively) are less potent. In contrast, DSBPs, but not DTBPs, enhance GABA(A)-R function and are general anesthetics. 2,6-DTBP retained propofol's selectivity for HCN1 over HCN2-HCN4. In a peripheral nerve ligation model of neuropathic pain, 2,6-DTBP and subhypnotic propofol are antihyperalgesic. The findings are consistent with these alkylphenols exerting analgesia via non-GABA(A)-R targets and suggest that antagonism of central HCN1 channels may be of limited importance to general anesthesia. Alkylphenols are hydrophobic, and thus potential modifiers of lipid bilayers, but their effects on HCN channels are due to direct drug-channel interactions because they have little bilayer-modifying effect at therapeutic concentrations. The alkylphenol antihyperalgesic target may be HCN1 channels in the damaged peripheral nervous system.

Evolutionary genomics reveals the premetazoan origin of opposite gating polarity in animal-type voltage-gated ion channels

Electrical signaling in animals ensures the rapid and accurate transmission of information, often carried by voltage-gated Na+, Ca2+ and K+ channels that are activated by membrane depolarization. In heart and neurons, a distinct type of ion channel called the hyperpolarization-activated, cyclic nucleotide-regulated (HCN) channel is activated by membrane hyperpolarization. Recent genomic studies have revealed that animal-type voltage-gated Na+ channels (Liebeskind BJ, et al. 2011. Proc Natl Acad Sci U S A. 108:9154) had evolved in choanoflagellates, one of the unicellular relatives of animals. To date, HCN channels have been considered to be animal-specific. Here, we demonstrate the presence of an HCN channel homolog (SroHCN) in the choanoflagellate protist Salpingoeca rosetta. SroHCN contains highly conserved functional domains and sequence motifs that are correlated with the unique biophysical activities of HCN channels. These findings provide novel genomic insights into the evolution of complex electrical signaling before the emergence of multicellular animals.

Fisetin Regulation of HCN2 and Eag1 Channels Reveals Conserved Gating Mechanisms

Hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels are pace-making channels that contribute to rhythmic activity in the heart and brain. All HCN-family channels contain a large intracellular domain in their carboxy terminal region which includes a cyclic nucleotide binding domain (CNBD) and a C-linker that connects it to the pore. We recently reported that the flavonoids luteolin and myricetin regulate Eag1 channels by binding a domain in their carboxy-terminal region that shares sequence and structural similarity with the CNBD of HCN2. In a screen of flavonoids, we identified fisetin as another potent regulator of Eag1. Fisetin shifted the voltage-dependence of Eag1 channels toward more hyperpolarizing potentials and slowed channel deactivation. Here we demonstrate that fisetin also regulates the kinetic and steady state properties of HCN2 channels. We found that 30 μM fisetin sped activation at −140 mV, decreasing the time constant from 1.17 ± 0.10 s to 0.80 ± 0.8 s (N=10). Additionally, 30 μM fisetin shifted the Vhalf of the conductance-voltage relationship from −127.8 ± 0.7 mV, to −120.4 ± 0.7 mV (N=10). Fisetin shifted the voltage-dependence of Eag and HCN channels in opposite directions, indicating that it is not simply interacting with the voltage sensor. Furthermore we found that fisetin and cAMP had non-additive effects on HCN2 channel gating. Using a FRET-based binding assay, we showed that fisetin bound to the purified CNBD of HCN2. Additionally, fisetin did not speed activation or shift the voltage-dependence of HCN2 channels lacking their CNBD. Together these data indicate that the CNBD mediates fisetin regulation of HCN2 channels. Based on the similar regulation of Eag and HCN channels by fisetin we propose that the mechanisms underlying ligand-mediated gating is well-conserved amongst these channels.

Association of adaptor protein TRIP8b with clathrin

TPR-containing Rab8b-interacting protein (TRIP8b) is a brain-specific hydrophilic cytosolic protein that contains tetratricopeptide repeats (TPRs). Previous studies revealed interaction of this protein via its TPR-containing domain with Rab8b small GTPase, hyperpolarization-activated cyclic nucleotide-regulated channel (HCN) channels and G protein-coupled receptor calcium-independent receptor of α-latrotoxin. We identified clathrin as a major component of eluates from the TRIP8b affinity matrix. In the present study, by in vitro-binding analysis we demonstrate a direct interaction between clathrin and TRIP8b. The clathrin-binding site was localized in the N-terminal (non-TPR containing) part of the TRIP8b molecule that contains two short motifs involved in the clathrin binding. In transfected HEK293 cells, co-expression of HCN1 with TRIP8b resulted in translocation of the channels from the cell surface to large intracellular puncta where both TRIP8b and clathrin were concentrated. These puncta co-localized partially with an early endosome marker and strongly overlapped with lysosome staining reagent. When HCN1 was co-expressed with a clathrin-non-binding mutant of TRIP8b, clathrin did not translocate to HCN1 and TRIP8b-containing puncta, suggesting that TRIP8b interacts with HCN and clathrin independently. We found TRIP8b present in the fraction of clathrin-coated vesicles purified from brain tissues. Stripping the clathrin coat proteins from the vesicles with Tris alkaline buffer resulted in concomitant release of TRIP8b. Our data suggest complex regulatory functions of TRIP8b in neuronal endocytosis through independent interaction with membrane proteins and components of the clathrin coat.

Can biologic pacemakers respond to physiologic emotional arousal?

Can biologic pacemakers respond to physiologic emotional arousal?

TRIP8b Splice Variants Form a Family of Auxiliary Subunits that Regulate Gating and Trafficking of HCN Channels in the Brain

Hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels, which generate the I(h) current, mediate a number of important brain functions. The HCN1 isoform regulates dendritic integration in cortical pyramidal neurons and provides an inhibitory constraint on both working memory in prefrontal cortex and spatial learning and memory in the hippocampus. Altered expression of HCN1 following seizures may contribute to the development of temporal lobe epilepsy. Yet the regulatory networks and pathways governing HCN channel expression and function in the brain are largely unknown. Here, we report the presence of nine alternative N-terminal splice forms of the brain-specific cytoplasmic protein TRIP8b and demonstrate the differential effects of six isoforms to downregulate or upregulate HCN1 surface expression. Furthermore, we find that all TRIP8b isoforms inhibit channel opening by shifting activation to more negative potentials. TRIP8b thus functions as an auxiliary subunit that provides a mechanism for the dynamic regulation of HCN1 channel expression and function.

Pathway and Endpoint Free Energy Calculations for Cyclic Nucleotide Binding to HCN Channels

cAMP and cGMP differentially bind to and regulate a variety of proteins, including cyclic nucleotide-gated (CNG) channels and hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels. Previous site-directed mutagenesis studies have isolated two conserved residues that are critical for enabling certain channels to selectively bind cGMP relative to cAMP. However, no definitive mechanism has been identified that explains the preferential activation of other channels by cAMP. Here we apply computational binding free energy methods, including thermodynamic integration, linear interaction energy, and continuum electrostatic calculations, to gain insights into the mechanisms of cyclic nucleotide selectivity. Consistent with experimental observations, computational results for the cAMP-selective HCN channels show that the binding free energy of cAMP is lower (more favorable) than that of cGMP. Surprisingly, cAMP selectivity is not due to its preferential contacts with protein, but rather reflects the greater hydration energy of cGMP relative to cAMP, resulting in a greater energetic cost for cGMP binding.

Propofol inhibits HCN1 pacemaker channels by selective association with the closed states of the membrane embedded channel core

Activation of native IH pacemaker channels and channels formed on heterologous expression of some isoforms of their pore forming HCN (hyperpolarization-activated, cyclic nucleotide-regulated) subunits is inhibited by the intravenous general anaesthetic propofol (2,6-diisopropylphenol). Here, we show that inhibition of homomeric HCN1 channels is mediated through anaesthetic association with the membrane embedded channel core, a domain that is highly conserved between this isoform and the relatively insensitive HCN2 and 4 subunits. Decoupling of HCN channel gating from cAMP and internal protons reveals that changes in these second messengers are neither necessary nor sufficient to account for propofol's actions. Modelling of the equilibrium and kinetic behaviour of HCN1 channels in the absence and presence of anaesthetic reveals that (1) gating is best described by models wherein closed and open states communicate via a voltage-independent reaction with no significant equilibrium occupancy of a deactivated open state at non-permissive voltages, and (2) propofol modifies gating by preferentially associating with closed-resting and closed-activated states but a low affinity interaction with the activated open state shapes the effect of the drug under physiological conditions. Our findings illuminate the mechanism of HCN channel gating and provide a framework that will facilitate development of propofol derivates that have altered pharmacological properties and therapeutic potentials.

Modulation of cyclic nucleotide-regulated HCN channels by PIP2 and receptors coupled to phospholipase C

Recent results indicate that phosphoinositides, including phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), directly enhance the opening of hyperpolarization-activated, cyclic nucleotide-regulated (HCN) channels by shifting their activation gating to more positive voltages. This contrasts with the action of phosphoinositides to inhibit the opening of the related cyclic nucleotide-gated (CNG) channels involved in sensory signaling. We both review previous studies and present new experiments that investigate whether HCN channels may be regulated by dynamic changes in PI(4,5)P(2) levels caused by the receptor-mediated activation of phospholipase C (PLC). We coexpressed HCN1 or HCN2 channels in Xenopus oocytes with the PLC-coupled bradykinin BK(2) receptor, the muscarinic M1 receptor, or the TrkA receptor. Activation of all three receptors produced a positive shift in HCN channel voltage gating, the opposite of the effect expected for PI(4,5)P(2) depletion. This action was not caused by alterations in cAMP as the effect was preserved in HCN mutant channels that fail to bind cAMP. The receptor effects were mediated by PLC activity, but did not depend on signaling through the downstream products of PI(4,5)P(2) hydrolysis: IP(3) or diacylglycerol (DAG). Importantly, the modulatory effects on gating were blocked by inhibitors of phosphatidylinositol (PI) kinases, suggesting a role for increased PI(4,5)P(2) synthesis. Finally, we found that bradykinin exerted a similar PI kinase-dependent effect on the gating of native HCN channels in cardiac sinoatrial node cells, suggesting that this pathway may represent a novel, physiologically relevant mechanism for enhancing HCN channel function.

Presynaptic effectors contributing to cAMP-induced synaptic potentiation inDrosophila

cAMP analogs and activation of adenylyl cyclase by forskolin strongly potentiate synaptic transmission at the Drosophila neuromuscular junction. These effects are generally attributed to activation of cAMP-dependent protein kinase. Recent reports on crustacean and mammalian synapses have implicated other cAMP-dependent effectors in synaptic potentiation. Drosophila neuromuscular junctions were tested for effects of two known cAMP-dependent effectors: hyperpolarization-activated, cyclic nucleotide-regulated channels (HCNCs) and guanine nucleotide exchange protein activated by cAMP (Epac). Forskolin-induced enhancement of synaptic transmission was drastically reduced by a blocker of HCNCs, but not completely eliminated. A specific agonist for Epac modestly enhanced synaptic potentials. This agonist also stabilized their amplitudes in the presence of a blocker of HCNCs. The observations implicate HCNCs and Epac in cAMP-dependent potentiation that does not require cAMP-dependent protein kinase, indicating that additional previously unexplored factors contribute to synaptic plasticity in Drosophila. Genetic and molecular techniques available for Drosophila can be used to define the underlying molecular basis for cAMP-dependent synaptic potentiation.