ABSTRACT
Hyperpolarization-activated cation-nonselective (HCN) channels regulate electrical activity in the brain and heart in a cAMP-dependent manner. The voltage-gating of these channels is mediated by a transmembrane (TM) region but is additionally regulated by direct binding of cAMP to a cyclic nucleotide-binding (CNB) fold in the cytoplasmic C-terminal region. Cyclic AMP potentiation has been explained by an autoinhibition model which views the unliganded CNB fold as an inhibitory module whose influence is disrupted by cAMP binding. However, the HCN2 subtype uses two other CNB fold-mediated mechanisms called open-state trapping and Quick-Activation to respectively slow the deactivation kinetics and speed the activation kinetics, against predictions of an autoinhibition model. To test how these multiple mechanisms are influenced by the TM region, we replaced the TM region of HCN2 with that of HCN4. This HCN4 TM-replacement preserved cAMP potentiation but augmented the magnitude of autoinhibition by the unliganded CNB fold; it moreover disrupted open-state trapping and Quick-Activation so that autoinhibition became the dominant mechanism contributed by the C-terminal region to determine kinetics. Truncation within the CNB fold partially relieved this augmented autoinhibition. This argues against the C-terminal region acting like a portable module with consistent effects on TM regions of different subtypes. Our findings provide evidence that functional interactions between the HCN2 TM region and C-terminal region govern multiple CNB fold-mediated mechanisms, implying that the molecular mechanisms of autoinhibition, open-state trapping, and Quick-Activation include participation of TM region structures.
Subject(s)
Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/metabolism , Protein Interaction Domains and Motifs , Cell Membrane/metabolism , Cyclic AMP/metabolism , Cytoplasm/metabolism , Electrophysiological Phenomena , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/agonists , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/antagonists & inhibitors , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/chemistry , Ion Channel Gating , Kinetics , Ligands , Protein BindingABSTRACT
cAMP acts as a second messenger in many cellular processes. Three protein types mainly mediate cAMP-induced effects: PKA, exchange protein directly activated by cAMP (Epac), and cyclic nucleotide-modulated channels (cyclic nucleotide-gated or hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels). Discrimination among these cAMP signaling pathways requires specific targeting of only one protein. Previously, cAMP modifications at position N6 of the adenine ring (PKA) and position 2'-OH of the ribose (Epac) have been used to produce target-selective compounds. However, cyclic nucleotide-modulated ion channels were usually outside of the scope of these previous studies. These channels are widely distributed, so possible channel cross-activation by PKA- or Epac-selective agonists warrants serious consideration. Here we demonstrate the agonistic effects of three PKA-selective cAMP derivatives, N6-phenyladenosine-3',5'-cyclic monophosphate (N6-Phe-cAMP), N6-benzyladenosine-3',5'-cyclic monophosphate (N6-Bn-cAMP), and N6-benzoyl-adenosine-3',5'-cyclic monophosphate (N6-Bnz-cAMP), on murine HCN2 pacemaker channels. Electrophysiological characterization in Xenopus oocytes revealed that these derivatives differ in apparent affinities depending on the modification type but that their efficacy and effects on HCN2 activation kinetics are similar to those of cAMP. Docking experiments suggested a pivotal role of Arg-635 at the entrance of the binding pocket in HCN2, either causing stabilizing cation-π interactions with the aromatic ring in N6-Phe-cAMP or N6-Bn-cAMP or a steric clash with the aromatic ring in N6-Bnz-cAMP. A reduced apparent affinity of N6-Phe-cAMP toward the variants R635A and R635E strengthened that notion. We conclude that some PKA activators also effectively activate HCN2 channels. Hence, when studying PKA-mediated cAMP signaling with cAMP derivatives in a native environment, activation of HCN channels should be considered.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP/metabolism , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/agonists , Amino Acid Sequence , Amino Acid Substitution , Animals , Arginine/metabolism , Binding Sites , Enzyme Activation , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/chemistry , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/metabolism , Ion Channel Gating , Kinetics , Ligands , Mice , Molecular Docking Simulation , Oocytes/metabolism , XenopusABSTRACT
Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are tetrameric non-specific cation channels in the plasma membrane that are activated by either cAMP or cGMP binding to specific binding domains incorporated in each subunit. Typical apparent affinities of these channels for these cyclic nucleotides range from several hundred nanomolar to tens of micromolar. Here we synthesized and characterized novel cAMP and cGMP derivatives by substituting either hydrophobic alkyl chains or similar-sized more hydrophilic heteroalkyl chains to the 8-position of the purine ring with the aim to obtain full agonists of higher potency. The compounds were tested in homotetrameric CNGA2, heterotetrameric CNGA2:CNGA4:CNGB1b and homotetrameric HCN2 channels. We show that nearly all compounds are full agonists and that longer alkyl chains systematically increase the apparent affinity, at the best more than 30 times. The effects are stronger in CNG than HCN2 channels which, however, are constitutively more sensitive to cAMP. Kinetic analyses reveal that the off-rate is significantly slowed by the hydrophobic alkyl chains. Molecular dynamics simulations and free energy calculations suggest that an intricate enthalpy - entropy compensation underlies the higher apparent affinity of the derivatives with the longer alkyl chains, which is shown to result from a reduced loss of configurational entropy upon binding.
Subject(s)
Cyclic AMP/analogs & derivatives , Cyclic AMP/pharmacology , Cyclic GMP/analogs & derivatives , Cyclic GMP/pharmacology , Cyclic Nucleotide-Gated Cation Channels/agonists , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/agonists , Animals , Cyclic Nucleotide-Gated Cation Channels/metabolism , Entropy , Humans , Hydrophobic and Hydrophilic Interactions , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/metabolism , Mice , Molecular Dynamics Simulation , Rats , Thermodynamics , XenopusABSTRACT
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels have a key role in the control of cellular excitability. HCN2, a subgroup of the HCN family channels, are heavily expressed in small dorsal root ganglia (DRG) neurons and their activation seems to be important in the determination of pain intensity. Intracellular elevation of cAMP levels activates HCN-mediated current (Ih) and small DRG neurons excitability. GPR35, a Gi/o coupled receptor, is highly expressed in small DRG neurons, and we hypothesized that its activation, mediated by endogenous or exogenous ligands, could lead to pain control trough a reduction of Ih current. Patch clamp recordings were carried out in primary cultures of rat DRG neurons and the effects of GPR35 activation on Ih current and neuronal excitability were studied in control conditions and after adenylate cyclase activation with either forskolin or prostaglandin E2 (PGE2). We found that both kynurenic acid (KYNA) and zaprinast, the endogenous and synthetic GPR35 agonist respectively, were able to antagonize the forskolin-induced depolarization of resting membrane potential by reducing Ih-mediated depolarization. Similar results were obtained when PGE2 was used to activate adenylate cyclase and to increase Ih current and the overall neuronal excitability. Finally, we tested the analgesic effect of both GPR35 agonists in an in vivo model of PGE2-induced thermal hyperalgesia. In accord with the hypothesis, both KYNA and zaprinast showed a dose dependent analgesic effect. In conclusion, GPR35 activation leads to a reduced excitability of small DRG neurons in vitro and causes a dose-dependent analgesia in vivo. GPR35 agonists, by reducing adenylate cyclase activity and inhibiting Ih in DRG neurons may represent a promising new group of analgesic drugs.
Subject(s)
Analgesia/methods , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/physiology , Kynurenic Acid/therapeutic use , Purinones/therapeutic use , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/metabolism , Animals , Animals, Newborn , Cells, Cultured , Dose-Response Relationship, Drug , Ganglia, Spinal/drug effects , Ganglia, Spinal/metabolism , Hyperalgesia/drug therapy , Hyperalgesia/metabolism , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/agonists , Kynurenic Acid/pharmacology , Purinones/pharmacology , Rats , Rats, WistarABSTRACT
Bisoprolol, an antagonist of ß1-adrenergic receptors, is effective in reducing the morbidity and mortality in patients with heart failure (HF). It has been found that HF is accompanied with dysfunction of the sinoatrial node (SAN). However, whether bisoprolol reverses the decreased SAN function in HF and how the relevant ion channels in SAN change were relatively less studied. SAN function and messenger RNA (mRNA) expression of sodium channels and hyperpolarization-activated cyclic nucleotide-gated (HCN) channel subunits were assessed in sham-operated rats, abdominal arterio-venous shunt (volume overload)-induced HF rats, and bisoprolol- treated HF rats. SAN cells of rats were isolated by laser capture microdissection. Quantitative real-time PCR analysis was used to quantify mRNA expression of sodium channels and HCN channel subunits in SAN. Intrinsic heart rate declined and sinus node recovery time prolonged in HF rats, indicating the suppressed SAN function, which could be improved by bisoprolol treatment. Nav1.1, Nav1.6, and HCN4 mRNA expressions were reduced in SAN in HF rats compared with that in control rats. Treatment with bisoprolol could reverse both the SAN function and the Nav1.1, Nav1.6, and HCN4 mRNA expression partially. These data indicated that bisoprolol is effective in HF treatment partially due to improved SAN function by reversing the down-regulation of sodium channels (Nav1.1 and Nav1.6) and HCN channel (HCN4) subunits in SAN in failing hearts.
Subject(s)
Adrenergic beta-1 Receptor Antagonists/therapeutic use , Bisoprolol/therapeutic use , Cardiotonic Agents/therapeutic use , Disease Models, Animal , Heart Failure/drug therapy , Ion Channels/agonists , Sinoatrial Node/drug effects , Animals , Atrial Remodeling/drug effects , Gene Expression Regulation/drug effects , Heart Failure/metabolism , Heart Failure/pathology , Heart Failure/physiopathology , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/agonists , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/genetics , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/metabolism , Ion Channels/genetics , Ion Channels/metabolism , Laser Capture Microdissection , Male , NAV1.1 Voltage-Gated Sodium Channel/chemistry , NAV1.1 Voltage-Gated Sodium Channel/genetics , NAV1.1 Voltage-Gated Sodium Channel/metabolism , NAV1.6 Voltage-Gated Sodium Channel/chemistry , NAV1.6 Voltage-Gated Sodium Channel/genetics , NAV1.6 Voltage-Gated Sodium Channel/metabolism , Potassium Channels/agonists , Potassium Channels/genetics , Potassium Channels/metabolism , Protein Subunits/agonists , Protein Subunits/genetics , Protein Subunits/metabolism , RNA, Messenger/metabolism , Random Allocation , Rats, Sprague-Dawley , Sinoatrial Node/metabolism , Sinoatrial Node/pathology , Sinoatrial Node/physiopathologyABSTRACT
Neuronal hyperexcitability in the anterior cingulate cortex (ACC) is considered as one of the most important pathological changes responsible for the chronification of neuropathic pain. However, the underlying mechanisms remain elusive. In the present study, we investigated the possible mechanisms using a rat model of chronic constriction injury (CCI) to the sciatic nerve. We found a substantial decrease in hyperpolarization-activated/cyclic nucleotide-gated (HCN) currents in layer 5 pyramidal neurons (L5 PNs) in ACC slices, which dramatically increased the excitability of these neurons. This effect could be mimicked in sham slices by activating group 1 metabotropic glutamate receptors, and be blocked in CCI slices by inhibiting metabotropic glutamate receptor subtype 1 (mGluR1). Next, the inhibition of HCN currents was reversed by a protein kinase C (PKC) inhibitor, followed by a reduced neuronal hyperexcitability. Furthermore, HCN channel subtype 1 (HCN1) level was significantly reduced after CCI, whereas mGluR1 level increased. These changes were mainly observed in L5 of the ACC, where HCN1 and mGluR1 were highly colocalized. For behavioral tests, intra-ACC microinjection of mGluR1-shRNA suppressed the CCI-induced behavioral hypersensitivity, particularly thermal hyperalgesia, but not aversive behavior, and this effect was attenuated by the pre-blockade of HCN channels. Taken together, the neuronal hyperexcitability of ACC L5 PNs likely results from an upregulation of mGluR1 and a downstream pathway involving PKC activation and a downregulation of HCN1 in the early phase of neuropathic pain. These alterations may at least in part contribute to the development of behavioral hypersensitivity in CCI rats.
Subject(s)
Gyrus Cinguli/drug effects , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/drug effects , Neurons/drug effects , Potassium Channels/drug effects , Receptors, AMPA/agonists , Animals , Behavior, Animal/drug effects , Constriction, Pathologic/drug therapy , Enzyme Inhibitors/pharmacology , Gyrus Cinguli/cytology , Hyperalgesia/drug therapy , Hyperalgesia/psychology , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/agonists , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/antagonists & inhibitors , In Vitro Techniques , Male , Neuralgia/physiopathology , Patch-Clamp Techniques , Potassium Channels/agonists , Protein Kinase C/antagonists & inhibitors , Pyramidal Cells/drug effects , RNA, Small Interfering , Rats , Rats, Sprague-DawleyABSTRACT
The high-voltage spindles (HVSs), one of the characteristic oscillations that include theta frequencies in the basal ganglia (BG)-cortical system, are involved in immobile behavior and show increasing power in Parkinson's disease (PD). Our previous results suggested that the D2 dopamine receptor might be involved in HVSs modulations in a rat model of PD. Membrane resonance is one of the cellular mechanisms of network oscillation; therefore, we investigated how dopamine modulates the theta frequency membrane resonance of neurons in the subthalamic nucleus (STN), a central pacemaker of BG, and whether such changes in STN neurons subsequently alter HVSs in the BG-cortical system. In particular, we tested whether dopamine modulates HVSs through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels-dependent membrane resonance in STN neurons. We found that an antagonist of D2 receptors, but not of D1 receptors, inhibited membrane resonance and HCN currents of STN neurons through a G-protein activity in acute brain slices. Our further in vivo experiments using local injection of a D2 receptor antagonist or an HCN blocker in STNs of free-moving rats showed an increase in HVSs power and correlation in the BG-cortical system. Local injection of lamotrigine, an HCN agonist, counteracted the effect induced by the D2 antagonist. Taken together, our results revealed a potential cellular mechanism underlying HVSs activity modulation in the BG-cortical system, i.e. tuning HCN activities in STN neurons through dopamine D2 receptors. Our findings might lead to a new direction in PD treatment by providing promising new drug targets for HVSs activity modulation.
Subject(s)
Cerebral Cortex/cytology , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/drug effects , Neurons/drug effects , Receptors, Dopamine D2/drug effects , Subthalamic Nucleus/cytology , Animals , Basal Ganglia/drug effects , Benzazepines/pharmacology , Brain Chemistry/drug effects , Cerebral Cortex/drug effects , Dopamine/pharmacology , Dopamine Antagonists/pharmacology , Electrophysiological Phenomena/drug effects , Excitatory Amino Acid Antagonists/pharmacology , GTP-Binding Proteins/metabolism , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/agonists , Lamotrigine , Male , Parkinson Disease, Secondary/chemically induced , Parkinson Disease, Secondary/pathology , Rats , Rats, Sprague-Dawley , Subthalamic Nucleus/drug effects , Theta Rhythm/drug effects , Triazines/pharmacologyABSTRACT
Amyloid beta (Aß) accumulation plays an important role in the pathogenesis of Alzheimer's disease (AD) by changing the neuronal excitability. However, the cellular mechanisms by which accumulation of Aß affects intrinsic neuronal properties are not well understood. The effect of bilateral intra-frontal cortex Aß (1-42) peptide injection on the intrinsic excitability of hippocampal CA1 pyramidal neurons with particular focus on the contribution of hyperpolarization-activated (Ih) channel currents was examined using whole-cell patch-clamp recording. Passive avoidance memory impairment and morphological changes in rats receiving intra-frontal Aß treatment were observed, which was associated with significant changes both in passive and active intrinsic electrical membrane properties of CA1 pyramidal neurons. Electrophysiological recording showed a significant decrease in neuronal excitability associated with an augmentation in the first spike after-hyperpolarization (AHP) amplitude. In addition, the depolarizing sag voltage was altered in neurons recorded from Aß-treated group. In voltage-clamp condition, a hyperpolarizing activated inward current sensitive to ZD7288 and capsaicin was significantly increased in neurons from Aß-treated rats. The Ih current density was increased and the activation curve was shifted toward less negative potential in the Aß-treated group as compared to control group. The enhancing effect of Aß treatment on Ih current was confirmed by showing upregulation of the mRNA of HCN1 channel in the CA1 pyramidal layer of hippocampi. These findings suggest the contribution of Ih and possibly TRPV1 channel currents to the changes induced by Aß treatment in the intrinsic membrane properties, which, in turn, may provide therapeutic targets for treatment of AD.
Subject(s)
Alzheimer Disease/chemically induced , Alzheimer Disease/pathology , Amyloid beta-Peptides/toxicity , CA1 Region, Hippocampal/pathology , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/metabolism , Neurons/physiology , Alzheimer Disease/complications , Animals , Avoidance Learning/drug effects , Avoidance Learning/physiology , Capsaicin/analogs & derivatives , Capsaicin/pharmacology , Disease Models, Animal , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/agonists , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/antagonists & inhibitors , In Vitro Techniques , Male , Memory Disorders/etiology , Nerve Net/drug effects , Nerve Net/physiology , Neurons/drug effects , Patch-Clamp Techniques , Pyrimidines/agonists , Pyrimidines/pharmacology , Rats , Rats, Wistar , Sensory System Agents/pharmacology , TRPV Cation Channels/agonists , TRPV Cation Channels/antagonists & inhibitorsSubject(s)
Adrenergic beta-Antagonists/therapeutic use , Benzazepines/therapeutic use , Cardiomyopathies/drug therapy , Heart Rate/drug effects , Cardiomyopathies/chemically induced , Cardiomyopathies/complications , Cardiomyopathies/pathology , Humans , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/agonists , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/antagonists & inhibitors , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/metabolism , Ivabradine , Lipopolysaccharides , Myocardium/metabolism , Myocardium/pathology , Sepsis/chemically induced , Sepsis/complications , Sepsis/drug therapy , Sepsis/pathologyABSTRACT
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.