Structural and functional approaches to studying cAMP regulation of HCN channels

2021 ◽  
Author(s):  
Andrea Saponaro ◽  
Gerhard Thiel ◽  
Anna Moroni
Keyword(s):  
Cyclic Nucleotide ◽  
Metal Ion ◽  
Molecular Mechanisms ◽  
Signal Transmission ◽  
Functional Results ◽  
Mechanistic Explanation ◽  
Hcn Channels ◽  
Membrane Domains ◽  
Open Probability ◽  
Recent Advancement

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are primarily activated by voltage and further modulated by cAMP. While cAMP binding alone does not open the channel, its presence facilitates the action of voltage, increasing channel open probability. Functional results indicate that the membrane-based voltage sensor domain (VSD) communicates with the cytosolic cyclic nucleotide-binding domain (CNBD), and vice-versa. Yet, a mechanistic explanation on how this could occur in structural terms is still lacking. In this review, we will discuss the recent advancement in understanding the molecular mechanisms connecting the VSD with the CNBD in the tetrameric organization of HCN channels unveiled by the 3D structures of HCN1 and HCN4. Data show that the HCN domain transmits cAMP signal to the VSD by bridging the cytosolic to the membrane domains. Furthermore, a metal ion coordination site connects the C-linker to the S4–S5 linker in HCN4, further facilitating cAMP signal transmission to the VSD in this isoform.

scholarly journals The HCN Channel Voltage Sensor Undergoes A Large Downward Motion During Hyperpolarization

2019 ◽  
Author(s):  
Gucan Dai ◽  
Teresa K. Aman ◽  
Frank DiMaio ◽  
William N. Zagotta
Keyword(s):  
Ion Channels ◽  
Metal Ion ◽  
Voltage Sensor ◽  
The Body ◽  
Resting Membrane Potential ◽  
Hcn Channels ◽  
Hcn Channel ◽  
Open Probability ◽  
Membrane Hyperpolarization ◽  
Transmembrane Voltage

Voltage-gated ion channels (VGICs) underlie almost all electrical signaling in the body1. They change their open probability in response to changes in transmembrane voltage, allowing permeant ions to flow across the cell membrane. Ion flow through VGICs underlies numerous physiological processes in excitable cells1. In particular, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which operate at the threshold of excitability, are essential for pacemaking activity, resting membrane potential, and synaptic integration2. VGICs contain a series of positively-charged residues that are displaced in response to changes in transmembrane voltage, resulting in a conformational change that opens the pore3–6. These voltage-sensing charges, which reside in the S4 transmembrane helix of the voltage-sensor domain (VSD)3 and within the membrane’s electric field, are thought to move towards the inside of the cell (downwards) during membrane hyperpolarization7. HCN channels are unique among VGICs because their open probability is increased by membrane hyperpolarization rather than depolarization8–10. The mechanism underlying this “reverse gating” is still unclear. Moreover, although many X-ray crystal and cryo-EM structures have been solved for the depolarized state of the VSD, including that of HCN channels11, no structures have been solved at hyperpolarized voltages. Here we measure the precise movement of the charged S4 helix of an HCN channel using transition metal ion fluorescence resonance energy transfer (tmFRET). We show that the S4 undergoes a significant (~10 Å) downward movement in response to membrane hyperpolarization. Furthermore, by applying constraints determined from tmFRET experiments to Rosetta modeling, we reveal that the carboxyl-terminal part of the S4 helix exhibits an unexpected tilting motion during hyperpolarization activation. These data provide a long-sought glimpse of the hyperpolarized state of a functioning VSD and also a framework for understanding the dynamics of reverse gating in HCN channels. Our methods can be broadly applied to probe short-distance rearrangements in other ion channels and membrane proteins.

scholarly journals Pathways of inter- and intrasubunit allosteric signaling in the C-linker disk and cyclic nucleotide-binding domain of HCN2 channels

2020 ◽  
Author(s):  
Christopher Pfleger ◽  
Jana Kusch ◽  
Mahesh Kondapuram ◽  
Tina Schwabe ◽  
Christian Sattler ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Cyclic Nucleotide ◽  
Signal Transmission ◽  
Nucleotide Binding ◽  
Binding Domain ◽  
Hcn Channels ◽  
Nucleotide Binding Domain ◽  
Membrane Hyperpolarization ◽  
Cyclic Nucleotide Binding ◽  
Cyclic Nucleotide Binding Domain

AbstractOpening 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 disk (CL). Confocal patch-clamp fluorometry revealed a 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 towards 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.

Abstract 15907: Disruption of HCN2 Leads to Laterality Defects and Cardiac Arrhythmia

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Bin Ye ◽  
Greg Taycher ◽  
Tim Hacker ◽  
Leslie Chan ◽  
Nian-Qing Shi
Keyword(s):  
Molecular Mechanisms ◽  
Ventricular Myocytes ◽  
Heart Defects ◽  
Cell Types ◽  
Conditional Knockout ◽  
Hcn Channels ◽  
Special Diet ◽  
Ultrasound Study ◽  
Central Nervous Systems ◽  
Laterality Defects

Background: Congenital heart defects (CHDs) encompass a large numbers of cardiovascular malformations, and remain the major cause of infant mortality among all types of birth defects. However, molecular mechanisms underlying CHDs remain elusive, largely owning to the complexity of the diseases and lack of animal models that can reproduce the pathophysiological conditions in a laboratory setting. The hyperpolarization-activated, cyclic nucleotide-gated cation channels (HCN) are responsible for generating spontaneous pacemaker activities in cardiac and central nervous systems. These channels are also detected in other cell types such as ventricular myocytes. HCN currents recorded from neonatal cells have an activation threshold of -70 mV while those recorded from adult cells are activated at -110 mV. This difference indicates that HCN activity might be important in early development. However, roles of HCN channels in cardiogenesis and development are not fully understood. Methods and Results: We created a HCN2 conditional knockout (HCN2KO) model in which the full-length HCN2 was disrupted. Two KO lines were subsequently derived from this model. The first line was weaned by 21 days and they all died by 4-5 wks of age. Maternal ultrasound study revealed that these KO mice developed fetal arrhythmia and had an underdeveloped left side in their hearts. The second line was able to live on under our special diet/care. These mice displayed a slower growth rate (1.1±0.2 g/wk) and lower body weight (15.1±2.0 g) relative to their age-matched WT controls (2.2±0.1 g/wk and 27.5±1.5 g; n=9-10, p<0.05). Echocardiography and tissue staining data suggested that KO hearts had laterality defects compared to their size-matched WT controls. The survived KO mice were able to maintain cardiac function by developing much thicker anterior and posterior walls to sustain blood-pumping (n=6, p<0.05). Electrocardiographic results indicated that the average heart rate recorded from KO mice was ~100 bpm slower than their age-matched WT controls (n=5-6, p<0.05). Conclusions: These novel findings indicate that HCN2 is indispensable in mouse cardiogenesis and development. Our KO models are therefore innovative platforms for future CHD research.

scholarly journals Discovery and Characterization of a Distinct Cyclic Nucleotide Binding Pocket in HCN Channels

2014 ◽  
Vol 106 (2) ◽  
pp. 627a
Author(s):  
Anna Moroni ◽  
Marco Lolicato ◽  
Annalisa Bucchi ◽  
Cristina Arrigoni ◽  
Stefano Zucca ◽  
...  
Keyword(s):  
Cyclic Nucleotide ◽  
Nucleotide Binding ◽  
Binding Pocket ◽  
Hcn Channels ◽  
Cyclic Nucleotide Binding

scholarly journals Hyperpolarization-activated cyclic-nucleotide-gated channels potentially modulate axonal excitability at different thresholds

2017 ◽  
Vol 118 (6) ◽  
pp. 3044-3050 ◽  
Author(s):  
Dinushi Weerasinghe ◽  
Parvathi Menon ◽  
Steve Vucic
Keyword(s):  
Cyclic Nucleotide ◽  
Neurological Diseases ◽  
Lower Threshold ◽  
Resting Membrane Potential ◽  
Hcn Channels ◽  
Motor Axons ◽  
Cyclic Nucleotide Gated Channels ◽  
Sensory Axons ◽  
Current Threshold ◽  
Axonal Excitability

Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels mediate differences in sensory and motor axonal excitability at different thresholds in animal models. Importantly, HCN channels are responsible for voltage-gated inward rectifying ( Ih) currents activated during hyperpolarization. The Ih currents exert a crucial role in determining the resting membrane potential and have been implicated in a variety of neurological disorders, including neuropathic pain. In humans, differences in biophysical properties of motor and sensory axons at different thresholds remain to be elucidated and could provide crucial pathophysiological insights in peripheral neurological diseases. Consequently, the aim of this study was to characterize sensory and motor axonal function at different threshold. Median nerve motor and sensory axonal excitability studies were undertaken in 15 healthy subjects (45 studies in total). Tracking targets were set to 20, 40, and 60% of maximum for sensory and motor axons. Hyperpolarizing threshold electrotonus (TEh) at 90–100 ms was significantly increased in lower threshold sensory axons times ( F = 11.195, P < 0.001). In motor axons, the hyperpolarizing current/threshold ( I/ V) gradient was significantly increased in lower threshold axons ( F = 3.191, P < 0.05). The minimum I/ V gradient was increased in lower threshold motor and sensory axons. In conclusion, variation in the kinetics of HCN isoforms could account for the findings in motor and sensory axons. Importantly, assessing the function of HCN channels in sensory and motor axons of different thresholds may provide insights into the pathophysiological processes underlying peripheral neurological diseases in humans, particularly focusing on the role of HCN channels with the potential of identifying novel treatment targets. NEW & NOTEWORTHY Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels, which underlie inward rectifying currents ( Ih), appear to mediate differences in sensory and motor axonal properties. Inward rectifying currents are increased in lower threshold motor and sensory axons, although different HCN channel isoforms appear to underlie these changes. While faster activating HCN channels seem to underlie Ih changes in sensory axons, slower activating HCN isoforms appear to be mediating the differences in Ih conductances in motor axons of different thresholds. The differences in HCN gating properties could explain the predilection for dysfunction of sensory and motor axons in specific neurological diseases.

scholarly journals Structural Similarity with Cholesterol Reveals Crucial Insights into Mechanisms Sustaining the Immunomodulatory Activity of the Mycotoxin Alternariol

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 847 ◽  
Author(s):  
Giorgia Del Favero ◽  
Raphaela M. Mayer ◽  
Luca Dellafiora ◽  
Lukas Janker ◽  
Laura Niederstaetter ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Structural Similarity ◽  
Immunomodulatory Activity ◽  
Continuous Exposure ◽  
Membrane Domains ◽  
Toll Like Receptor ◽  
Caveolin 1 ◽  
Domestic Environments ◽  
Opening Up ◽  
Immunomodulatory Potential

The proliferation of molds in domestic environments can lead to uncontrolled continuous exposure to mycotoxins. Even if not immediately symptomatic, this may result in chronic effects, such as, for instance, immunosuppression or allergenic promotion. Alternariol (AOH) is one of the most abundant mycotoxins produced by Alternaria alternata fungi, proliferating among others in fridges, as well as in humid walls. AOH was previously reported to have immunomodulatory potential. However, molecular mechanisms sustaining this effect remained elusive. In differentiated THP-1 macrophages, AOH hardly altered the secretion of pro-inflammatory mediators when co-incubated with lipopolysaccharide (LPS), opening up the possibility that the immunosuppressive potential of the toxin could be related to an alteration of a downstream pro-inflammatory signaling cascade. Intriguingly, the mycotoxin affected the membrane fluidity in macrophages and it synergistically reacted with the cholesterol binding agent MβCD. In silico modelling revealed the potential of the mycotoxin to intercalate in cholesterol-rich membrane domains, like caveolae, and immunofluorescence showed the modified interplay of caveolin-1 with Toll-like Receptor (TLR) 4. In conclusion, we identified the structural similarity with cholesterol as one of the key determinants of the immunomodulatory potential of AOH.

scholarly journals Pharmacological Overview of the BGP-15 Chemical Agent as a New Drug Candidate for the Treatment of Symptoms of Metabolic Syndrome

Molecules ◽  
2020 ◽  
Vol 25 (2) ◽  
pp. 429
Author(s):  
Ágota Pető ◽  
Dóra Kósa ◽  
Pálma Fehér ◽  
Zoltán Ujhelyi ◽  
Dávid Sinka ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Parp Inhibitor ◽  
Drug Candidate ◽  
Chemical Agent ◽  
Membrane Domains ◽  
Protective Effects ◽  
Pharmacological Effects ◽  
Research Groups ◽  
Insulin Sensitizer ◽  
Beneficial Effects

BGP-15 is a new insulin sensitizer drug candidate, which was developed by Hungarian researchers. In recent years, numerous research groups have studied its beneficial effects. It is effective in the treatment of insulin resistance and it has protective effects in Duchenne muscular dystrophy, diastolic dysfunction, tachycardia, heart failure, and atrial fibrillation, and it can alleviate cardiotoxicity. BGP-15 exhibits chemoprotective properties in different cytostatic therapies, and has also proven to be photoprotective. It can additionally have advantageous effects in mitochondrial-stress-related diseases. Although the precise mechanism of the effect is still unknown to us, we know that the molecule is a PARP inhibitor, chaperone co-inducer, reduces ROS production, and is able to remodel the organization of cholesterol-rich membrane domains. In the following review, our aim was to summarize the investigated molecular mechanisms and pharmacological effects of this potential API. The main objective was to present the wide pharmacological potentials of this chemical agent.

scholarly journals Dual Effect of Tamoxifen on Arterial KCa Channels Does Not Depend on the Presence of the β1 Subunit

2005 ◽  
Vol 280 (23) ◽  
pp. 21739-21747 ◽  
Author(s):  
Guillermo J. Pérez
Keyword(s):  
Molecular Mechanisms ◽  
Single Channel ◽  
Protective Role ◽  
Wild Type ◽  
Open Probability ◽  
Dual Effect ◽  
Knock Out ◽  
Kca Channels ◽  
Molecular Specificity ◽  
Excised Patches

Tamoxifen has been reported to directly activate large conductance calcium-activated potassium (KCa) channels through the KCa β1 subunit, suggesting a cardio-protective role of this compound. The present study using knock-out (KO) mice for the KCa channel β1 subunit was aimed at understanding the molecular mechanisms of the effects of tamoxifen on arterial smooth muscle KCa channels. Single channel studies were conducted in excised patches from cerebral artery myocytes from both wild-type and KO animals. The present data demonstrated that tamoxifen can inhibit arterial KCa channels due to a major decrease in channel open probability (Po), a mechanism different from the reduction in single channel amplitude reported previously and also observed in the present work. A tamoxifen-induced decrease in Po was present in arterial KCa channels from both wild-type and β1 KO animals. This inhibition was concentration-dependent and partially reversible with a half-maximal concentration constant IC50 of 2.6 μm. The effect of tamoxifen was actually dual Single channel kinetic analysis showed that tamoxifen shortens both mean closed time and mean open time; the latter is probably due to an intermediate duration voltage-independent blocking mechanism. Thus, tamoxifen block would predominate when KCa channel Po is >0.1–0.2, limiting the maximum Po, whereas a leftward shift in voltage or Ca2+ activation curves can be observed for Po values lower than those values. This dual effect of tamoxifen appears to be independent of the β1 subunit. The molecular specificity of tamoxifen, or eventually other xenoestrogen derivatives, for the KCa channel β1 subunit is uncertain.

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