Cannabidiol potentiates hyperpolarization-activated cyclic nucleotide-gated (HCN4) channels.

Cannabidiol (CBD), the main non-psychotropic phytocannabinoid produced by the Cannabis sativa plant, blocks a variety of cardiac ion channels. We aimed to identify whether CBD regulated the cardiac pacemaker channel or the hyperpolarization-activated cyclic nucleotide-gated channel (HCN4). HCN4 channels are important for the generation of the action potential in the sinoatrial node of the heart and increased heart rate in response to ß-adrenergic stimulation. HCN4 channels were expressed in HEK 293T cells, and the effect of CBD application was examined using a whole-cell patch clamp. We found that CBD depolarized the V1/2 of activation in holo-HCN4 channels, with an EC50 of 1.6 µM, without changing the current density. CBD also sped activation kinetics by approximately threefold. CBD potentiation of HCN4 channels occurred via binding to the closed state of the channel. We found that CBD's mechanism of action was distinct from cAMP, as CBD also potentiated apo-HCN4 channels. The addition of an exogenous PIP2 analog did not alter the ability of CBD to potentiate HCN4 channels, suggesting that CBD also acts using a unique mechanism from the known HCN4 potentiator PIP2. Lastly, to gain insight into CBD's mechanism of action, computational modeling and targeted mutagenesis were used to predict that CBD binds to a lipid-binding pocket at the C-terminus of the voltage sensor. CBD represents the first FDA-approved drug to potentiate HCN4 channels, and our findings suggest a novel starting point for drug development targeting HCN4 channels.

Multifocal Ectopic Purkinje Premature Contractions due to neutralization of an SCN5A negative charge: structural insights into the gating pore hypothesis.

Background: We identified a novel SCN5A variant, E171Q, in a neonate with very frequent ectopy and reduced ejection fraction which normalized after arrhythmia suppression by flecainide. This clinical picture is consistent with multifocal ectopic Purkinje-related premature contractions (MEPPC). Most previous reports of MEPPC have implicated SCN5A variants such as R222Q that neutralize positive charges in the S4 voltage sensor helix of the channel protein NaV1.5 and generate a gating pore current. Methods and Results: E171 is a highly conserved negatively-charged residue located in the S2 transmembrane helix of NaV1.5 domain I. E171 is a key component of the Gating Charge Transfer Center, a region thought to be critical for normal movement of the S4 voltage sensor helix. We used heterologous expression, CRISPR-edited induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), and molecular dynamics simulations to demonstrate that E171Q generates a gating pore current, which was suppressed by a low concentration of flecainide (IC50 = 0.71±0.07 µM). R222Q shifts voltage dependence of activation and inactivation in a negative direction but we observed positive shifts with E171Q. E171Q iPSC-CMs demonstrated abnormal spontaneous activity and prolonged action potentials. Molecular dynamics simulations revealed that both R222Q and E171Q proteins generate a water-filled permeation pathway that underlies generation of the gating pore current. Conclusion: Previously identified MEPPC-associated variants that create gating pore currents are located in positively-charged residues in the S4 voltage sensor and generate negative shifts in the voltage dependence of activation and inactivation. We demonstrate that neutralizing a negatively charged S2 helix residue in the Gating Charge Transfer Center generates positive shifts but also create a gating pore pathway. These findings implicate the gating pore pathway as the primary functional and structural determinant of MEPPC and widen the spectrum of variants that are associated with gating pore-related disease in voltage-gated ion channels.

ARumenamides: A novel class of potential antiarrhythmic compounds.

Background: Most therapeutics targeting cardiac voltage-gated sodium channels (Nav1.5) attenuate the sodium current (INa) conducted through the pore of the protein. Whereas these drugs may be beneficial for disease states associated with gain-of-function (GoF) in Nav1.5, few attempts have been made to therapeutically treat loss-of-function (LoF) conditions. The primary impediment to designing efficacious therapies for LoF is a tendency for drugs to occlude the Nav1.5 central pore. We hypothesized that molecular candidates with a high affinity for the fenestrations would potentially reduce pore block. Methods and Results: Virtual docking was performed on 21 compounds, selected based on their affinity for the fenestrations in Nav1.5, which included a class of sulfonamides and carboxamides we identify as ARumenamide (AR). Six ARs, AR-051, AR-189, AR-674, AR-802, AR-807 and AR-811, were further docked against Nav1.5 built on NavAb and rNav1.5. Based on the virtual docking results, these particular ARs have a high affinity for Domain III-IV and Domain VI-I fenestrations. Upon functional characterization, a trend was observed in the effects of the six ARs on INa. An inverse correlation was established between the aromaticity of the AR's functional moieties and compound block. Due to its aromaticity, AR-811 blocked INa the least compared with other aromatic ARs, which also decelerated fast inactivation onset. AR-674, with its aliphatic functional group, significantly suppresses INa and enhances use-dependence in Nav1.5. AR-802 and AR-811, in particular, decelerated fast inactivation kinetics in the most common Brugada Syndrome Type 1 and Long-QT Syndrome Type 3 mutant, E1784K, without affecting peak or persistent INa. Conclusion: Our hypothesis that LoF in Nav1.5 may be therapeutically treated was supported by the discovery of ARs, which appear to preferentially block the fenestrations. ARs with aromatic functional groups as opposed to aliphatic groups efficaciously maintained Nav1.5 availability. We predict that these bulkier side groups may have a higher affinity for the hydrophobic milieu of the fenestrations, remaining there rather than in the central pore of the channel. Future refinements of AR compound structures and additional validation by molecular dynamic simulations and screening against more Brugada variants will further support their potential benefits in treating certain LoF cardiac arrhythmias.

Cytoplasmic Autoinhibition in HCN Channels is Regulated by the Transmembrane Region.

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.

Methodological-conceptual problems in the study of chimpanzees' folk physics: how studies with adult humans can help.

In three experiments, we examined humans' folk physics (i.e., a naturally occurring and spontaneous understanding of the physical world), using variations of problems used to study chimpanzees' folk physics. Presented with trap-tube problems in two experiments, adult humans showed an unnecessary bias to insert a stick into the end of the tube farthest from the reward to push it out the other end. When presented with trap-table problems with ineffective trapping holes, people unnecessarily avoided the side with the hole. The similarity of humans' and chimpanzees' behavior on these tasks highlights methodological and conceptual problems in studies of chimpanzees' folk physics and suggests alternative explanations for their behavior.