Say Cheese: Structure of the Cardiac Electrical Engine Is Captured.

Voltage-gated sodium channel (Nav)1.5 is the predominantly expressed sodium channel in the myocardium. Mutations in the gene encoding Nav1.5 are associated with several types of cardiac arrhythmias. In their recent study, Jiang et al. provide a detailed structure of the rat Nav1.5, with major implications regarding its physiology, pharmacology, and pathophysiology.

Late sodium current: incomplete inactivation triggers seizures, myotonias, arrhythmias, and pain syndromes.

Acquired and inherited dysfunction in voltage-gated sodium channels underlies a wide range of diseases. In addition to defects in trafficking and expression, sodium channelopathies are caused by dysfunction in one or several gating properties, for instance activation or inactivation. Disruption of channel inactivation leads to increased late sodium current, which is a common defect in seizure disorders, cardiac arrhythmias skeletal muscle myotonia and pain. An increase in late sodium current leads to repetitive action potentials in neurons and skeletal muscles, and prolonged action potential duration in the heart. In this Topical Review, we compare the effects of late sodium current in brain, heart, skeletal muscle and peripheral nerves.

Inhibitory effects of cannabidiol on voltage-dependent sodium currents.

Cannabis sativa contains many related compounds known as phytocannabinoids. The main psychoactive and nonpsychoactive compounds are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), respectively. Much of the evidence for clinical efficacy of CBD-mediated antiepileptic effects has been from case reports or smaller surveys. The mechanisms for CBD's anticonvulsant effects are unclear and likely involve noncannabinoid receptor pathways. CBD is reported to modulate several ion channels, including sodium channels (Nav). Evaluating the therapeutic mechanisms and safety of CBD demands a richer understanding of its interactions with central nervous system targets. Here, we used voltage-clamp electrophysiology of HEK-293 cells and iPSC neurons to characterize the effects of CBD on Nav channels. Our results show that CBD inhibits hNav1.1-1.7 currents, with an IC50 of 1.9-3.8 µm, suggesting that this inhibition could occur at therapeutically relevant concentrations. A steep Hill slope of ∼3 suggested multiple interactions of CBD with Nav channels. CBD exhibited resting-state blockade, became more potent at depolarized potentials, and also slowed recovery from inactivation, supporting the idea that CBD binding preferentially stabilizes inactivated Nav channel states. We also found that CBD inhibits other voltage-dependent currents from diverse channels, including bacterial homomeric Nav channel (NaChBac) and voltage-gated potassium channel subunit Kv2.1. Lastly, the CBD block of Nav was temperature-dependent, with potency increasing at lower temperatures. We conclude that CBD's mode of action likely involves 1) compound partitioning in lipid membranes, which alters membrane fluidity affecting gating, and 2) undetermined direct interactions with sodium and potassium channels, whose combined effects are loss of channel excitability.

Case studies in neuroscience: a novel amino acid duplication in the NH2-terminus of the brain sodium channel NaV1.1 underlying Dravet syndrome.

Dravet syndrome is a severe form of childhood epilepsy characterized by frequent temperature-sensitive seizures and delays in cognitive development. In the majority (80%) of cases, Dravet syndrome is caused by mutations in the SCN1A gene, encoding the voltage-gated sodium channel NaV1.1, which is abundant in the central nervous system. Dravet syndrome can be caused by either gain-of-function mutation or loss of function in NaV1.1, making it necessary to characterize each novel mutation. Here we use a combination of patch-clamp recordings and immunocytochemistry to characterize the first known NH2-terminal amino acid duplication mutation found in a patient with Dravet syndrome, M72dup. M72dup does not significantly alter rate of fast inactivation recovery or rate of fast inactivation onset at any measured membrane potential. M72dup significantly shifts the midpoint of the conductance voltage relationship to more hyperpolarized potentials. Most interestingly, M72dup significantly reduces peak current of NaV1.1 and reduces membrane expression. This suggests that M72dup acts as a loss-of-function mutation primarily by impacting the ability of the channel to localize to the plasma membrane.NEW & NOTEWORTHY Genetic screening of a patient with Dravet syndrome revealed a novel mutation in SCN1A. Of over 700 SCN1A mutations known to cause Dravet syndrome, M72dup is the first to be identified in the NH2-terminus of NaV1.1. We studied M72dup using patch-clamp electrophysiology and immunocytochemistry. M72dup causes a decrease in membrane expression of NaV1.1 and overall loss of function, consistent with the role of the NH2-terminal region in membrane trafficking of NaV1.1.

SCN5A mutations in 442 neonates and children: genotype-phenotype correlation and identification of higher-risk subgroups.

Aims: To clarify the clinical characteristics and outcomes of children with SCN5A-mediated disease and to improve their risk stratification. Methods and results: A multicentre, international, retrospective cohort study was conducted in 25 tertiary hospitals in 13 countries between 1990 and 2015. All patients ≤16 years of age diagnosed with a genetically confirmed SCN5A mutation were included in the analysis. There was no restriction made based on their clinical diagnosis. A total of 442 children {55.7% boys, 40.3% probands, median age: 8.0 [interquartile range (IQR) 9.5] years} from 350 families were included; 67.9% were asymptomatic at diagnosis. Four main phenotypes were identified: isolated progressive cardiac conduction disorders (25.6%), overlap phenotype (15.6%), isolated long QT syndrome type 3 (10.6%), and isolated Brugada syndrome type 1 (1.8%); 44.3% had a negative electrocardiogram phenotype. During a median follow-up of 5.9 (IQR 5.9) years, 272 cardiac events (CEs) occurred in 139 (31.5%) patients. Patients whose mutation localized in the C-terminus had a lower risk. Compound genotype, both gain- and loss-of-function SCN5A mutation, age ≤1 year at diagnosis in probands and age ≤1 year at diagnosis in non-probands were independent predictors of CE. Conclusion: In this large paediatric cohort of SCN5A mutation-positive subjects, cardiac conduction disorders were the most prevalent phenotype; CEs occurred in about one-third of genotype-positive children, and several independent risk factors were identified, including age ≤1 year at diagnosis, compound mutation, and mutation with both gain- and loss-of-function.

pH Modulation of Voltage-Gated Sodium Channels.

Changes in blood and tissue pH accompany physiological and pathophysiological conditions including exercise, cardiac ischemia, ischemic stroke, and cocaine ingestion. These conditions are known to trigger the symptoms of electrical diseases in patients carrying sodium channel mutations. Protons cause a diverse set of changes to sodium channel gating, which generally lead to decreases in the amplitude of the transient sodium current and increases in the fraction of non-inactivating channels that pass persistent currents. These effects are shared with disease-causing mutants in neuronal, skeletal muscle, and cardiac tissue and may be compounded in mutants that impart greater proton sensitivity to sodium channels, suggesting a role of protons in triggering acute symptoms of electrical disease.In this chapter, we review the mechanisms of proton block of the sodium channel pore and a suggested mode of action by which protons alter channel gating. We discuss the available data on isoform specificity of proton effects and tissue level effects. Finally, we review the role that protons play in disease and our own recent studies on proton-sensitizing mutants in cardiac and skeletal muscle sodium channels.

Differential calcium sensitivity in NaV 1.5 mixed syndrome mutants.

KEY POINTS: SCN5a mutations may express gain-of-function (Long QT Syndrome-3), loss-of-function (Brugada Syndrome 1) or both (mixed syndromes), depending on the mutation and environmental triggers. One such trigger may be an increase in cytosolic calcium, accompanying exercise. Many mixed syndromes mutants, including ∆KPQ, E1784K, 1795insD and Q1909R, are found in calcium-sensitive regions. Elevated cytosolic calcium attenuates gain-of-function properties in ∆KPQ, 1795insD and Q1909R, but not in E1784K. By contrast, elevated cytosolic calcium further exacerbates gain-of-function in E1784K by destabilizing slow inactivation. Action potential modelling, using a modified O'Hara Rudy model, suggests that elevated heart rate rescues action potential duration in ∆KPQ, 1795insD and Q1909R, but not in E1784K. Action potential simulations suggest that E1784K carriers have an increased intracellular sodium-to-calcium ratio under bradycardia and tachycardia conditions. Elevated cytosolic calcium, which is common during high heart rates, ameliorates or exacerbates the mixed syndrome phenotype depending on the genetic signature. ABSTRACT: Inherited arrhythmias may arise from mutations in the gene for SCN5a, which encodes the cardiac voltage-gated sodium channel, NaV 1.5. Mutants in NaV 1.5 result in Brugada Syndrome (BrS1), Long-QT Syndrome (LQT3) or mixed syndromes (an overlap of BrS1/LQT3). Exercise is a potential arrhythmogenic trigger in mixed syndromes. We aimed to determine the effects of elevated cytosolic calcium, which is common during exercise, in mixed syndrome NaV 1.5 mutants. We used whole-cell patch clamp to assess the biophysical properties of NaV 1.5 wild-type (WT), ∆KPQ, E1784K, 1795insD and Q1909R mutants in human embryonic kidney 293 cells transiently transfected with the NaV 1.5 α subunit (WT or mutants), ß1 subunit and enhanced green fluorescent protein. Voltage-dependence and kinetics were measured at cytosolic calcium levels of approximately 0, 500 and 2500 nm. In silico, action potential (AP) model simulations were performed using a modified O'Hara Rudy model. Elevated cytosolic calcium attenuates the late sodium current in ∆KPQ, 1795insD and Q1909R, but not in E1784K. Elevated cytosolic calcium restores steady-state slow inactivation (SSSI) to the WT-form in Q1909R, but depolarized SSSI in E1784K. Our AP simulations showed a frequency-dependent reduction of AP duration in ∆KPQ, 1795insD and Q1909R carriers. In E1784K, AP duration is relatively prolonged at both low and high heart rates, resulting in a sodium overload. Cellular perturbations during exercise may affect BrS1/LQT3 patients differently depending on their individual genetic signature. Thus, exercise may be therapeutic or may be an arrhythmogenic trigger in some SCN5a patients.

Differential thermosensitivity in mixed syndrome cardiac sodium channel mutants.

Cardiac arrhythmias are often associated with mutations in SCN5A the gene that encodes the cardiac paralogue of the voltage-gated sodium channel, NaV 1.5. The NaV 1.5 mutants R1193Q and E1784K give rise to both long QT and Brugada syndromes. Various environmental factors, including temperature, may unmask arrhythmia. We sought to determine whether temperature might be an arrhythmogenic trigger in these two mixed syndrome mutants. Whole-cell patch clamp was used to measure the biophysical properties of NaV 1.5 WT, E1784K and R1193Q mutants. Recordings were performed using Chinese hamster ovary (CHOk1) cells transiently transfected with the NaV 1.5 α subunit (WT, E1784K, or R1193Q), ß1 subunit, and eGFP. The channels' voltage-dependent and kinetic properties were measured at three different temperatures: 10ºC, 22ºC, and 34ºC. The E1784K mutant is more thermosensitive than either WT or R1193Q channels. When temperature is elevated from 22°C to 34°C, there is a greater increase in late INa and use-dependent inactivation in E1784K than in WT or R1193Q. However, when temperature is lowered to 10°C, the two mutants show a decrease in channel availability. Action potential modelling using Q10 fit values, extrapolated to physiological and febrile temperatures, show a larger transmural voltage gradient in E1784K compared to R1193Q and WT with hyperthermia. The E1784K mutant is more thermosensitive than WT or R1193Q channels. This enhanced thermosensitivity may be a mechanism for arrhythmogenesis in patients with E1784K sodium channels.

Proton sensors in the pore domain of the cardiac voltage-gated sodium channel.

Protons impart isoform-specific modulation of inactivation in neuronal, skeletal muscle, and cardiac voltage-gated sodium (Na(V)) channels. Although the structural basis of proton block in Na(V) channels has been well described, the amino acid residues responsible for the changes in Na(V) kinetics during extracellular acidosis are as yet unknown. We expressed wild-type (WT) and two pore mutant constructs (H880Q and C373F) of the human cardiac Na(V) channel, Na(V)1.5, in Xenopus oocytes. C373F and H880Q both attenuated proton block, abolished proton modulation of use-dependent inactivation, and altered pH modulation of the steady-state and kinetic parameters of slow inactivation. Additionally, C373F significantly reduced the maximum probability of use-dependent inactivation and slow inactivation, relative to WT. H880Q also significantly reduced the maximum probability of slow inactivation and shifted the voltage dependence of activation and fast inactivation to more positive potentials, relative to WT. These data suggest that Cys-373 and His-880 in Na(V)1.5 are proton sensors for use-dependent and slow inactivation and have implications in isoform-specific modulation of Na(V) channels.

Introduction to sodium channels.

Voltage-gated sodium channels (VGSCs) are present in many tissue types within the human body including both cardiac and neuronal tissues. Like other channels, VGSCs activate, deactivate, and inactivate in response to changes in membrane potential. VGSCs also have a similar structure to other channels: 24 transmembrane segments arranged into four domains that surround a central pore. The structure and electrical activity of these channels allows them to create and respond to electrical signals in the body. Because of their distribution throughout the body, VGSCs are implicated in a variety of diseases including epilepsy, cardiac arrhythmias, and neuropathic pain. As such the study of these channels is essential. This brief review will introduce sodium channel structure, physiology, and pathophysiology.

Proton modulation of cardiac I Na: a potential arrhythmogenic trigger.

Voltage-gated sodium (NaV) channels generate the upstroke and mediate duration of the ventricular action potential, thus they play a critical role in mediating cardiac excitability. Cardiac ischemia triggers extracellular pH to drop as low as pH 6.0, within just 10 min of its onset. Heightened proton concentrations reduce sodium conductance and alter the gating parameters of the cardiac-specific voltage-gated sodium channel, NaV1.5. Most notably, acidosis destabilizes fast inactivation, which plays a critical role in regulating action potential duration. The changes in NaV1.5 channel gating contribute to cardiac dysfunction during ischemia that can cause syncope, cardiac arrhythmia, and even sudden cardiac death. Understanding NaV channel modulation by protons is paramount to treatment and prevention of the deleterious effects of cardiac ischemia and other triggers of cardiac acidosis.

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.

Molecular Pharmacology of Selective NaV1.6 and Dual NaV1.6/NaV1.2 Channel Inhibitors that Suppress Excitatory Neuronal Activity Ex Vivo.

Voltage-gated sodium channel (NaV) inhibitors are used to treat neurological disorders of hyperexcitability such as epilepsy. These drugs act by attenuating neuronal action potential firing to reduce excitability in the brain. However, all currently available NaV-targeting antiseizure medications nonselectively inhibit the brain channels NaV1.1, NaV1.2, and NaV1.6, which potentially limits the efficacy and therapeutic safety margins of these drugs. Here, we report on XPC-7724 and XPC-5462, which represent a new class of small molecule NaV-targeting compounds. These compounds specifically target inhibition of the NaV1.6 and NaV1.2 channels, which are abundantly expressed in excitatory pyramidal neurons. They have a > 100-fold molecular selectivity against NaV1.1 channels, which are predominantly expressed in inhibitory neurons. Sparing NaV1.1 preserves the inhibitory activity in the brain. These compounds bind to and stabilize the inactivated state of the channels thereby reducing the activity of excitatory neurons. They have higher potency, with longer residency times and slower off-rates, than the clinically used antiseizure medications carbamazepine and phenytoin. The neuronal selectivity of these compounds is demonstrated in brain slices by inhibition of firing in cortical excitatory pyramidal neurons, without impacting fast spiking inhibitory interneurons. XPC-5462 also suppresses epileptiform activity in an ex vivo brain slice seizure model, whereas XPC-7224 does not, suggesting a possible requirement of Nav1.2 inhibition in 0-Mg2+- or 4-AP-induced brain slice seizure models. The profiles of these compounds will facilitate pharmacological dissection of the physiological roles of NaV1.2 and NaV1.6 in neurons and help define the role of specific channels in disease states. This unique selectivity profile provides a new approach to potentially treat disorders of neuronal hyperexcitability by selectively downregulating excitatory circuits.

Mechanisms underlying the antiarrhythmic effect of ARumenamide-787 in experimental models of the J wave syndromes and hypothermia.

BACKGROUND: Brugada (BrS) and early repolarization syndromes (ERS), the so-called J wave syndromes (JWS), are associated with life-threatening ventricular arrhythmias. Pharmacologic approaches to therapy are currently limited. In this study, we examine the effects of ARumenamide-787 (AR-787) to suppress the electrocardiographic and arrhythmic manifestations of JWS and hypothermia. METHODS: We studied the effects of AR-787 on INa and IKr in HEK-293 cells stably expressing the α- and ß1-subunits of the cardiac (NaV1.5) sodium channel and hERG channel, respectively. In addition, we studied its effect on Ito, INa and ICa in dissociated canine ventricular myocytes along with action potentials and ECG from coronary-perfused right (RV) and left (LV) ventricular wedge preparations. The Ito agonist, NS5806 (5-10 µM), ICa blocker, verapamil (2.5 µM), and INa blocker, ajmaline (2.5 µM), were used to mimic the genetic defects associated with JWS and to induce the electrocardiographic and arrhythmic manifestations of JWS (prominent J waves/ST segment elevation, phase 2 reentry and polymorphic VT/VF) in canine ventricular wedge preparations. RESULTS: AR-787 (1, 10 and 50 µM) exerted pleiotropic effects on cardiac ion channels. The predominant effect was inhibition of the transient outward current (Ito) and enhancement of the sodium channel current (INa), with lesser effects to inhibit IKr and augment calcium channel current (ICa). AR-787 diminished the electrocardiographic J wave and prevented and/or suppressed all arrhythmic activity in canine RV and LV experimental models of BrS, ERS and hypothermia. CONCLUSIONS: Our findings point to AR-787 as promising candidate for the pharmacologic treatment of JWS and hypothermia.

A thermoprotective role of the sodium channel ß1 subunit is lost with the ß1 (C121W) mutation.

PURPOSE: A mutation in the ß(1) subunit of the voltage-gated sodium (Na(V)) channel, ß(1) (C121W), causes genetic epilepsy with febrile seizures plus (GEFS+), a pediatric syndrome in which febrile seizures are the predominant phenotype. Previous studies of molecular mechanisms underlying neuronal hyperexcitability caused by this mutation were conducted at room temperature. The prevalence of seizures during febrile states in patients with GEFS+, however, suggests that the phenotypic consequence of ß(1) (C121W) may be exacerbated by elevated temperature. We investigated the putative mechanism underlying seizure generation by the ß(1) (C121W) mutation with elevated temperature. METHODS: Whole-cell voltage clamp experiments were performed at 22 and 34°C using Chinese Hamster Ovary (CHO) cells expressing the α subunit of neuronal Na(V) channel isoform, Na(V) 1.2. Voltage-dependent properties were recorded from CHO cells expressing either Na(V) 1.2 alone, Na(V) 1.2 plus wild-type (WT) ß(1) subunit, or Na(V) 1.2 plus ß(1) (C121W). KEY FINDINGS: Our results suggest WT ß(1) is protective against increased channel excitability induced by elevated temperature; protection is lost in the absence of WT ß(1) or with expression of ß(1) (C121W). At 34°C, Na(V) 1.2 + ß(1) (C121W) channel excitability increased compared to NaV1.2 + WT ß(1) by the following mechanisms: decreased use-dependent inactivation, increased persistent current and window current, and delayed onset of, and accelerated recovery from, fast inactivation. SIGNIFICANCE: Temperature-dependent changes found in our study are consistent with increased neuronal excitability of GEFS+ patients harboring C121W. These results suggest a novel seizure-causing mechanism for ß(1) (C121W): increased channel excitability at elevated temperature.

Action potential generation requires a high sodium channel density in the axon initial segment.

The axon initial segment (AIS) is a specialized region in neurons where action potentials are initiated. It is commonly assumed that this process requires a high density of voltage-gated sodium (Na(+)) channels. Paradoxically, the results of patch-clamp studies suggest that the Na(+) channel density at the AIS is similar to that at the soma and proximal dendrites. Here we provide data obtained by antibody staining, whole-cell voltage-clamp and Na(+) imaging, together with modeling, which indicate that the Na(+) channel density at the AIS of cortical pyramidal neurons is approximately 50 times that in the proximal dendrites. Anchoring of Na(+) channels to the cytoskeleton can explain this discrepancy, as disruption of the actin cytoskeleton increased the Na(+) current measured in patches from the AIS. Computational models required a high Na(+) channel density (approximately 2,500 pS microm(-2)) at the AIS to account for observations on action potential generation and backpropagation. In conclusion, action potential generation requires a high Na(+) channel density at the AIS, which is maintained by tight anchoring to the actin cytoskeleton.

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.

Cannabidiol increases gramicidin current in human embryonic kidney cells: An observational study.

Gramicidin is a monomeric protein that is thought to non-selectively conduct cationic currents and water. Linear gramicidin is considered an antibiotic. This function is considered to be mediated by the formation of pores within the lipid membrane, thereby killing bacterial cells. The main non-psychoactive active constituent of the cannabis plant, cannabidiol (CBD), has recently gained interest, and is proposed to possess various potential therapeutic properties, including being an antibiotic. We previously determined that CBD's activity on ion channels could be, in part, mediated by altering membrane biophysical properties, including elasticity. In this study, our goal was to determine the empirical effects of CBD on gramicidin currents in human embryonic kidney (HEK) cells, seeking to infer potential direct compound-protein interactions. Our results indicate that gramicidin, when applied to the extracellular HEK cell membrane, followed by CBD perfusion, increases the gramicidin current.

Cannabidiol and Sodium Channel Pharmacology: General Overview, Mechanism, and Clinical Implications.

Voltage-gated sodium (Nav) channels initiate action potentials in excitable tissues. Altering these channels' function can lead to many pathophysiological conditions. Nav channels are composed of several functional and structural domains that could be targeted pharmacologically as potential therapeutic means against various neurological conditions. Mutations in Nav channels have been suggested to underlie various clinical syndromes in different tissues and in association with conditions ranging from epileptic to muscular problems. Treating those mutations that increase the excitability of Nav channels requires inhibitors that could effectively reduce channel firing. The main non-psychotropic constituent of the cannabis plant, cannabidiol (CBD), has recently gained interest as a viable compound to treat some of the conditions that are associated with Nav malfunctions. In this review, we discuss an overview of Nav channels followed by an in-depth description of the interactions of CBD and Nav channels. We conclude with some clinical implications of CBD use against Nav hyperexcitability based on a series of preclinical studies published to date, with a focus on Nav/CBD interactions.