Nav Channel Isoforms Research Articles

Interaction between scorpion toxins and the cardiac sodium channel isoform Nav1.5: overlooked role of the membrane

Interaction between scorpion toxins and the cardiac sodium channel isoform Nav1.5: overlooked role of the membrane

ST-2560, a selective inhibitor of the NaV1.7 sodium channel, affects nocifensive and cardiovascular reflexes in non-human primates.

The voltage-gated sodium channel isoform NaV1.7 is a high-interest target for the development of non-opioid analgesics due to its preferential expression in pain-sensing neurons. NaV1.7 is also expressed in autonomic neurons, yet its contribution to involuntary visceral reflexes has received limited attention. The small molecule inhibitor ST-2560 was advanced into pain behaviour and cardiovascular models to understand the pharmacodynamic effects of selective inhibition of NaV1.7. Potency of ST-2560 at NaV1.7 and off-target ion channels was evaluated by whole-cell patch-clamp electrophysiology. Effects on nocifensive reflexes were assessed in non-human primate (NHP) behavioural models, employing the chemical capsaicin and mechanical stimuli. Cardiovascular parameters were monitored continuously in freely-moving, telemetered NHPs following administration of vehicle and ST-2560. ST-2560 is a potent inhibitor (IC50 = 39 nM) of NaV1.7 in primates with ≥1000-fold selectivity over other isoforms of the human NaV1.x family. Following systemic administration, ST-2560 (0.1-0.3 mg·kg-1, s.c.) suppressed noxious mechanical- and chemical-evoked reflexes at free plasma concentrations threefold to fivefold above NaV1.7 IC50. ST-2560 (0.1-1.0 mg·kg-1, s.c.) also produced changes in haemodynamic parameters, most notably a 10- to 20-mmHg reduction in systolic and diastolic arterial blood pressure, at similar exposures. Acute pharmacological inhibition of NaV1.7 is antinociceptive, but also has the potential to impact the cardiovascular system. Further work is merited to understand the role of NaV1.7 in autonomic ganglia involved in the control of heart rate and blood pressure, and the effect of selective NaV1.7 inhibition on cardiovascular function.

Discovery of Selective Inhibitors of NaV1.7 Templated on Saxitoxin as Therapeutics for Pain.

The voltage-gated sodium channel isoform NaV1.7 has drawn widespread interest as a target for non-opioid, investigational new drugs to treat pain. Selectivity over homologous, off-target sodium channel isoforms, which are expressed in peripheral motor neurons, the central nervous system, skeletal muscle and the heart, poses a significant challenge to the development of small molecule inhibitors of NaV1.7. Most inhibitors of NaV1.7 disclosed to date belong to a class of aryl and acyl sulfonamides that preferentially bind to an inactivated conformation of the channel. By taking advantage of a sequence variation unique to primate NaV1.7 in the extracellular pore of the channel, a series of bis-guanidinium analogues of the natural product, saxitoxin, has been identified that are potent against the resting conformation of the channel. A compound of interest, 25, exhibits >600-fold selectivity over off-target sodium channel isoforms and is efficacious in a preclinical model of acute pain.

Depolarizing NaV and Hyperpolarizing KV Channels Are Co-Trafficked in Sensory Neurons.

Neuronal excitability relies on coordinated action of functionally distinction channels. Voltage-gated sodium (NaV) and potassium (KV) channels have distinct but complementary roles in firing action potentials: NaV channels provide depolarizing current while KV channels provide hyperpolarizing current. Mutations and dysfunction of multiple NaV and KV channels underlie disorders of excitability, including pain and epilepsy. Modulating ion channel trafficking may offer a potential therapeutic strategy for these diseases. A fundamental question, however, is whether these channels with distinct functional roles are transported independently or packaged together in the same vesicles in sensory axons. We have used Optical Pulse-Chase Axonal Long-distance imaging to investigate trafficking of NaV and KV channels and other axonal proteins from distinct functional classes in live rodent sensory neurons (from male and female rats). We show that, similar to NaV1.7 channels, NaV1.8 and KV7.2 channels are transported in Rab6a-positive vesicles, and that each of the NaV channel isoforms expressed in healthy, mature sensory neurons (NaV1.6, NaV1.7, NaV1.8, and NaV1.9) is cotransported in the same vesicles. Further, we show that multiple axonal membrane proteins with different physiological functions (NaV1.7, KV7.2, and TNFR1) are cotransported in the same vesicles. However, vesicular packaging of axonal membrane proteins is not indiscriminate, since another axonal membrane protein (NCX2) is transported in separate vesicles. These results shed new light on the development and organization of sensory neuron membranes, revealing complex sorting of axonal proteins with diverse physiological functions into specific transport vesicles.SIGNIFICANCE STATEMENT Normal neuronal excitability is dependent on precise regulation of membrane proteins, including NaV and KV channels, and imbalance in the level of these channels at the plasma membrane could lead to excitability disorders. Ion channel trafficking could potentially be targeted therapeutically, which would require better understanding of the mechanisms underlying trafficking of functionally diverse channels. Optical Pulse-chase Axonal Long-distance imaging in live neurons permitted examination of the specificity of ion channel trafficking, revealing co-packaging of axonal proteins with opposing physiological functions into the same transport vesicles. This suggests that additional trafficking mechanisms are necessary to regulate levels of surface channels, and reveals an important consideration for therapeutic strategies that target ion channel trafficking for the treatment of excitability disorders.

Pharmacologically Targeting the Fibroblast Growth Factor 14 Interaction Site on the Voltage-Gated Na+ Channel 1.6 Enables Isoform-Selective Modulation.

Voltage-gated Na+ (Nav) channels are the primary molecular determinant of the action potential. Among the nine isoforms of the Nav channel α subunit that have been described (Nav1.1-Nav1.9), Nav1.1, Nav1.2, and Nav1.6 are the primary isoforms expressed in the central nervous system (CNS). Crucially, these three CNS Nav channel isoforms display differential expression across neuronal cell types and diverge with respect to their subcellular distributions. Considering these differences in terms of their localization, the CNS Nav channel isoforms could represent promising targets for the development of targeted neuromodulators. However, current therapeutics that target Nav channels lack selectivity, which results in deleterious side effects due to modulation of off-target Nav channel isoforms. Among the structural components of the Nav channel α subunit that could be pharmacologically targeted to achieve isoform selectivity, the C-terminal domains (CTD) of Nav channels represent promising candidates on account of displaying appreciable amino acid sequence divergence that enables functionally unique protein–protein interactions (PPIs) with Nav channel auxiliary proteins. In medium spiny neurons (MSNs) of the nucleus accumbens (NAc), a critical brain region of the mesocorticolimbic circuit, the PPI between the CTD of the Nav1.6 channel and its auxiliary protein fibroblast growth factor 14 (FGF14) is central to the generation of electrical outputs, underscoring its potential value as a site for targeted neuromodulation. Focusing on this PPI, we previously developed a peptidomimetic derived from residues of FGF14 that have an interaction site on the CTD of the Nav1.6 channel. In this work, we show that whereas the compound displays dose-dependent effects on the activity of Nav1.6 channels in heterologous cells, the compound does not affect Nav1.1 or Nav1.2 channels at comparable concentrations. In addition, we show that the compound correspondingly modulates the action potential discharge and the transient Na+ of MSNs of the NAc. Overall, these results demonstrate that pharmacologically targeting the FGF14 interaction site on the CTD of the Nav1.6 channel is a strategy to achieve isoform-selective modulation, and, more broadly, that sites on the CTDs of Nav channels interacted with by auxiliary proteins could represent candidates for the development of targeted therapeutics.

Targeted Augmentation of Nuclear Gene Output (TANGO) of Scn1a rescues parvalbumin interneuron excitability and reduces seizures in a mouse model of Dravet Syndrome

Dravet Syndrome (DS) is a severe developmental and epileptic encephalopathy typically caused by loss-of-function de novo mutations in the SCN1A gene which encodes the voltage-gated sodium channel isoform NaV1.1. Decreased NaV1.1 expression results in impaired excitability of inhibitory interneurons and seizure onset. To date, there are no clinically available treatments for DS that directly address the core mechanism of disease; reduced NaV1.1 expression levels in interneurons. Recently, Targeted Augmentation of Nuclear Gene Output (TANGO) of SCN1A by the antisense oligonucleotide (ASO) STK-001, was shown to increase Scn1a mRNA levels, increase NaV1.1 protein expression, reduce seizures, and improve survival in the Scn1a+/− mouse model of DS. However, it remains unknown whether STK-001 treatment rescues the reduced intrinsic excitability of parvalbumin-positive (PV) inhibitory interneurons associated with DS. In this study, we demonstrate that STK-001 treatment reduces seizures, prolongs survival, and rescues PV interneuron excitability in Scn1a+/− mice to levels observed in WT littermates. Together, these results support the notion that TANGO-mediated augmentation of NaV1.1 levels directly targets and rescues one of the core disease mechanisms of DS.

Detrimental proarrhythmogenic interaction of Ca2+/calmodulin-dependent protein kinase II and NaV1.8 in heart failure

An interplay between Ca2+/calmodulin-dependent protein kinase IIδc (CaMKIIδc) and late Na+ current (INaL) is known to induce arrhythmias in the failing heart. Here, we elucidate the role of the sodium channel isoform NaV1.8 for CaMKIIδc-dependent proarrhythmia. In a CRISPR-Cas9-generated human iPSC-cardiomyocyte homozygous knock-out of NaV1.8, we demonstrate that NaV1.8 contributes to INaL formation. In addition, we reveal a direct interaction between NaV1.8 and CaMKIIδc in cardiomyocytes isolated from patients with heart failure (HF). Using specific blockers of NaV1.8 and CaMKIIδc, we show that NaV1.8-driven INaL is CaMKIIδc-dependent and that NaV1.8-inhibtion reduces diastolic SR-Ca2+ leak in human failing cardiomyocytes. Moreover, increased mortality of CaMKIIδc-overexpressing HF mice is reduced when a NaV1.8 knock-out is introduced. Cellular and in vivo experiments reveal reduced ventricular arrhythmias without changes in HF progression. Our work therefore identifies a proarrhythmic CaMKIIδc downstream target which may constitute a prognostic and antiarrhythmic strategy.

Pharmacological Inhibition of Wee1 Kinase Selectively Modulates the Voltage-Gated Na+ Channel 1.2 Macromolecular Complex.

Voltage-gated Na+ (Nav) channels are a primary molecular determinant of the action potential (AP). Despite the canonical role of the pore-forming α subunit in conferring this function, protein–protein interactions (PPI) between the Nav channel α subunit and its auxiliary proteins are necessary to reconstitute the full physiological activity of the channel and to fine-tune neuronal excitability. In the brain, the Nav channel isoforms 1.2 (Nav1.2) and 1.6 (Nav1.6) are enriched, and their activities are differentially regulated by the Nav channel auxiliary protein fibroblast growth factor 14 (FGF14). Despite the known regulation of neuronal Nav channel activity by FGF14, less is known about cellular signaling molecules that might modulate these regulatory effects of FGF14. To that end, and building upon our previous investigations suggesting that neuronal Nav channel activity is regulated by a kinase network involving GSK3, AKT, and Wee1, we interrogate in our current investigation how pharmacological inhibition of Wee1 kinase, a serine/threonine and tyrosine kinase that is a crucial component of the G2-M cell cycle checkpoint, affects the Nav1.2 and Nav1.6 channel macromolecular complexes. Our results show that the highly selective inhibitor of Wee1 kinase, called Wee1 inhibitor II, modulates FGF14:Nav1.2 complex assembly, but does not significantly affect FGF14:Nav1.6 complex assembly. These results are functionally recapitulated, as Wee1 inhibitor II entirely alters FGF14-mediated regulation of the Nav1.2 channel, but displays no effects on the Nav1.6 channel. At the molecular level, these effects of Wee1 inhibitor II on FGF14:Nav1.2 complex assembly and FGF14-mediated regulation of Nav1.2-mediated Na+ currents are shown to be dependent upon the presence of Y158 of FGF14, a residue known to be a prominent site for phosphorylation-mediated regulation of the protein. Overall, our data suggest that pharmacological inhibition of Wee1 confers selective modulatory effects on Nav1.2 channel activity, which has important implications for unraveling cellular signaling pathways that fine-tune neuronal excitability.

Somatostatin-Positive Interneurons Contribute to Seizures in SCN8A Epileptic Encephalopathy.

SCN8A epileptic encephalopathy is a devastating epilepsy syndrome caused by mutant SCN8A, which encodes the voltage-gated sodium channel NaV1.6. To date, it is unclear if and how inhibitory interneurons, which express NaV1.6, influence disease pathology. Using both sexes of a transgenic mouse model of SCN8A epileptic encephalopathy, we found that selective expression of the R1872W SCN8A mutation in somatostatin (SST) interneurons was sufficient to convey susceptibility to audiogenic seizures. Patch-clamp electrophysiology experiments revealed that SST interneurons from mutant mice were hyperexcitable but hypersensitive to action potential failure via depolarization block under normal and seizure-like conditions. Remarkably, GqDREADD-mediated activation of WT SST interneurons resulted in prolonged electrographic seizures and was accompanied by SST hyperexcitability and depolarization block. Aberrantly large persistent sodium currents, a hallmark of SCN8A mutations, were observed and were found to contribute directly to aberrant SST physiology in computational modeling and pharmacological experiments. These novel findings demonstrate a critical and previously unidentified contribution of SST interneurons to seizure generation not only in SCN8A epileptic encephalopathy, but epilepsy in general.SIGNIFICANCE STATEMENTSCN8A epileptic encephalopathy is a devastating neurological disorder that results from de novo mutations in the sodium channel isoform Nav1.6. Inhibitory neurons express NaV1.6, yet their contribution to seizure generation in SCN8A epileptic encephalopathy has not been determined. We show that mice expressing a human-derived SCN8A variant (R1872W) selectively in somatostatin (SST) interneurons have audiogenic seizures. Physiological recordings from SST interneurons show that SCN8A mutations lead to an elevated persistent sodium current which drives initial hyperexcitability, followed by premature action potential failure because of depolarization block. Furthermore, chemogenetic activation of WT SST interneurons leads to audiogenic seizure activity. These findings provide new insight into the importance of SST inhibitory interneurons in seizure initiation, not only in SCN8A epileptic encephalopathy, but for epilepsy broadly.

Development of Allosteric Modulators of Voltage-Gated Na+ Channels: A Novel Approach for an Old Target.

Given their primacy in governing the action potential (AP) of excitable cells, voltage-gated Na+ (Nav) channels are important pharmacological targets of therapeutics for a diverse array of clinical indications. Despite historically being a traditional drug target, therapeutics targeting Nav channels lack isoform selectivity, giving rise to off-target side effects. To develop isoform-selective modulators of Nav channels with improved target-specificity, the identification and pharmacological targeting of allosteric sites that display structural divergence among Nav channel isoforms represents an attractive approach. Despite the high homology among Nav channel α subunit isoforms (Nav1.1-Nav1.9), there is considerable amino acid sequence divergence among their constituent C-terminal domains (CTD), which enables structurally and functionally specific protein: protein interactions (PPI) with auxiliary proteins. Although pharmacological targeting of such PPI interfaces between the CTDs of Nav channels and auxiliary proteins represents an innovate approach for developing isoform-selective modulators of Nav channels, appreciable modulation of PPIs using small molecules has conventionally been difficult to achieve. After briefly discussing the challenges of modulating PPIs using small molecules, this current frontier review that follows subsequently expounds on approaches for circumventing such difficulties in the context of developing small molecule modulators of PPIs between transmembrane ion channels and their auxiliary proteins. In addition to broadly discussing such approaches, the implementation of such approaches is specifically discussed in the context of developing small molecule modulators between the CTD of Nav channels and auxiliary proteins. Developing allosteric modulators of ion channels by targeting their PPI interfaces with auxiliary proteins represents an innovative and promising strategy in ion channel drug discovery that could expand the "druggable genome" and usher in first-in-class PPI-targeting therapeutics for a multitude of channelopathies.

Activation of reverse Na+-Ca2+ exchanger by skeletal Na+ channel isoform increases excitation-contraction coupling efficiency in rabbit cardiomyocytes.

Our prior work has shown that Na+ current (INa) affects sarcoplasmic reticular (SR) Ca2+ release by activating early reverse of the Na+-Ca2+ exchanger (NCX). The resulting Ca2+ entry primes the dyadic cleft, which appears to increase Ca2+ channel coupling fidelity. It has been shown that the skeletal isoform of the voltage-gated Na+ channel (Nav1.4) is the main tetrodotoxin (TTX)-sensitive Nav isoform expressed in adult rabbit ventricular cardiomyocytes. Here, I tested the hypothesis that it is also the principal isoform involved in the priming mechanism. Action potentials (APs) were evoked in isolated rabbit ventricular cells loaded with fluo-4, and simultaneously recorded Ca2+ transients before and after the application of either relatively low doses of TTX (100 nM), the specific Nav1.4 inhibitor μ-Conotoxin GIIIB or the specific Nav1.1 inhibitor ICA 121430. Although APs changes after the application of each drug reflected the relative abundance of each isoform, the effects of TTX and GIIIB on SR Ca2+ release (measured as the transient maximum upstroke velocity) were no different. Furthermore, this reduction in SR Ca2+ release was comparable with the value that we obtained previously when total INa was inactivated with a ramp applied under voltage clamp. Finally, SR Ca2+ release was unaltered by the same ramp in the presence of TTX or GIIB. In contrast, application of ICA had no effect of SR Ca2+ release. These results suggest that Nav1.4 is the main Nav isoform involved in regulating the efficiency of excitation-contraction coupling in rabbit cardiomyocytes by priming the junction via activation of reverse-mode NCX.NEW & NOTEWORTHY A number of studies suggest that the Na+-Ca2+ exchanger (NCX) activated by Na+ currents is involved in the process of excitation-contraction (EC) coupling in cardiac ventricular myocytes. Although insufficient to trigger sarcoplasmic Ca2+ release alone, the Ca2+ entering through reverse NCX during an action potential can prime the dyadic cleft and increase the Ca2+ current coupling fidelity. Using specific Na+ inhibitors in this study, we show that in rabbit ventricular cells the skeletal Na+ channel isoform (Nav1.4) is the main isoform responsible for this priming. Our study provides insights into a mechanism that may have an increased relevance where EC coupling is remodeled.

SCN10A-knock-out improves survival and proarrhythmia in a transgenic heart failure mouse model

Abstract Background In heart failure (HF) both Ca2+/Calmodulin-dependent protein-kinase II (CaMKII) and late sodium current (INaL) are known to contribute to arrhythmogenesis as they contribute to action-potential (AP) prolongation and the occurrence of early- (EADs) and delayed afterdepolarizations (DADs). Further, augmented CaMKII and INaL maintain a vicious cycle as they both can activate each other. We recently found that the sodium channel isoform NaV1.8 is upregulated in HF and hypertrophy and that it is involved in INaL-generation. In the current study we investigated the effects of NaV1.8-knock-out (KO) on HF-progression and arrhythmogenesis in a CaMKII-overexpressing HF mouse model. Methods/Results CaMKII overexpressing mice (CaMKII+/T) were crossbred with NaV1.8-KO mice (SCN10A−/−). To our surprise knock-out of NaV1.8 in CaMKII+/T mice (SCN10A−/−/CaMKII+/T) significantly improved survival (median survival 103 days vs 74.5 CaMKII+/T, p<0.01). CaMKII+/T mice exhibited a strong HF phenotype compared to WT with increased heart-weight to tibia length ratio as well as reduced ejection fraction and left-ventricular end-diastolic diameter obtained by echocardiography. However, these structural parameters did not differ between SCN10A−/−/CaMKII+/T and CaMKII+/T. Therefore, cellular electrophysiology experiments were performed in isolated cardiomyocytes for a better understanding of the observed improvement in survival. INaL, measured by patch-clamp technique, was significantly augmented in CaMKII+/T vs WT and SCN10A−/−, while SCN10A−/−/CaMKII+/T showed significantly less INaL than CaMKII+/T alone. Further, AP-duration (APD) was significantly reduced in SCN10A−/−/CaMKII+/T vs CaMKII+/T while AP-amplitude, resting membrane-potential and upstroke velocity (dv/dtmax) remained unchanged. In addition, the occurrence of afterdepolarizations was significantly lower in SCN10A−/−/ CaMKII+/T vs CaMKII+/T. Confocal microscopy using the dye Fluo-4AM was performed and significantly less diastolic Ca2+-waves occurred in SCN10A−/−/CaMKII+/T compared to CaMKII+/T. In order to analyze an organ-specific SCN10A-KO, we generated homozygous SCN10A-KO lines of induced pluripotent stem cells by using CRISPR/Cas9 technology. 2-month old iPSC-cardiomyocytes lacking NaV1.8 were treated with low dose isoprenaline and showed significantly less INaL, thereby serving as a final proof of the relevant role of this Na+-channel on INaL-generation in the cardiomyocyte. Conclusion We found a survival benefit by selective knock-out of the neuronal sodium channel isoform NaV1.8 in a proarrhythmic HF mouse model with augmented CaMKII expression. However, in our model NaV1.8-knock-out showed no effects on HF progression, while cellular proarrhythmic triggers were attenuated. Taken together with our findings in IPS-cardiomyocytes treated with the CRSIPR/Cas9 technology NaV1.8 plays a significant role for the generation of INaL and cellular arrhythmogenic triggers in the cardiomyocyte. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Deutsche Stiftung für Herzforschung

Discovery of a selective, state-independent inhibitor of NaV1.7 by modification of guanidinium toxins

The voltage-gated sodium channel isoform NaV1.7 is highly expressed in dorsal root ganglion neurons and is obligatory for nociceptive signal transmission. Genetic gain-of-function and loss-of-function NaV1.7 mutations have been identified in select individuals, and are associated with episodic extreme pain disorders and insensitivity to pain, respectively. These findings implicate NaV1.7 as a key pharmacotherapeutic target for the treatment of pain. While several small molecules targeting NaV1.7 have been advanced to clinical development, no NaV1.7-selective compound has shown convincing efficacy in clinical pain applications. Here we describe the discovery and characterization of ST-2262, a NaV1.7 inhibitor that blocks the extracellular vestibule of the channel with an IC50 of 72 nM and greater than 200-fold selectivity over off-target sodium channel isoforms, NaV1.1–1.6 and NaV1.8. In contrast to other NaV1.7 inhibitors that preferentially inhibit the inactivated state of the channel, ST-2262 is equipotent in a protocol that favors the resting state of the channel, a protocol that favors the inactivated state, and a high frequency protocol. In a non-human primate study, animals treated with ST-2262 exhibited reduced sensitivity to noxious heat. These findings establish the extracellular vestibule of the sodium channel as a viable receptor site for the design of selective ligands targeting NaV1.7.

Cell-Adhesion Properties of β-Subunits in the Regulation of Cardiomyocyte Sodium Channels.

Voltage-gated sodium (Nav) channels drive the rising phase of the action potential, essential for electrical signalling in nerves and muscles. The Nav channel α-subunit contains the ion-selective pore. In the cardiomyocyte, Nav1.5 is the main Nav channel α-subunit isoform, with a smaller expression of neuronal Nav channels. Four distinct regulatory β-subunits (β1–4) bind to the Nav channel α-subunits. Previous work has emphasised the β-subunits as direct Nav channel gating modulators. However, there is now increasing appreciation of additional roles played by these subunits. In this review, we focus on β-subunits as homophilic and heterophilic cell-adhesion molecules and the implications for cardiomyocyte function. Based on recent cryogenic electron microscopy (cryo-EM) data, we suggest that the β-subunits interact with Nav1.5 in a different way from their binding to other Nav channel isoforms. We believe this feature may facilitate trans-cell-adhesion between β1-associated Nav1.5 subunits on the intercalated disc and promote ephaptic conduction between cardiomyocytes.

Contribution of the neuronal sodium channel NaV1.8 to sodium- and calcium-dependent cellular proarrhythmia

ObjectiveIn myocardial pathology such as heart failure a late sodium current (INaL) augmentation is known to be involved in conditions of arrhythmogenesis. However, the underlying mechanisms of the INaL generation are not entirely understood. By now evidence is growing that non-cardiac sodium channel isoforms could also be involved in the INaL generation. The present study investigates the contribution of the neuronal sodium channel isoform NaV1.8 to arrhythmogenesis in a clearly-defined setting of enhanced INaL by using anemone toxin II (ATX-II) in the absence of structural heart disease. MethodsElectrophysiological experiments were performed in order to measure INaL, action potential duration (APD), SR-Ca2+-leak and cellular proarrhythmic triggers in ATX-II exposed wild-type (WT) and SCN10A−/− mice cardiomyocytes. In addition, WT cardiomyocytes were stimulated with ATX-II in the presence or absence of NaV1.8 inhibitors. INCX was measured by using the whole cell patch clamp method. ResultsIn WT cardiomyocytes exposure to ATX-II augmented INaL, prolonged APD, increased SR-Ca2+-leak and induced proarrhythmic triggers such as early afterdepolarizations (EADs) and Ca2+-waves. All of them could be significantly reduced by applying NaV1.8 blockers PF-01247324 and A-803467. Both blockers had no relevant effects on cellular electrophysiology of SCN10A−/− cardiomyocytes. Moreover, in SCN10A−/−-cardiomyocytes, the ATX-II-dependent increase in INaL, SR-Ca2+-leak and APD prolongation was less than in WT and comparable to the results which were obtained with WT cardiomyocytes being exposed to ATX-II and NaV1.8 inhibitors in parallel. Moreover, we found a decrease in reverse mode NCX current and reduced CaMKII-dependent RyR2-phosphorylation after application of PF-01247324 as an underlying explanation for the Na+-mediated Ca2+-dependent proarrhythmic triggers. ConclusionThe current findings demonstrate that NaV1.8 is a significant contributor for INaL-induced arrhythmic triggers. Therefore, NaV1.8 inhibition under conditions of an enhanced INaL constitutes a promising antiarrhythmic strategy which merits further investigation.

Nav1.6 promotes inflammation and neuronal degeneration in a mouse model of multiple sclerosis

BackgroundIn multiple sclerosis (MS) and in the experimental autoimmune encephalomyelitis (EAE) model of MS, the Nav1.6 voltage-gated sodium (Nav) channel isoform has been implicated as a primary contributor to axonal degeneration. Following demyelination Nav1.6, which is normally co-localized with the Na+/Ca2+ exchanger (NCX) at the nodes of Ranvier, associates with β-APP, a marker of neural injury. The persistent influx of sodium through Nav1.6 is believed to reverse the function of NCX, resulting in an increased influx of damaging Ca2+ ions. However, direct evidence for the role of Nav1.6 in axonal degeneration is lacking.MethodsIn mice floxed for Scn8a, the gene that encodes the α subunit of Nav1.6, subjected to EAE we examined the effect of eliminating Nav1.6 from retinal ganglion cells (RGC) in one eye using an AAV vector harboring Cre and GFP, while using the contralateral either injected with AAV vector harboring GFP alone or non-targeted eye as control.ResultsIn retinas, the expression of Rbpms, a marker for retinal ganglion cells, was found to be inversely correlated to the expression of Scn8a. Furthermore, the gene expression of the pro-inflammatory cytokines Il6 (IL-6) and Ifng (IFN-γ), and of the reactive gliosis marker Gfap (GFAP) were found to be reduced in targeted retinas. Optic nerves from targeted eyes were shown to have reduced macrophage infiltration and improved axonal health.ConclusionTaken together, our results are consistent with Nav1.6 promoting inflammation and contributing to axonal degeneration following demyelination.

P1596Interaction of CaMKII and NaV1.8 modulates cardiac electrophysiology in human heart failure

Abstract Introduction In human heart failure, electrical remodeling contributes to the risk of arrhythmia generation. Increased expression of Ca/Calmodulin-dependent protein kinase IIδ (CaMKIIδ) and an enhanced persistent Na current (INaL) have been linked to arrhythmogenesis. CaMKIIδ increases INaL via regulation of sodium channels thereby contributing to arrhythmias through early- and delayed-afterdepolarizations (EADs and DADs). Genome-wide association studies (GWAS) have described the implication of the neuronal sodium channel isoform NaV1.8 (SCN10A) in cardiac electrophysiology showing modulation in cardiac conduction. We showed that the expression of the isoform Nav1.8 is significantly increased in human failing cardiomyocytes and contributes substantially to the enhanced INaL. Purpose We investigated a potential interaction of CaMKIIδ and NaV1.8 and thereby its role in arrhythmia generation and electrophysiology in human and murine failing hearts. Methods Cardiomyocytes were isolated from explanted failing hearts and CaMKIIδ transgenic (TG) mice. We performed immunostainings and co-immunoprecipitation (Co-IP) to show interactions of CaMKIIδ and Nav1.8 in isolated cardiomyocytes and homogenates. Whole-cell patch clamp experiments were conducted in isolated human and murine ventricular cardiomyocytes. Additionally, Ca2+ transients were measured using epifluorescence microscopy with the Ca2+ dye fura-2 (10μmol/L) whereas Ca2+ sparks measurements were performed by using confocal microscopy with the Ca2+ dye fluo-4 (10μmol/L). PF-01247324 is a novel specific NaV1.8 inhibitor (orally bioavailable; 1 μmol/L) and autocamtide inhibitory peptide (AIP, 1 μmol/L) was used to inhibit CaMKIIδ. Results Co-immunoprecipitation experiments revealed an association of CaMKIIδ and Nav1.8 in human homogenates compared to healthy controls. Furthermore, immunohistochemistry stainings in isolated human cardiomyocytes showed a co-localization of CaMKIIδ and NaV1.8 at the intercalated disc and t-tubules. We observed a significant reduction of INaL integral and proarrhythmic SR-Ca2+ spark frequency (CaSpF) after addition of either PF-01247324 or the CaMKIIδ inhibitor AIP in failing human and murine ventricular cardiomyocytes. When PF-01247324 and AIP were added together, the decrease in INaL integral and CaSpF was comparable to PF-01247324 alone in human failing cardiomyocytes. Inhibition of NaV1.8 did not show an effect on Ca2+ transient amplitude or Ca2+ transient decay at different stimulation frequencies in CaMKIIδ TG cardiomyocytes. Conclusion Our results demonstrate the significance of both CaMKIIδ and NaV1.8 in INaL generation and their detrimental interaction. This data suggest that increased CaMKIIδ activity plays a substantial role for the activation of NaV1.8-mediated late sodium current and SR-Ca2+ leak.

Sympathetic efferent neurons are less sensitive than nociceptors to 4 Hz sinusoidal stimulation.

Sinusoidal current stimuli preferentially activate C-nociceptors. Sodium channel isoforms NaV1.7 and NaV1.8 have been implicated in this. Sympathetic efferent neurons lack NaV1.8 and were explored upon sinusoidal activation. Quantitative Sudomotor Axon Reflex Test (QSART) was performed in hairy (n=16) and glabrous (n=12) skin. Responses of sympathetic efferents (n=10) and nociceptive afferents (n=21) to sinusoidal current stimulation (4Hz, 0.05-0.15mA) were recorded in humans by microneurography (n=11). Activation of sympathetic units upon supra-threshold sinusoidal currents (>0.8mA) was recorded in pigs (n=8). Sinusoidal stimuli (4Hz, 0.4mA) evoked weak sweat output (30ml/h/m2 ) in hairy skin compared to rectangular pulses (4Hz, 5mA, 53ml/h/m2 , p<.00001, ANOVA). No change in sweat output was recorded from glabrous skin to sine wave stimuli. Sinusoidal current at intensities ranging from 0.05 to 0.15mA activated almost all (85%) nociceptors but only 40% of sympathetic units in human. Stimuli lead to a significantly lower activation in sympathetic versus nociceptive fibres as measured by activity-dependent slowing (ADS) of conduction (sympathetic efferents average ADS 100±0.2% vs. C-nociceptors average ADS 113±4%, p<.003, ANOVA). Sympathetic efferent neurons are less apt to convert slow depolarizations into action potentials as compared to nociceptors. Distinctive sodium channel expression patterns between nociceptors and sympathetic efferent neurons may account for this difference. Sinusoidal stimulation therefore provokes weak sweat responses and provides no alternative for clinical assessment of autonomic function. C-nociceptors in hairy skin are activated by 4Hz sinusoidal current stimulation at lower intensities than myelinated fibres. Sympathetic efferent neurons-albeit also unmyelinated-are less responsive to sinusoidal activation than nociceptors within the same skin area. Cutaneous sympathetic efferent neurons apparently are less apt than nociceptors to convert slow depolarization into action potentials.

Challenges and Opportunities for Therapeutics Targeting the Voltage-Gated Sodium Channel Isoform NaV1.7.

Voltage-gated sodium ion channel subtype 1.7 (NaV1.7) is a high interest target for the discovery of non-opioid analgesics. Compelling evidence from human genetic data, particularly the finding that persons lacking functional NaV1.7 are insensitive to pain, has spurred considerable effort to develop selective inhibitors of this Na+ ion channel target as analgesic medicines. Recent clinical setbacks and disappointing performance of preclinical compounds in animal pain models, however, have led to skepticism around the potential of selective NaV1.7 inhibitors as human therapeutics. In this Perspective, we discuss the attributes and limitations of recently disclosed investigational drugs targeting NaV1.7 and review evidence that, by better understanding the requirements for selectivity and target engagement, the opportunity to deliver effective analgesic medicines targeting NaV1.7 endures.

Phoneutria nigriventer spider toxin PnTx2-1 (δ-Ctenitoxin-Pn1a) is a modulator of sodium channel gating

Spider venoms are complex mixtures of biologically active components with potentially interesting applications for drug discovery or for agricultural purposes. The spider Phoneutria nigriventer is responsible for a number of envenomations with sometimes severe clinical manifestations in humans. A more efficient treatment requires a comprehensive knowledge of the venom composition and of the action mechanism of the constituting components. PnTx2-1 (also called δ-ctenitoxin-Pn1a) is a 53-amino-acid-residue peptide isolated from the venom fraction PhTx2. Although PnTx2-1 is classified as a neurotoxin, its molecular target has remained unknown. This study describes the electrophysiological characterization of PnTx2-1 as a modulator of voltage-gated sodium channels. PnTx2-1 is investigated for its activity on seven mammalian NaV-channel isoforms, one insect NaV channel and one arachnid NaV channel. Furthermore, comparison of the activity of both PnTx2-1 and PnTx2-6 on NaV1.5 channels reveals that this family of Phoneutria toxins modulates the cardiac NaV channel in a bifunctional manner, resulting in an alteration of the inactivation process and a reduction of the sodium peak current.