Biophysical mechanisms underlying tefluthrin-induced modulation of gating changes and resurgent current generation in the human Nav1.4 channel.

Human skeletal muscle contraction is triggered by activation of Nav1.4 channels. Nav1.4 channels can generate resurgent currents by channel reopening at hyperpolarized potentials through a gating transition dependent on the intracellular Navß4 peptide in the physiological conditions. Tefluthrin (TEF) is a pyrethroid insecticide that can disrupt electrical signaling in nerves and skeletal muscle, resulting in seizures, muscle spasms, fasciculations, and mental confusion. TEF can also induce tail currents through other voltage-gated sodium channels in the absence of Navß4 peptide, suggesting that muscle spasms may be caused by resurgent currents. Further, intracellular Navß4 peptide and extracellular TEF may show competitive or synergistic effects; however, their binding sites are still unknown. To address these issues, electrophysiological recordings were performed on CHO-K1 cells expressing Nav1.4 channels with intracellular Navß4 peptide, extracellular TEF, or both. TEF and Navß4 peptide induced a hyperpolarizing shift of activation and inactivation curves in the Nav1.4 channel. TEF also substantially prolonged the inactivation time constants, while simultaneous application of Navß4 peptide partially reversed this effect. Resurgent currents were enhanced by TEF and Navß4 peptide at negative potentials, but TEF more potently enhances resurgent currents and dampens decay of resurgent currents. With longer depolarization, peak resurgent currents decay was fastest with the TEF alone. Molecular docking suggested that TEF and Navß4 peptide binding site(s) are not in the narrowest part of the channel pore, but rather in the bundle-crossing regions and in the domain linkers, respectively. TEF can induce resurgent currents independently and synergistically with Navß4 peptide, which may explain the muscle spasms observed in TEF intoxication.

Inhibition of resurgent Na+ currents by rufinamide.

Na+ channels are essential for the genesis of action potentials in most neurons. After opening by membrane depolarization, Na+ channels enter a series of inactivated states (e.g. the fast, intermediate, and slow inactivated states; or If, Ii, and Is). The inactivated Na+ channel may recover via the open state upon membrane repolarization, giving rise to "resurgent" Na+ currents which could be critical for densely repetitive or burst discharges. We incubated CHO-K1 cells transfected with human NaV1.7 cDNA and measured resurgent currents with whole-cell patch recordings. We found Ii is the major inactivated state responsible for the genesis of resurgent currents. Rufinamide, in therapeutic concentrations, could selectively bind to Ii to slow the recovery process and dose-dependently inhibit resurgent currents. The other Na+ channel-inhibiting antiseizure medications (ASM), such as phenytoin and lacosamide (selectively binds to If and Is, separately), fail to show a similar inhibitory effect in clinically relevant concentrations. Resurgent currents are decreased with lengthening of the prepulse, presumably because of redistribution of the channel from Ii to If. Rufinamide could accentuate the decrease to mimic a use-dependent inhibitory effect. The molecular action of slowing of recovery from inactivation by binding to Ii also explains the highly correlative inhibitory effect of rufinamide on both transient and resurgent Na+ currents. The modest but correlative inhibition of both currents may make a novel synergistic effect and thus strong-enough suppression of pathological repetitive and especially burst discharges. Rufinamide may thus have a unique spectrum of therapeutic applications for disorders with excessive neural excitabilities.

Effects of transient, persistent, and resurgent sodium currents on excitability and spike regularity in vestibular ganglion neurons.

Vestibular afferent neurons occur as two populations, regular and irregular, that provide distinct information about head motions. Differences in spike timing regularity are correlated with the different sensory responses important for vestibular processing. Relative to irregular afferents, regular afferents have more sustained firing patterns in response to depolarizing current steps, are more excitable, and have different complements of ion channels. Models of vestibular regularity and excitability emphasize the influence of increased expression of low-voltage-activated potassium currents in irregular neurons. We investigated the potential impact of different modes of voltage-gated sodium (NaV) current (transient, persistent, and resurgent) in cell bodies from vestibular ganglion neurons (VGNs), dissociated and cultured overnight. We hypothesized that regular VGNs would show the greatest impact of persistent (non-inactivating) NaV currents and of resurgent NaV currents, which flow when NaV channels are blocked and then unblocked. Whole-cell patch clamp experiments showed that much of the NaV current modes is carried by NaV1.6 channels. With simulations, we detected little substantial effect in any model VGN of persistent or resurgent modes on regularity of spike timing driven by postsynaptic current trains. For simulated irregular neurons, we also saw little effect on spike rate or firing pattern. For simulated regular VGNs, adding resurgent current changed the detailed timing of spikes during a current step, while the small persistent conductance (less than10% of transient NaV conductance density) strongly depolarized resting potential, altered spike waveform, and increased spike rate. These results suggest that persistent and resurgent NaV current can have a greater effect on the regular VGNs than on irregular VGNs, where low-voltage-activated K conductances dominate.

Dataset of electrophysiological patch-clamp recordings of the effect of the compounds deltamethrin, ATx-II and ß4-peptide on human cardiac Nav1.5 sodium channel gating properties.

This article describes the effect of the pyrethroid insecticide deltamethrin on the cardiac voltage-gated sodium channel Nav1.5. Two concentrations of deltamethrin were used and the effects were compared with those of the sea anemone toxin ATx-II and ß4-peptide, which is the C-terminus of the Nav channel ß-subunit. Activation, fast inactivation, deactivation, persistent currents and resurgent currents of Nav1.5 channels were assessed in the presence of these compounds. The data display not only the effect of separately applied compounds on Nav1.5 channels but also investigates how combinations of these substances affect Nav1.5 channel gating properties. The dataset presented in this article is related to the research article "Mechanism underlying hooked resurgent-like tail currents induced by an insecticide in human cardiac Nav1.5″ (Sarah Thull, Cristian Neacsu, Andrias O. O'Reilly, Stefanie Bothe, Ralf Hausmann, Tobias Huth, Jannis Meents, Angelika Lampert, doi: 10.1016/j.taap.2020.11501), that investigates the effect of the pyrethroid insecticide deltamethrin on Nav channel gating properties and explains the mechanism underlying hooked, resurgent-like tail currents induced by deltamethrin in Nav1.5 channels.

Differential Inhibition of Human Nav1.2 Resurgent and Persistent Sodium Currents by Cannabidiol and GS967.

Many epilepsy patients are refractory to conventional antiepileptic drugs. Resurgent and persistent currents can be enhanced by epilepsy mutations in the Nav1.2 channel, but conventional antiepileptic drugs inhibit normal transient currents through these channels, along with aberrant resurgent and persistent currents that are enhanced by Nav1.2 epilepsy mutations. Pharmacotherapies that specifically target aberrant resurgent and/or persistent currents would likely have fewer unwanted side effects and be effective in many patients with refractory epilepsy. This study investigated the effects of cannbidiol (CBD) and GS967 (each at 1 µM) on transient, resurgent, and persistent currents in human embryonic kidney (HEK) cells stably expressing wild-type hNav1.2 channels. We found that CBD preferentially inhibits resurgent currents over transient currents in this paradigm; and that GS967 preferentially inhibits persistent currents over transient currents. Therefore, CBD and GS967 may represent a new class of more targeted and effective antiepileptic drugs.

Changes of Resurgent Na+ Currents in the Nav1.4 Channel Resulting from an SCN4A Mutation Contributing to Sodium Channel Myotonia.

Myotonia congenita (MC) is a rare disorder characterized by stiffness and weakness of the limb and trunk muscles. Mutations in the SCN4A gene encoding the alpha-subunit of the voltage-gated sodium channel Nav1.4 have been reported to be responsible for sodium channel myotonia (SCM). The Nav1.4 channel is expressed in skeletal muscles, and its related channelopathies affect skeletal muscle excitability, which can manifest as SCM, paramyotonia and periodic paralysis. In this study, the missense mutation p.V445M was identified in two individual families with MC. To determine the functional consequences of having a mutated Nav1.4 channel, whole-cell patch-clamp recording of transfected Chinese hamster ovary cells was performed. Evaluation of the transient Na+ current found that a hyperpolarizing shift occurs at both the activation and inactivation curves with an increase of the window currents in the mutant channels. The Nav1.4 channel's co-expression with the Navß4 peptide can generate resurgent Na+ currents at repolarization following a depolarization. The magnitude of the resurgent currents is higher in the mutant than in the wild-type (WT) channel. Although the decay kinetics are comparable between the mutant and WT channels, the time to the peak of resurgent Na+ currents in the mutant channel is significantly protracted compared with that in the WT channel. These findings suggest that the p.V445M mutation in the Nav1.4 channel results in an increase of both sustained and resurgent Na+ currents, which may contribute to hyperexcitability with repetitive firing and is likely to facilitate recurrent myotonia in SCM patients.

Mechanism underlying hooked resurgent-like tail currents induced by an insecticide in human cardiac Nav1.5.

Voltage-gated sodium channels are responsible not only for the fast upstroke of the action potential, but they also modify cellular excitability via persistent and resurgent currents. Insecticides act via permanently opening sodium channels to immobilize the animals. Cellular recordings performed decades ago revealed distinctly hooked tail currents induced by these compounds. Here, we applied the classical type-II pyrethroid deltamethrin on human cardiac Nav1.5 and observed resurgent-like currents at very negative potentials in the absence of any pore-blocker, which resemble those hooked tail currents. We show that deltamethrin dramatically slows both fast inactivation and deactivation of Nav1.5 and thereby induces large persistent currents. Using the sea anemone toxin ATx-II as a tool to prevent all inactivation-related processes, resurgent-like currents were eliminated while persistent currents were preserved. Our experiments suggest that, in deltamethrin-modified channels, recovery from inactivation occurs faster than delayed deactivation, opening a brief window for sodium influx and leading to hooked, resurgent-like currents, in the absence of an open channel blocker. Thus, we now explain with pharmacological methods the biophysical gating changes underlying the deltamethrin induced hooked tail currents. SUMMARY: The pyrethroid deltamethrin induces hooked resurgent-like tail currents in human cardiac voltage-gated Nav1.5 channels. Using deltamethrin and ATx-II, we identify additional conducting channel states caused by a faster recovery from inactivation compared to the deltamethrin-induced delayed deactivation.

Distinct functional alterations in SCN8A epilepsy mutant channels.

KEY POINTS: Mutations in the SCN8A gene cause early infantile epileptic encephalopathy. We characterize a new epilepsy-related SCN8A mutation, R850Q, in the human SCN8A channel and present gain-of-function properties of the mutant channel. Systematic comparison of R850Q with three other SCN8A epilepsy mutations, T761I, R1617Q and R1872Q, identifies one common dysfunction in resurgent current, although these mutations alter distinct properties of the channel. Computational simulations in two different neuron models predict an increased excitability of neurons carrying these mutations, which explains the over-excitation that underlies seizure activities in patients. These data provide further insight into the mechanism of SCN8A-related epilepsy and reveal subtle but potentially important distinction of functional characterization performed in the human vs. rodent channels. ABSTRACT: SCN8A is a novel causal gene for early infantile epileptic encephalopathy. It is well accepted that gain-of-function mutations in SCN8A underlie the disorder, although the remarkable heterogeneity of its clinical presentation and poor treatment response demand a better understanding of the disease mechanisms. Here, we characterize a new epilepsy-related SCN8A mutation, R850Q, in human Nav1.6. We show that it is a gain-of-function mutation, with a hyperpolarizing shift in voltage dependence of activation, a two-fold increase of persistent current and a slowed decay of resurgent current. We systematically compare its biophysics with three other SCN8A epilepsy mutations, T767I, R1617Q and R1872Q, in the human Nav1.6 channel. Although all of these mutations are gain-of-function, the mutations affect different aspects of channel properties. One commonality that we discovered is an alteration of resurgent current kinetics, although the mechanisms by which resurgent currents are augmented remain unclear for all of the mutations. Computational simulations predict an increased excitability of neurons carrying these mutations with differential enhancement by open channel blockade.

Resurgent and Gating Pore Currents Induced by De Novo SCN2A Epilepsy Mutations.

Over 150 mutations in the SCN2A gene, which encodes the neuronal Nav1.2 protein, have been implicated in human epilepsy cases. Of these, R1882Q and R853Q are two of the most commonly reported mutations. This study utilized voltage-clamp electrophysiology to characterize the biophysical effects of the R1882Q and R853Q mutations on the hNav1.2 channel, including their effects on resurgent current and gating pore current, which are not typically investigated in the study of Nav1.2 channel mutations. HEK cells transiently transfected with DNA encoding either wild-type (WT) or mutant hNav1.2 revealed that the R1882Q mutation induced a gain-of-function phenotype, including slowed fast inactivation, depolarization of the voltage dependence of inactivation, and increased persistent current. In this model system, the R853Q mutation primarily produced loss-of-function effects, including reduced transient current amplitude and density, hyperpolarization of the voltage dependence of inactivation, and decreased persistent current. The presence of a Navß4 peptide (KKLITFILKKTREK-OH) in the pipette solution induced resurgent currents, which were increased by the R1882Q mutation and decreased by the R853Q mutation. Further study of the R853Q mutation in Xenopus oocytes indicated a reduced surface expression and revealed a robust gating pore current at negative membrane potentials, a function absent in the WT channel. This not only shows that different epileptogenic point mutations in hNav1.2 have distinct biophysical effects on the channel, but also illustrates that individual mutations can have complex consequences that are difficult to identify using conventional analyses. Distinct mutations may, therefore, require tailored pharmacotherapies in order to eliminate seizures.

Identification of Persistent and Resurgent Sodium Currents in Spiral Ganglion Neurons Cultured from the Mouse Cochlea.

In spiral ganglion neurons (SGNs), the afferent single units of the auditory nerve, high spontaneous and evoked firing rates ensure preservation of the temporal code describing the key features of incoming sound. During postnatal development, the spatiotemporal distribution of ion channel subtypes contributes to the maturation of action potential generation in SGNs, and to their ability to generate spike patterns that follow rapidly changing inputs. Here we describe tetrodotoxin (TTX)-sensitive Na+ currents in SGNs cultured from mice, whose properties may support this fast spiking behavior. A subthreshold persistent Na+ current (INaP) and a resurgent Na+ current (INaR) both emerged prior to the onset of hearing and became more prevalent as hearing matured. Navß4 subunits, which are proposed to play a key role in mediating INaR elsewhere in the nervous system, were immunolocalized to the first heminode where spikes are generated in the auditory nerve, and to perisomatic nodes of Ranvier. ATX-II, a sea anemone toxin that slows classical Na+ channel inactivation selectively, enhanced INaP five-fold and INaR three-fold in voltage clamp recordings. In rapidly-adapting SGNs under current clamp, ATX-II increased the likelihood of firing additional action potentials. The data identify INaP and INaR as novel regulators of excitability in SGNs, and consistent with their roles in other neuronal types, we suggest that these nonclassical Na+ currents may contribute to the control of refractoriness in the auditory nerve.

FHF2 isoforms differentially regulate Nav1.6-mediated resurgent sodium currents in dorsal root ganglion neurons.

Nav1.6 and Nav1.6-mediated resurgent currents have been implicated in several pain pathologies. However, our knowledge of how fast resurgent currents are modulated in neurons is limited. Our study explored the potential regulation of Nav1.6-mediated resurgent currents by isoforms of fibroblast growth factor homologous factor 2 (FHF2) in an effort to address the gap in our knowledge. FHF2 isoforms colocalize with Nav1.6 in peripheral sensory neurons. Cell line studies suggest that these proteins differentially regulate inactivation. In particular, FHF2A mediates long-term inactivation, a mechanism proposed to compete with the open-channel blocker mechanism that mediates resurgent currents. On the other hand, FHF2B lacks the ability to mediate long-term inactivation and may delay inactivation favoring open-channel block. Based on these observations, we hypothesized that FHF2A limits resurgent currents, whereas FHF2B enhances resurgent currents. Overall, our results suggest that FHF2A negatively regulates fast resurgent current by enhancing long-term inactivation and delaying recovery. In contrast, FHF2B positively regulated resurgent current and did not alter long-term inactivation. Chimeric constructs of FHF2A and Navß4 (likely the endogenous open channel blocker in sensory neurons) exhibited differential effects on resurgent currents, suggesting that specific regions within FHF2A and Navß4 have important regulatory functions. Our data also indicate that FHFAs and FHF2B isoform expression are differentially regulated in a radicular pain model and that associated neuronal hyperexcitability is substantially attenuated by a FHFA peptide. As such, these findings suggest that FHF2A and FHF2B regulate resurgent current in sensory neurons and may contribute to hyperexcitability associated with some pain pathologies.

A gain-of-function mutation in Nav1.6 in a case of trigeminal neuralgia.

Idiopathic trigeminal neuralgia (TN) is a debilitating pain disorder characterized by episodic unilateral facial pain along the territory of branches of the trigeminal nerve. Human painful disorders, but not TN, have been linked to gain-of-function mutations in peripheral voltage-gated sodium channels (NaV1.7, NaV1.8 and NaV1.9). Gain-of-function mutations in NaV1.6, which is expressed in myelinated and unmyelinated CNS and peripheral nervous system neurons and supports neuronal high-frequency firing, have been linked to epilepsy but not to pain. Here, we describe an individual who presented with evoked and spontaneous paroxysmal unilateral facial pain, and carried a diagnosis of TN. Magnetic resonance imaging showed unilateral neurovascular compression, consistent with pain in areas innervated by the second branch of the trigeminal nerve. Genetic analysis as part of a phase 2 clinical study in patients with TN conducted by Convergence Pharmaceuticals Ltd revealed a previously undescribed de novo missense mutation in NaV1.6 (c.A406G; p.Met136Val). Whole-cell voltage-clamp recordings show that the Met136Val mutation significantly increases peak current density (1.5-fold) and resurgent current (1.6-fold) without altering gating properties. Current-clamp studies in trigeminal ganglion (TRG) neurons showed that Met136Val increased the fraction of high-firing neurons, lowered the current threshold and increased the frequency of evoked action potentials in response to graded stimuli. Our results demonstrate a novel NaV1.6 mutation in TN, and show that this mutation potentiates transient and resurgent sodium currents and leads to increased excitability in TRG neurons. We suggest that this gain-of-function NaV1.6 mutation may exacerbate the pathophysiology of vascular compression and contribute to TN.

Ion channel regulation by ß-secretase BACE1 - enzymatic and non-enzymatic effects beyond Alzheimer's disease.

ß-site APP-cleaving enzyme 1 (BACE1) has become infamous for its pivotal role in the pathogenesis of Alzheimer's disease (AD). Consequently, BACE1 represents a prime target in drug development. Despite its detrimental involvement in AD, it should be quite obvious that BACE1 is not primarily present in the brain to drive mental decline. In fact, additional functions have been identified. In this review, we focus on the regulation of ion channels, specifically voltage-gated sodium and KCNQ potassium channels, by BACE1. These studies provide evidence for a highly unexpected feature in the functional repertoire of BACE1. Although capable of cleaving accessory channel subunits, BACE1 exerts many of its physiologically significant effects through direct, non-enzymatic interactions with main channel subunits. We discuss how the underlying mechanisms can be conceived and develop scenarios how the regulation of ion conductances by BACE1 might shape electric activity in the intact and diseased brain and heart.

Aberrant epilepsy-associated mutant Nav1.6 sodium channel activity can be targeted with cannabidiol.

Mutations in brain isoforms of voltage-gated sodium channels have been identified in patients with distinct epileptic phenotypes. Clinically, these patients often do not respond well to classic anti-epileptics and many remain refractory to treatment. Exogenous as well as endogenous cannabinoids have been shown to target voltage-gated sodium channels and cannabidiol has recently received attention for its potential efficacy in the treatment of childhood epilepsies. In this study, we further investigated the ability of cannabinoids to modulate sodium currents from wild-type and epilepsy-associated mutant voltage-gated sodium channels. We first determined the biophysical consequences of epilepsy-associated missense mutations in both Nav1.1 (arginine 1648 to histidine and asparagine 1788 to lysine) and Nav1.6 (asparagine 1768 to aspartic acid and leucine 1331 to valine) by obtaining whole-cell patch clamp recordings in human embryonic kidney 293T cells with 200 µM Navß4 peptide in the pipette solution to induce resurgent sodium currents. Resurgent sodium current is an atypical near threshold current predicted to increase neuronal excitability and has been implicated in multiple disorders of excitability. We found that both mutations in Nav1.6 dramatically increased resurgent currents while mutations in Nav1.1 did not. We then examined the effects of anandamide and cannabidiol on peak transient and resurgent currents from wild-type and mutant channels. Interestingly, we found that cannabidiol can preferentially target resurgent sodium currents over peak transient currents generated by wild-type Nav1.6 as well as the aberrant resurgent and persistent current generated by Nav1.6 mutant channels. To further validate our findings, we examined the effects of cannabidiol on endogenous sodium currents from striatal neurons, and similarly we found an inhibition of resurgent and persistent current by cannabidiol. Moreover, current clamp recordings show that cannabidiol reduces overall action potential firing of striatal neurons. These findings suggest that cannabidiol could be exerting its anticonvulsant effects, at least in part, through its actions on voltage-gated sodium channels, and resurgent current may be a promising therapeutic target for the treatment of epilepsy syndromes.

Protein kinase C enhances human sodium channel hNav1.7 resurgent currents via a serine residue in the domain III-IV linker.

Resurgent sodium currents likely play a role in modulating neuronal excitability. Here we studied whether protein kinase C (PKC) activation can increase resurgent currents produced by the human sodium channel hNav1.7. We found that a PKC agonist significantly enhanced hNav1.7-mediated resurgent currents and this was prevented by PKC antagonists. The enhancing effects were replicated by two phosphorylation-mimicking mutations and were prevented by a phosphorylation-deficient mutation at a conserved PKC phosphorylation site (Serine 1479). Our results suggest that PKC can increase sodium resurgent currents through phosphorylation of a conserved Serine residue located in the domain III-IV linker of sodium channels.