Voltage-gated sodium channels

Epilepsy is a brain disorder characterized by seizures and convulsions. The basis of epilepsy is an increase in neuronal excitability that, in some cases, may be caused by functional defects in neuronal voltage gated sodium channels, Nav1.1 and Nav1.2. The effects of antiepileptic drugs (AEDs) as effective therapies for epilepsy have been characterized by extensive research. Most of the classic AEDs targeting Nav share a common mechanism of action by stabilizing the channel’s fast-inactivated state. In contrast, novel AEDs, such as lacosamide, stabilize the slow-inactivated state in neuronal Nav1.1 and Nav1.7 isoforms. This paper reviews the different mechanisms by which this stabilization occurs to determine new methods for treatment.     

Alternative splicing modulates inactivation of type 1 voltage-gated sodium channels by toggling an amino acid in the first S3-S4 linker

Voltage-gated sodium channels underlie the upstroke of action potentials and are fundamental to neuronal excitability. Small changes in the behavior of these channels are sufficient to change neuronal firing and trigger seizures. These channels are subject to highly conserved alternative splicing, affecting the short linker between the third transmembrane segment (S3) and the voltage sensor (S4) in their first domain. The biophysical consequences of this alternative splicing are incompletely understood. Here we focus on type 1 sodium channels (Nav1.1) that are implicated in human epilepsy. We show that the functional consequences of alternative splicing are highly sensitive to recording conditions, including the identity of the major intracellular anion and the recording temperature. In particular, the inactivation kinetics of channels containing the alternate exon 5N are more sensitive to intracellular fluoride ions and to changing temperature than channels containing exon 5A. Moreover, Nav1.1 channels containing exon 5N recover from inactivation more rapidly at physiological temperatures. Three amino acids differ between exons 5A and 5N. However, the changes in sensitivity and stability of inactivation were reproduced by a single conserved change from aspartate to asparagine in channels containing exon 5A, which was sufficient to make them behave like channels containing the complete exon 5N sequence. These data suggest that splicing at this site can modify the inactivation of sodium channels and reveal a possible interaction between splicing and anti-epileptic drugs that stabilize sodium channel inactivation.     

Mutant LGI1 inhibits seizure-induced trafficking of Kv4.2 potassium channels

Activity-dependent redistribution of ion channels mediates neuronal circuit plasticity and homeostasis, and could provide pro-epileptic or compensatory anti-epileptic responses to a seizure. Thalamocortical neurons transmit sensory information to the cerebral cortex and through reciprocal corticothalamic connections are intensely activated during a seizure. Therefore, we assessed whether a seizure alters ion channel surface expression and consequent neurophysiologic function of thalamocortical neurons. We report a seizure triggers a rapid (<2h) decrease of excitatory postsynaptic current (EPSC)-like current-induced phasic firing associated with increased transient A-type K(+) current. Seizures also rapidly redistributed the A-type K(+) channel subunit Kv4.2 to the neuronal surface implicating a molecular substrate for the increased K(+) current. Glutamate applied in vitro mimicked the effect, suggesting a direct effect of glutamatergic transmission. Importantly, leucine-rich glioma-inactivated-1 (LGI1), a secreted synaptic protein mutated to cause human partial epilepsy, regulated this seizure-induced circuit response. Human epilepsy-associated dominant-negative-truncated mutant LGI1 inhibited the seizure-induced suppression of phasic firing, increase of A-type K(+) current, and recruitment of Kv4.2 surface expression (in vivo and in vitro). The results identify a response of thalamocortical neurons to seizures involving Kv4.2 surface recruitment associated with dampened phasic firing. The results also identify impaired seizure-induced increases of A-type K(+) current as an additional defect produced by the autosomal dominant lateral temporal lobe epilepsy gene mutant that might contribute to the seizure disorder.     

Post-Treatment with Voltage-Gated Na+ Channel Blocker Attenuates Kainic Acid-Induced Apoptosis in Rat Primary Hippocampal Neurons

Injection of rats with kainic acid (KA), a non-N-methyl-D-aspartate (NMDA) type glutamate receptor agonist, induces recurrent (delayed) convulsive seizures and subsequently hippocampal neurodegeneration, which is reminiscent of human epilepsy. The protective effect of anti-epileptic drugs on seizure-induced neuronal injury is well known; however, molecular basis of this protective effect has not yet been elucidated. In this study, we investigated the effect and signaling mediators of voltage-gated Na(+) channel blockers (Lamotrigine, Rufinamide, Oxcarbazepine, Valproic Acid, and Zonisamide) on KA-induced apoptosis in rat primary hippocampal neurons. Exposure of hippocampal neurons to 10 μM KA for 24 h caused significant increases in morphological and biochemical features of apoptosis, as determined by Wright staining and ApopTag assay, respectively. Analyses showed increases in expression and activity of cysteine proteases, production of reactive oxygen species (ROS), intracellular free [Ca(2+)], and Bax:Bcl-2 ratio during apoptosis. Cells exposed to KA for 15 min were then treated with Lamotrigine, Rufinamide, Oxcarbazepine, Valproic Acid, or Zonisamide. Post-treatment with one of these anti-epileptic drugs (500 nM) attenuated production of ROS and prevented apoptosis in hippocampal neurons. Lamotrigine, Rufinamide, and Oxcarbazepine appeared to be less protective when compared with Valproic Acid or Zonisamide. This difference may be due to blockade of T-type Ca(2+) channels also by Valproic Acid and Zonisamide. Our findings thus suggest that the anti-epileptic drugs that block both Na(+) channels and Ca(2+) channels are significantly more effective than agents that block only Na(+) channels for attenuating seizure-induced hippocampal neurodegeneration.     

Dynamic compartmentalization of the voltage-gated sodium channels in axons

One of the major physiological roles of the neuronal voltage-gated sodium channel is to generate action potentials at the axon hillock/initial segment and to ensure propagation along myelinated or unmyelinated fibers to nerve terminal. These processes require a precise distribution of sodium channels accumulated at high density in discrete subdomains of the nerve membrane. In neurons, information relevant to ion channel trafficking and compartmentalization into sub-domains of the plasma membrane is far from being elucidated. Besides, whereas information on dendritic targeting is beginning to emerge, less is known about the mechanisms leading to the polarized distribution of proteins in axon. To obtain a better understanding of how neurons selectively target sodium channels to discrete subdomains of the nerve, we addressed the question as to whether any of the large intracellular regions of Nav1.2 contain axonal sorting and/or clustering signals. We first obtained evidence showing that addition of the cytoplasmic carboxy-terminal region of Nav1.2 restricted the distribution of a dendritic-axonal reporter protein to axons of hippocampal neurons. The analysis of mutants revealed that a di-leucine-based motif mediates chimera compartmentalization in axons and its elimination in soma and dendrites by endocytosis. The analysis of the others generated chimeras showed that the determinant conferring sodium channel clustering at the axonal initial segment is contained within the cytoplasmic loop connecting domains II-III of Nav1.2. Expression of a soluble Nav1.2 II-III linker protein led to the disorganization of endogenous sodium channels. The motif was sufficient to redirect a somatodendritic potassium channel to the axonal initial segment, a process involving association with ankyrin G. Thus, it is conceivable that concerted action of the two determinants is required for sodium channel compartmentalization in axons.     

Drosophila as a model for epilepsy: bss is a gain-of-function mutation in the para sodium channel gene that leads to seizures

We report the identification of bang senseless (bss), a Drosophila melanogaster mutant exhibiting seizure-like behaviors, as an allele of the paralytic (para) voltage-gated Na(+) (Na(V)) channel gene. Mutants are more prone to seizure episodes than normal flies because of a lowered seizure threshold. The bss phenotypes are due to a missense mutation in a segment previously implicated in inactivation, termed the "paddle motif" of the Na(V) fourth homology domain. Heterologous expression of cDNAs containing the bss(1) lesion, followed by electrophysiology, shows that mutant channels display altered voltage dependence of inactivation compared to wild type. The phenotypes of bss are the most severe of the bang-sensitive mutants in Drosophila and can be ameliorated, but not suppressed, by treatment with anti-epileptic drugs. As such, bss-associated seizures resemble those of pharmacologically resistant epilepsies caused by mutation of the human Na(V) SCN1A, such as severe myoclonic epilepsy in infants or intractable childhood epilepsy with generalized tonic-clonic seizures.     

Differential expression of K+ channel mRNAs in the rat brain and down-regulation in the hippocampus following seizures.

K+ channels are major determinants of membrane excitability. Differences in neuronal excitability within the nervous system may arise from differential expression of K+ channel genes, regulated spatially in a cell type-specific manner, or temporally in response to neuronal activity. We have compared the distribution of mRNAs of three K+ channel genes, Kv1.1, Kv1.2, and Kv4.2 in rat brain, and examined activity-dependent changes following treatment with the convulsant drug pentylenetetrazole. Both regional and cell type-specific differences of K+ channel gene expression were found. In addition, seizure activity caused a reduction of Kv1.2 and Kv4.2 mRNAs in the dentate granule cells of the hippocampus, raising the possibility that K+ channel gene regulation may play a role in long-term neuronal plasticity.     

Mitochondrial dysfunction in epilepsy

Mitochondrial dysfunction has been identified as one potential cause of epileptic seizures. Impaired mitochondrial function has been reported for the seizure focus of patients with temporal lobe epilepsy and Ammon's horn sclerosis and of adult and immature animal models of epilepsy. Since mitochondrial oxidative phosphorylation provides the major source of ATP in neurons and mitochondria participate in cellular Ca(2+) homeostasis and generation of reactive oxygen species, their dysfunction strongly affects neuronal excitability and synaptic transmission. Therefore, mitochondrial dysfunction is proposed to be highly relevant for seizure generation. Additionally, mitochondrial dysfunction is known to trigger neuronal cell death, which is a prominent feature of therapy-resistant epilepsy. For this reason mitochondria have to be considered as promising targets for neuroprotective strategies in epilepsy.     

Determinants of voltage-gated sodium channel clustering in neurons

In mammalian neurons, the generation and propagation of the action potential result from the presence of dense clusters of voltage-gated sodium channels (Nav) at the axonal initial segment (AIS) and nodes of Ranvier. In these two structures, the assembly of specific supra-molecular complexes composed of numerous partners, such as cytoskeletal scaffold proteins and signaling proteins ensures the high concentration of Nav channels. Understanding how neurons regulate the expression and discrete localization of Nav channels is critical to understanding the diversity of normal neuronal function as well as neuronal dysfunction caused by defects in these processes. Here, we review the mechanisms establishing the clustering of Nav channels at the AIS and in the node and discuss how the alterations of Nav channel clustering can lead to certain pathophysiologies.     

Hippocampal neurons and glia in epileptic EL mice

Reactive changes in hippocampal astrocytes are frequently encountered in association with temporal lobe epilepsy in humans and with drug or kindling-induced seizures in animal models. These reactive changes generally involve increases in astrocyte size and number and often occur together with neuronal loss and synaptic rearrangements. In addition to producing astrocytic changes, seizure activity can also produce reactive changes in microglia, the resident macrophages of brain. In this study, we examined the effects of recurrent seizure activity on hippocampal neurons and glia in the epileptic EL mouse, a natural model of human multifactorial idiopathic epilepsy and complex partial seizures. Timm staining was used to evaluate infrapyramidal mossy fiber organization and the optical dissector method was used to count Nissl-stained neurons in hippocampus of adult (about one year of age) EL mice and nonepileptic C57BL/6J (B6) and DDY mice. Immunostaining forglial fibrillary acidic protein (GFAP) and Iba1, an actin cross-linking molecule restricted to macrophages and microglia, was used to evaluate astrocytes and microglia, respectively. The EL mice experienced about 25–30 complex partial seizures with secondary generalization during routine weekly cage changing. No significant differences were found among the mouse strains for Timm staining scores or for neuronal counts in the CA1 and CA3 pyramidal fields or in the hilus. However, the number of GFAP-positive astrocytes was significantly elevated in the stratum radiatum and hilus of EL mice, while microglia appeared hyper-ramified and were more intensely stained in EL mice than in the B6 or DDY mice in the hilus, parietal cortex, and pyriform cortex. The results indicate that recurrent seizure activity in EL mice is associated with abnormalities in hippocampal astrocytes and brain microglia, but is not associated with obvious neuronal loss or mossy fiber synaptic rearrangements. The EL mouse can be a useful model for evaluating neuron-glia interactions related to idiopathic epilepsy.     

ECS Dynamism and Its Influence on Neuronal Excitability and Seizures

Seizure activity is governed by changes in normal neuronal physiology that lead to a state of neuronal hyperexcitability and synchrony. There is a growing body of research and evidence suggesting that alterations in the volume fraction (α) of the brain’s extracellular space (ECS) have the ability to prolong or even initiate seizures. These ictogenic effects likely occur due to the ECS volume being critically important in determining both the concentration of neuroactive substances contained within it, such as ions and neurotransmitters, and the effect of electric field-mediated interactions between neurons. Changes in the size of the ECS likely both precede a seizure, assisting in its initiation, and occur during a seizure, assisting in its maintenance. Different cellular ion and water transporters and channels are essential mediators in determining neuronal excitability and synchrony and can do so through alterations in ECS volume and/or through non-ECS volume related mechanisms. This review will parse out the relationships between how the ECS volume changes during normal physiology and seizures, how those changes might alter neuronal physiology to promote seizures, and what ion and water transporters and channels are important in linking ECS volume changes and seizures.

     

Use-dependent potentiation of the Nav1.6 sodium channel

Nav1.2 and Nav1.6 are two voltage-gated sodium channel isoforms that are abundant in the adult central nervous system. These channels are expressed in different cells and localized in different neuronal regions, which may reflect functional specialization. To examine this possibility, we compared the properties of Nav1.2 and Nav1.6 in response to a rapid series of repetitive depolarizations. Currents through Nav1.6 coexpressed with beta1 demonstrated use-dependent potentiation during a rapid train of depolarizations. This potentiation was in contrast to the use-dependent decrease in current for Nav1.2 with beta1. The voltage dependence of potentiation correlated with the voltage dependence of activation, and it still occurred when fast inactivation was removed by mutation. Rapid stimulation accelerated a slow phase of activation in the Nav1.6 channel that had fast inactivation removed, resulting in faster channel activation. Although the Nav1.2 channel with fast inactivation removed also demonstrated slightly faster activation, that channel showed very pronounced slow inactivation compared to Nav1.6. These results indicate that potentiation of Nav1.6 sodium currents results from faster channel activation, and that this effect is masked by slow inactivation in Nav1.2. The data suggest that Nav1.6 might be more resistant to inactivation, which might be helpful for high-frequency firing at nodes of Ranvier compared to Nav1.2.     

Seizure-induced plasticity of h channels in entorhinal cortical layer III pyramidal neurons

The entorhinal cortex (EC) provides the predominant excitatory drive to the hippocampal CA1 and subicular neurons in chronic epilepsy. Discerning the mechanisms underlying signal integration within EC neurons is essential for understanding network excitability alterations involving the hippocampus during epilepsy. Twenty-four hours following a single seizure episode when there were no behavioral or electrographic seizures, we found enhanced spontaneous activity still present in the rat EC in vivo and in vitro. The increased excitability was accompanied by a profound reduction in I(h) in EC layer III neurons and a significant decline in HCN1 and HCN2 subunits that encode for h channels. Consequently, dendritic excitability was enhanced, resulting in increased neuronal firing despite hyperpolarized membrane potentials. The loss of I(h) and the increased neuronal excitability persisted for 1 week following seizures. Our results suggest that dendritic I(h) plays an important role in determining the excitability of EC layer III neurons and their associated neural networks.     

Age‐dependent expression of Nav1.9 channels in medial prefrontal cortex pyramidal neurons in rats

Developmental changes that occur in the prefrontal cortex during adolescence alter behavior. These behavioral alterations likely stem from changes in prefrontal cortex neuronal activity, which may depend on the properties and expression of ion channels. Nav1.9 sodium channels conduct a Na⁺ current that is TTX resistant with a low threshold and noninactivating over time. The purpose of this study was to assess the presence of Nav1.9 channels in medial prefrontal cortex (mPFC) layer II and V pyramidal neurons in young (20‐day old), late adolescent (60‐day old), and adult (6‐ to 7‐month old) rats. First, we demonstrated that layer II and V mPFC pyramidal neurons in slices obtained from young rats exhibited a TTX‐resistant, low‐threshold, noninactivating, and voltage‐dependent Na⁺ current. The mRNA expression of the SCN11a gene (which encodes the Nav1.9 channel) in mPFC tissue was significantly higher in young rats than in late adolescent and adult rats. Nav1.9 protein was immunofluorescently labeled in mPFC cells in slices and analyzed via confocal microscopy. Nav1.9 immunolabeling was present in layer II and V mPFC pyramidal neurons and was more prominent in the neurons of young rats than in the neurons of late adolescent and adult rats. We conclude that Nav1.9 channels are expressed in layer II and V mPFC pyramidal neurons and that Nav1.9 protein expression in the mPFC pyramidal neurons of late adolescent and adult rats is lower than that in the neurons of young rats. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1371–1384, 2017     

Calmodulin and Ca2+ control of voltage gated Na+ channels

The structures of the cytosolic portion of voltage activated sodium channels (CTNav) in complexes with calmodulin and other effectors in the presence and the absence of calcium provide information about the mechanisms by which these effectors regulate channel activity. The most studied of these complexes, those of Nav1.2 and Nav1.5, show details of the conformations and the specific contacts that are involved in channel regulation. Another voltage activated sodium channel, Nav1.4, shows significant calcium dependent inactivation, while its homolog Nav1.5 does not. The available structures shed light on the possible localization of the elements responsible for this effect. Mutations in the genes of these 3 Nav channels are associated with several disease conditions: Nav1.2, neurological conditions; Nav1.4, syndromes involving skeletal muscle; and Nav1.5, cardiac arrhythmias. Many of these disease-specific mutations are located at the interfaces involving CTNav and its effectors.     

The Fibroblast Growth Factor 14·Voltage-gated Sodium Channel Complex Is a New Target of Glycogen Synthase Kinase 3 (GSK3)

The FGF14 protein controls biophysical properties and subcellular distribution of neuronal voltage-gated Na⁺ (Nav) channels through direct binding to the channel C terminus. To gain insights into the dynamic regulation of this protein/protein interaction complex, we employed the split luciferase complementation assay to screen a small molecule library of kinase inhibitors against the FGF14·Nav1.6 channel complex and identified inhibitors of GSK3 as hits. Through a combination of a luminescence-based counter-screening, co-immunoprecipitation, patch clamp electrophysiology, and quantitative confocal immunofluorescence, we demonstrate that inhibition of GSK3 reduces the assembly of the FGF14·Nav channel complex, modifies FGF14-dependent regulation of Na⁺ currents, and induces dissociation and subcellular redistribution of the native FGF14·Nav channel complex in hippocampal neurons. These results further emphasize the role of FGF14 as a critical component of the Nav channel macromolecular complex, providing evidence for a novel GSK3-dependent signaling pathway that might control excitability through specific protein/protein interactions.     

Multifaceted Modulation of K+ Channels by Protein-tyrosine Phosphatase {epsilon} Tunes Neuronal Excitability

Non-receptor-tyrosine kinases (protein-tyrosine kinases) and non-receptor tyrosine phosphatases (PTPs) have been implicated in the regulation of ion channels, neuronal excitability, and synaptic plasticity. We previously showed that protein-tyrosine kinases such as Src kinase and PTPs such as PTPα and PTPε modulate the activity of delayed-rectifier K(+) channels (I(K)). Here we show cultured cortical neurons from PTPε knock-out (EKO) mice to exhibit increased excitability when compared with wild type (WT) mice, with larger spike discharge frequency, enhanced fast after-hyperpolarization, increased after-depolarization, and reduced spike width. A decrease in I(K) and a rise in large-conductance Ca(2+)-activated K(+) currents (mBK) were observed in EKO cortical neurons compared with WT. Parallel studies in transfected CHO cells indicate that Kv1.1, Kv1.2, Kv7.2/7.3, and mBK are plausible molecular correlates of this multifaceted modulation of K(+) channels by PTPε. In CHO cells, Kv1.1, Kv1.2, and Kv7.2/7.3 K(+) currents were up-regulated by PTPε, whereas mBK channel activity was reduced. The levels of tyrosine phosphorylation of Kv1.1, Kv1.2, Kv7.3, and mBK potassium channels were increased in the brain cortices of neonatal and adult EKO mice compared with WT, suggesting that PTPε in the brain modulates these channel proteins. Our data indicate that in EKO mice, the lack of PTPε-mediated dephosphorylation of Kv1.1, Kv1.2, and Kv7.3 leads to decreased I(K) density and enhanced after-depolarization. In addition, the deficient PTPε-mediated dephosphorylation of mBK channels likely contributes to enhanced mBK and fast after-hyperpolarization, spike shortening, and consequent increase in neuronal excitability observed in cortical neurons from EKO mice.     

Mutant bacterial sodium channels as models for local anesthetic block of eukaryotic proteins

Voltage gated sodium channels are the target of a range of local anesthetic, anti-epileptic and anti-arrhythmic compounds. But, gaining a molecular level understanding of their mode of action is difficult as we only have atomic resolution structures of bacterial sodium channels not their eukaryotic counterparts. In this study we used molecular dynamics simulations to demonstrate that the binding sites of both the local anesthetic benzocaine and the anti-epileptic phenytoin to the bacterial sodium channel NavAb can be altered significantly by the introduction of point mutations. Free energy techniques were applied to show that increased aromaticity in the pore of the channel, used to emulate the aromatic residues observed in eukaryotic Nav1.2, led to changes in the location of binding and dissociation constants of each drug relative to wild type NavAb. Further, binding locations and dissociation constants obtained for both benzocaine (660 μM) and phenytoin (1 μ M) in the mutant channels were within the range expected from experimental values obtained from drug binding to eukaryotic sodium channels, indicating that these mutant NavAb may be a better model for drug binding to eukaryotic channels than the wild type.