ABNORMAL EXPRESSION OF VOLTAGE-GATED SODIUM CHANNELS Nav1.7, Nav1.3 AND Nav1.8 IN TRIGEMINAL NEURALGIA

Voltage-gated sodium channels have been implicated in acute and chronic neuropathic pain. Among subtypes, Nav1.7 single mutations can cause congenital indifference to pain or chronic neuropathic pain syndromes, including paroxysmal ones. This channel is co-expressed with Nav1.8, which sustains the initial action potential; Nav1.3 is an embrionary channel which is expressed in neurons after injury, as in neuropathic conditions. Few studies are focused on the expression of these molecules in human tissues having chronic pain. Trigeminal neuralgia (TN) is an idiopathic paroxysmal pain treated with sodium channel blockers. The aim of this study was to investigate the expression of Nav1.3, Nav1.7 and Nav1.8 by RT-PCR in patients with TN, compared to controls. The gingival tissue was removed from the correspondent trigeminal area affected. We found that Nav1.7 was downregulated in TN (P=0.017) and Nav1.3 was upregulated in these patients (P=0.043). We propose a physiopathological mechanism for these findings. Besides vascular compression of TN, this disease might be also a channelopathy. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

Ubiquitylation of voltage-gated sodium channels.

Ion channel proteins are regulated by different types of posttranslational modifications. The focus of this review is the regulation of voltage-gated sodium channels (Navs) upon their ubiquitylation. The amiloride-sensitive epithelial sodium channel (ENaC) was the first ion channel shown to be regulated upon ubiquitylation. This modification results from the binding of ubiquitin ligase from the Nedd4 family to a protein-protein interaction domain, known as the PY motif, in the ENaC subunits. Many of the Navs have similar PY motifs, which have been demonstrated to be targets of Nedd4-dependent ubiquitylation, tagging them for internalization from the cell surface. The role of Nedd4-dependent regulation of the Nav membrane density in physiology and disease remains poorly understood. Two recent studies have provided evidence that Nedd4-2 is downregulated in dorsal root ganglion (DRG) neurons in both rat and mouse models of nerve injury-induced neuropathic pain. Using two different mouse models, one with a specific knockout of Nedd4-2 in sensory neurons and another where Nedd4-2 was overexpressed with the use of viral vectors, it was demonstrated that the neuropathy-linked neuronal hyperexcitability was the result of Nav1.7 and Nav1.8 overexpression due to Nedd4-2 downregulation. These studies provided the first in vivo evidence of the role of Nedd4-2-dependent regulation of Nav channels in a disease state. This ubiquitylation pathway may be involved in the development of symptoms and diseases linked to Nav-dependent hyperexcitability, such as pain, cardiac arrhythmias, epilepsy, migraine, and myotonias.

Sodium channels and mammalian sensory mechanotransduction.

BACKGROUND: Members of the degenerin/epithelial (DEG/ENaC) sodium channel family are mechanosensors in C elegans, and Nav1.7 and Nav1.8 voltage-gated sodium channel knockout mice have major deficits in mechanosensation. β and γENaC sodium channel subunits are present with acid sensing ion channels (ASICs) in mammalian sensory neurons of the dorsal root ganglia (DRG). The extent to which epithelial or voltage-gated sodium channels are involved in transduction of mechanical stimuli is unclear. RESULTS: Here we show that deleting β and γENaC sodium channels in sensory neurons does not result in mechanosensory behavioural deficits. We had shown previously that Nav1.7/Nav1.8 double knockout mice have major deficits in behavioural responses to noxious mechanical pressure. However, all classes of mechanically activated currents in DRG neurons are unaffected by deletion of the two sodium channels. In contrast, the ability of Nav1.7/Nav1.8 knockout DRG neurons to generate action potentials is compromised with 50% of the small diameter sensory neurons unable to respond to electrical stimulation in vitro. CONCLUSION: Behavioural deficits in Nav1.7/Nav1.8 knockout mice reflects a failure of action potential propagation in a mechanosensitive set of sensory neurons rather than a loss of primary transduction currents. DEG/ENaC sodium channels are not mechanosensors in mouse sensory neurons.

Regulation of Nav1.7 and Nav1.8 voltage-gated sodium channels by Nedd4-2 and β-subunits and its implication in neuropathic pain

In mammals, the presence of excitable cells in muscles, heart and nervous system is crucial and allows fast conduction of numerous biological information over long distances through the generation of action potentials (AP). Voltage-gated sodium channels (Navs) are key players in the generation and propagation of AP as they are responsible for the rising phase of the AP. Navs are heteromeric proteins composed of a large pore-forming a-subunit (Nav) and smaller ß-auxiliary subunits. There are ten genes encoding for Navl.l to Nav1.9 and NaX channels, each possessing its own specific biophysical properties. The excitable cells express differential combinations of Navs isoforms, generating a distinct electrophysiological signature. Noteworthy, only when anchored at the membrane are Navs functional and are participating in sodium conductance. In addition to the intrinsic properties of Navs, numerous regulatory proteins influence the sodium current. Some proteins will enhance stabilization of membrane Navs while others will favour internalization. Maintaining equilibrium between the two is of crucial importance for controlling cellular excitability. The E3 ubiquitin ligase Nedd4-2 is a well-characterized enzyme that negatively regulates the turnover of many membrane proteins including Navs. On the other hand, ß-subunits are known since long to stabilize Navs membrane anchoring. Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of Navs expressed in dorsal root ganglion (DRG) sensory neurons as highlighted in different animal models of neuropathic pain. Among Navs, Nav1.7 and Nav1.8 are abundantly and specifically expressed in DRG sensory neurons and have been recurrently incriminated in nociception and neuropathic pain development. Using the spared nerve injury (SNI) experimental model of neuropathic pain in mice, I observed a specific reduction of Nedd4-2 in DRG sensory neurons. This decrease subsequently led to an upregulation of Nav1.7 and Nav1.8 protein and current, in the axon and the DRG neurons, respectively, and was sufficient to generate neuropathic pain-associated hyperexcitability. Knocking out Nedd4-2 specifically in nociceptive neurons led to the same increase of Nav1.7 and Nav1.8 concomitantly with an increased thermal sensitivity in mice. Conversely, rescuing Nedd4-2 downregulation using viral vector transfer attenuated neuropathic pain mechanical hypersensitivity. This study demonstrates the significant role of Nedd4-2 in regulating cellular excitability in vivo and its involvement in neuropathic pain development. The role of ß-subunits in neuropathic pain was already demonstrated in our research group. Because of their stabilization role, the increase of ßl, ß2 and ß3 subunits in DRGs after SNI led to increased Navs anchored at the membrane. Here, I report a novel mechanism of regulation of a-subunits by ß- subunits in vitro; ßl and ß3-subunits modulate the glycosylation pattern of Nav1.7, which might account for stabilization of its membrane expression. This opens new perspectives for investigation Navs state of glycosylation in ß-subunits dependent diseases, such as in neuropathic pain. - Chez les mammifères, la présence de cellules excitables dans les muscles, le coeur et le système nerveux est cruciale; elle permet la conduction rapide de nombreuses informations sur de longues distances grâce à la génération de potentiels d'action (PA). Les canaux sodiques voltage-dépendants (Navs) sont des participants importants dans la génération et la propagation des PA car ils sont responsables de la phase initiale de dépolarisation du PA. Les Navs sont des protéines hétéromériques composées d'une grande sous-unité a (formant le pore du canal) et de petites sous-unités ß accompagnatrices. Il existe dix gènes qui codent pour les canaux sodiques, du Nav 1.1 au Nav 1.9 ainsi que NaX, chacun possédant des propriétés biophysiques spécifiques. Les cellules excitables expriment différentes combinaisons des différents isoformes de Navs, qui engendrent une signature électrophysiologique distincte. Les Navs ne sont fonctionnels et ne participent à la conductibilité du Na+, que s'ils sont ancrés à la membrane plasmique. En plus des propriétés intrinsèques des Navs, de nombreuses protéines régulatrices influencent également le courant sodique. Certaines protéines vont favoriser l'ancrage et la stabilisation des Navs exprimés à la membrane, alors que d'autres vont plutôt favoriser leur internalisation. Maintenir l'équilibre des deux processus est crucial pour contrôler l'excitabilité cellulaire. Dans ce contexte, Nedd4-2, de la famille des E3 ubiquitin ligase, est une enzyme bien caractérisée qui régule l'internalisation de nombreuses protéines, notamment celle des Navs. Inversement, les sous-unités ß sont connues depuis longtemps pour stabiliser l'ancrage des Navs à la membrane. La douleur neuropathique périphérique est une condition débilitante résultant d'une atteinte à un nerf. Elle est caractérisée par la dérégulation des Navs exprimés dans les neurones sensoriels du ganglion spinal (DRG). Ceci a été démontré à de multiples occasions dans divers modèles animaux de douleur neuropathique. Parmi les Navs, Nav1.7 et Nav1.8 sont abondamment et spécifiquement exprimés dans les neurones sensoriels des DRG et ont été impliqués de façon récurrente dans le développement de la douleur neuropathique. En utilisant le modèle animal de douleur neuropathique d'épargne du nerf sural (spared nerve injury, SNI) chez la souris, j'ai observé une réduction spécifique des Nedd4-2 dans les neurones sensoriels du DRG. Cette diminution avait pour conséquence l'augmentation de l'expression des protéines et des courants de Nav 1.7 et Nav 1.8, respectivement dans l'axone et les neurones du DRG, et était donc suffisante pour créer l'hyperexcitabilité associée à la douleur neuropathique. L'invalidation pour le gène codant pour Nedd4-2 dans une lignée de souris génétiquement modifiées a conduit à de similaires augmentations de Nav1.7 et Nav1.8, parallèlement à une augmentation à la sensibilité thermique. A l'opposé, rétablir une expression normale de Nedd4-2 en utilisant un vecteur viral a eu pour effet de contrecarrer le développement de l'hypersensibilité mécanique lié à ce modèle de douleur neuropathique. Cette étude démontre le rôle important de Nedd4-2 dans la régulation de l'excitabilité cellulaire in vivo et son implication dans le développement des douleurs neuropathiques. Le rôle des sous-unités ß dans les douleurs neuropathiques a déjà été démontré dans notre groupe de recherche. A cause de leur rôle stabilisateur, l'augmentation des sous-unités ßl, ß2 et ß3 dans les DRG après SNI, conduit à une augmentation des Navs ancrés à la membrane. Dans mon travail de thèse, j'ai observé un nouveau mécanisme de régulation des sous-unités a par les sous-unités ß in vitro. Les sous-unités ßl et ß3 régulent l'état de glycosylation du canal Nav1.7, et stabilisent son expression membranaire. Ceci ouvre de nouvelles perspectives dans l'investigation de l'état de glycosylation des Navs dans des maladies impliquant les sous-unités ß, notamment les douleurs neuropathiques.

CARDIAC AND MUSCLE CHANNELOPATHIES: ROLES AND REGULATION OF VOLTAGE-GATED SODIUM CHANNELS

Abstract :The contraction of the heart or skeletal muscles is mainly due to the propagation, through excitable cells, of an electrical influx called action potential (AP). The AP results from the sequential opening of ion channels that generate inward or outward currents through the cell membrane. Among all the channels involved, the voltage-gated sodium channel is responsible for the rising phase of the action potential. Ten genes encode the different isoforms of these channels (from Nav1.1 to Nav1.9 and an atypical channel named NavX). Nav1.4 and Nav1.5 are the main skeletal muscle and cardiac sodium channels respectively. Their importance for muscle and heart function has been highlighted by the description of mutations in their encoding genes SCN4A and SCNSA. They lead respectively to neuromuscular disorders such as myotonia or paralysis (for Nav1.4), and to cardiac arrhythmias that can deteriorate into sudden cardiac death (for Nav1.5).The general aim of my PhD work has been to study diseases linked with channels dysfunction, also called channelopathies. In that purpose, I investigated the function and the regulation of the muscle and cardiac voltage-gated sodium channels. During the two first studies, I characterized the effects of two mutations affecting Nav1.4 and Nav1.5 function. I used the HEK293 model cells to express wild-type or mutant channels and then studied their biophysical properties with the patch-clamp technique, in whole cell configuration. We found that the SCN4A mutation produced complex alterations of the muscle sodium channel function, that could explain the myotonic phenotype described in patients carrying the mutation. In the second study, the index case was an heterozygous carrier of a SCNSA mutation that leads to a "loss of function" of the channel. The decreased sodium current measured with mutated Nay 1.5 channels, at physiological temperature, was a one of the factors that could explain the observed Brugada syndrome. The last project aimed at identifying a new potential protein interacting with the cardiac sodium channel. We found that the protein SAP97 binds the three last amino-acids of the C-terminus of Na,, 1.5. Our results also indicated that silencing the expression of SAP97 in HEK293 cells decreased the sodium current. Sodium channels lacking their three last residues also produced a reduced INa. These preliminary results suggest that SAP97 is implicated in the regulation of sodium channel. Whether this effect is direct or imply the action of an adaptor protein remains to be investigated. Moreover, our group has previously shown that Nav1.5 channels are localized to lateral membranes of cardiomyocytes by the dystrophin multiprotein complex (DMC). This suggests that sodium channels are distributed in, at least, two different pools: one targeted at lateral membranes by DMC and the other at intercalated discs by another protein such as SAP97.These studies reveal that cardiac and muscle diseases may result from ion channel mutations but also from regulatory proteins affecting their regulation.Résumé :La contraction des muscles et du coeur est principalement due à la propagation, à travers les cellules excitables, d'un stimulus électrique appelé potentiel d'action (PA). C'est l'ouverture séquentielle de plusieurs canaux ioniques transmembranaires, permettant l'entrée ou la sortie d'ions dans la cellule, qui est à l'origine de ce PA. Parmi tous les canaux ioniques impliqués dans ce processus, les canaux sodiques dépendant du voltage sont responsables de la première phase du potentiel d'action. Les différentes isoformes de ces canaux (de Nav1.1 à Nav1.9 et NavX) sont codées par dix gènes distincts. Nav1.4 et Nav1.5 sont les principaux variants exprimés respectivement dans le muscle et le coeur. Plusieurs mutations ont été décrites dans les gènes qui codent pour ces deux canaux: SCN4A (pour Nav1.4) et SCNSA (pour Nav1.5). Elles sont impliquées dans des pathologies neuromusculaires telles que des paralysies ou myotonies (SCN4A) ou des arythmies cardiaques pouvant conduire à la mort subite cardiaque (SCNSA).Mon travail de thèse a consisté à étudier les maladies liées aux dysfonctionnements de ces canaux, aussi appelées canalopathies. J'ai ainsi analysé la fonction et la régulation des canaux sodiques dépendant du voltage dans le muscle squelettique et le coeur. A travers les deux premières études, j'ai ainsi pu examiner les conséquences de deux mutations affectant respectivement les canaux Nav1.4 et Nav1.5. Les canaux sauvages ou mutants ont été exprimés dans des cellules HEK293 afin de caractériser leurs propriétés biophysiques par la technique du patch clamp en configuration cellule entière. Nous avons pu déterminer que la mutation trouvée dans le gène SCN4A engendrait des modifications importantes de la fonction du canal musculaire. Ces altérations fournissent des indications nous permettant d'expliquer certains aspects de la myotonie observée chez les membres de la famille étudiée. Le patient présenté dans la deuxième étude était hétérozygote pour la mutation identifiée dans le gène SCNSA. La perte de fonction des canaux Nav1.5 ainsi engendrée, a été observée lors d'analyses à températures physiologiques. Elle représente l'un des éléments pouvant potentiellement expliquer le syndrome de Brugada du patient. La dernière étude a consisté à identifier une nouvelle protéine impliquée dans la régulation du canal sodique cardiaque. Nos expériences ont démontré que les trois derniers acides aminés de la partie C-terminale de Nav1.5 pouvaient interagir avec la protéine SAP97. Lorsque que l'expression de la SAP97 est réduite dans les cellules HEK293, cela induit une baisse importante du courant sodique. De même, les canaux tronqués de leurs trois derniers acides aminés génèrent un flux ionique réduit. Ces résultats préliminaires suggèrent que SAP97 est peut-être impliquée dans la régulation du canal Na,,1.5. Des expériences complémentaires permettront de déterminer si ces deux protéines interagissent directement ou si une protéine adaptatrice est nécessaire. De plus, nous avons préalablement montré que les canaux Nav1.5 étaient localisés au niveau de la membrane latérale des cardiomyocytes par le complexe multiprotéique de la dystrophine (DMC). Ceci suggère que les canaux sodiques peuvent être distribués dans un minimum de deux pools, l'un ciblé aux membranes latérales pax le DMC et l'autre dirigé vers les disques intercalaires par des protéines telles que SAP97.L'ensemble de ces études met en évidence que certaines maladies musculaires et cardiaques peuvent être la conséquence directe de mutations de canaux ioniques, mais que l'action de protéines auxiliaires peut aussi affecter leur fonction.

Dysregulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2 in neuropathic pain.

Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of voltage-gated sodium channels (Navs) expressed in dorsal root ganglion (DRG) sensory neurons. The mechanisms underlying the altered expression of Navs remain unknown. This study investigated the role of the E3 ubiquitin ligase NEDD4-2, which is known to ubiquitylate Navs, in the pathogenesis of neuropathic pain in mice. The spared nerve injury (SNI) model of traumatic nerve injury-induced neuropathic pain was used, and an Nav1.7-specific inhibitor, ProTxII, allowed the isolation of Nav1.7-mediated currents. SNI decreased NEDD4-2 expression in DRG cells and increased the amplitude of Nav1.7 and Nav1.8 currents. The redistribution of Nav1.7 channels toward peripheral axons was also observed. Similar changes were observed in the nociceptive DRG neurons of Nedd4L knockout mice (SNS-Nedd4L-/-). SNS-Nedd4L-/- mice exhibited thermal hypersensitivity and an enhanced second pain phase after formalin injection. Restoration of NEDD4-2 expression in DRG neurons using recombinant adenoassociated virus (rAAV2/6) not only reduced Nav1.7 and Nav1.8 current amplitudes, but also alleviated SNI-induced mechanical allodynia. These findings demonstrate that NEDD4-2 is a potent posttranslational regulator of Navs and that downregulation of NEDD4-2 leads to the hyperexcitability of DRG neurons and contributes to the genesis of pathological pain.

Post-translational modifications of voltage-gated sodium channels in chronic pain syndromes.

In the peripheral sensory nervous system the neuronal expression of voltage-gated sodium channels (Navs) is very important for the transmission of nociceptive information since they give rise to the upstroke of the action potential (AP). Navs are composed of nine different isoforms with distinct biophysical properties. Studying the mutations associated with the increase or absence of pain sensitivity in humans, as well as other expression studies, have highlighted Nav1.7, Nav1.8, and Nav1.9 as being the most important contributors to the control of nociceptive neuronal electrogenesis. Modulating their expression and/or function can impact the shape of the AP and consequently modify nociceptive transmission, a process that is observed in persistent pain conditions. Post-translational modification (PTM) of Navs is a well-known process that modifies their expression and function. In chronic pain syndromes, the release of inflammatory molecules into the direct environment of dorsal root ganglia (DRG) sensory neurons leads to an abnormal activation of enzymes that induce Navs PTM. The addition of small molecules, i.e., peptides, phosphoryl groups, ubiquitin moieties and/or carbohydrates, can modify the function of Navs in two different ways: via direct physical interference with Nav gating, or via the control of Nav trafficking. Both mechanisms have a profound impact on neuronal excitability. In this review we will discuss the role of Protein Kinase A, B, and C, Mitogen Activated Protein Kinases and Ca++/Calmodulin-dependent Kinase II in peripheral chronic pain syndromes. We will also discuss more recent findings that the ubiquitination of Nav1.7 by Nedd4-2 and the effect of methylglyoxal on Nav1.8 are also implicated in the development of experimental neuropathic pain. We will address the potential roles of other PTMs in chronic pain and highlight the need for further investigation of PTMs of Navs in order to develop new pharmacological tools to alleviate pain.

Novel wasp toxin discriminates between neuronal and cardiac sodium channels

Pompilidotoxins (PMTXs), derived from the venom of solitary wasp has been known to facilitate synaptic transmission in the lobster neuromuscular junction, and a recent further study from rat trigeminal neurons revealed that the toxin slows Na+ channel inactivation without modifying activation process. Here we report that beta -PMTX modifies rat brain type II Na+ channel alpha -subunit (rBII) expressed in human embryonic kidney cells but fails to act on the rat heart alpha -subunit (rH1) at similar concentrations. We constructed a series of chimeric mutants of rBII and rH1 Na+ channels and compared modification of the steady-state Na+ currents by beta -PMTX. We found that a difference in a single amino acid between Glu-1616 in rBII and Gln-1615 in rH1 at the extracellular loop of D4S3-S4 is crucial for the action of beta -PMTX. PMTXs, which are small peptides with 13 amino acids, would be a potential tool for exploring a new functional moiety of Na+ channels.

SAP97 and dystrophin macromolecular complexes determine two pools of cardiac sodium channels Nav1.5 in cardiomyocytes

The cardiac sodium channel Na(v)1.5 plays a key role in excitability and conduction. The 3 last residues of Na(v)1.5 (Ser-Ile-Val) constitute a PDZ-domain binding motif that interacts with the syntrophin-dystrophin complex. As dystrophin is absent at the intercalated discs, Na(v)1.5 could potentially interact with other, yet unknown, proteins at this site.

High salt intake down-regulates colonic mineralocorticoid receptors, epithelial sodium channels and 11β-hydroxysteroid dehydrogenase type 2

Besides the kidneys, the gastrointestinal tract is the principal organ responsible for sodium homeostasis. For sodium transport across the cell membranes the epithelial sodium channel (ENaC) is of pivotal relevance. The ENaC is mainly regulated by mineralocorticoid receptor mediated actions. The MR activation by endogenous 11β-hydroxy-glucocorticoids is modulated by the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). Here we present evidence for intestinal segment specific 11β-HSD2 expression and hypothesize that a high salt intake and/or uninephrectomy (UNX) affects colonic 11β-HSD2, MR and ENaC expression. The 11β-HSD2 activity was measured by means of 3H-corticosterone conversion into 3H-11-dehydrocorticosterone in Sprague Dawley rats on a normal and high salt diet. The activity increased steadily from the ileum to the distal colon by a factor of about 3, an observation in line with the relevance of the distal colon for sodium handling. High salt intake diminished mRNA and protein of 11β-HSD2 by about 50% (p<0.001) and reduced the expression of the MR (p<0.01). The functionally relevant ENaC-β and ENaC-γ expression, a measure of mineralocorticoid action, diminished by more than 50% by high salt intake (p<0.001). The observed changes were present in rats with and without UNX. Thus, colonic epithelial cells appear to contribute to the protective armamentarium of the mammalian body against salt overload, a mechanism not modulated by UNX.

Dysregulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2 in neuropathic pain

Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of voltage-gated sodium channels (Navs) expressed in dorsal root ganglion (DRG) sensory neurons. The mechanisms underlying the altered expression of Na(v)s remain unknown. This study investigated the role of the E3 ubiquitin ligase NEDD4-2, which is known to ubiquitylate Navs, in the pathogenesis of neuropathic pain in mice. The spared nerve injury (SNI) model of traumatic nerve injury-induced neuropathic pain was used, and an Na(v)1.7-specific inhibitor, ProTxII, allowed the isolation of Na(v)1.7-mediated currents. SNI decreased NEDD4-2 expression in DRG cells and increased the amplitude of Na(v)1.7 and Na(v)1.8 currents. The redistribution of Na(v)1.7 channels toward peripheral axons was also observed. Similar changes were observed in the nociceptive DRG neurons of Nedd4L knockout mice (SNS-Nedd4L(-/-)). SNS-Nedd4L(-/-) mice exhibited thermal hypersensitivity and an enhanced second pain phase after formalin injection. Restoration of NEDD4-2 expression in DRG neurons using recombinant adenoassociated virus (rAAV2/6) not only reduced Na(v)1.7 and Na(v)1.8 current amplitudes, but also alleviated SNI-induced mechanical allodynia. These findings demonstrate that NEDD4-2 is a potent posttranslational regulator of Na(v)s and that downregulation of NEDD4-2 leads to the hyperexcitability of DRG neurons and contributes to the genesis of pathological pain.

Ubiquitylation of voltage-gated sodium channels.

Ion channel proteins are regulated by different types of posttranslational modifications. The focus of this review is the regulation of voltage-gated sodium channels (Navs) upon their ubiquitylation. The amiloride-sensitive epithelial sodium channel (ENaC) was the first ion channel shown to be regulated upon ubiquitylation. This modification results from the binding of ubiquitin ligase from the Nedd4 family to a protein-protein interaction domain, known as the PY motif, in the ENaC subunits. Many of the Navs have similar PY motifs, which have been demonstrated to be targets of Nedd4-dependent ubiquitylation, tagging them for internalization from the cell surface. The role of Nedd4-dependent regulation of the Nav membrane density in physiology and disease remains poorly understood. Two recent studies have provided evidence that Nedd4-2 is downregulated in dorsal root ganglion (DRG) neurons in both rat and mouse models of nerve injury-induced neuropathic pain. Using two different mouse models, one with a specific knockout of Nedd4-2 in sensory neurons and another where Nedd4-2 was overexpressed with the use of viral vectors, it was demonstrated that the neuropathy-linked neuronal hyperexcitability was the result of Nav1.7 and Nav1.8 overexpression due to Nedd4-2 downregulation. These studies provided the first in vivo evidence of the role of Nedd4-2-dependent regulation of Nav channels in a disease state. This ubiquitylation pathway may be involved in the development of symptoms and diseases linked to Nav-dependent hyperexcitability, such as pain, cardiac arrhythmias, epilepsy, migraine, and myotonias.

Cellular hyper-excitability caused by mutations that alter the activation process of voltage-gated sodium channels.

Voltage-gated sodium channels (Nav) are widely expressed as macro-molecular complexes in both excitable and non-excitable tissues. In excitable tissues, the upstroke of the action potential is the result of the passage of a large and rapid influx of sodium ions through these channels. NaV dysfunction has been associated with an increasingly wide range of neurological, muscular and cardiac disorders. The purpose of this review is to summarize the recently identified sodium channel mutations that are linked to hyper-excitability phenotypes and associated with the alteration of the activation process of voltage gated sodium channels. Indeed, several clinical manifestations that demonstrate an alteration of tissue excitability were recently shown to be strongly associated with the presence of mutations that affect the activation process of the Nav. These emerging genotype-phenotype correlations have expanded the clinical spectrum of sodium channelopathies to include disorders which feature a hyper-excitability phenotype that may or may not be associated with a cardiomyopathy. The p.I141V mutation in SCN4A and SCN5A, as well as its homologous p.I136V mutation in SCN9A, are interesting examples of mutations that have been linked to inherited hyperexcitability myotonia, exercise-induced polymorphic ventricular arrhythmias and erythromelalgia, respectively. Regardless of which sodium channel isoform is investigated, the substitution of the isoleucine to valine in the locus 141 induces similar modifications in the biophysical properties of the Nav by shifting the voltage-dependence of steady state activation toward more negative potentials.