Voltage-gated Sodium Channel Family Research Articles

Calmodulin Interactions with Voltage-Gated Sodium Channels.

Calmodulin (CaM) is a small protein that acts as a ubiquitous signal transducer and regulates neuronal plasticity, muscle contraction, and immune response. It interacts with ion channels and plays regulatory roles in cellular electrophysiology. CaM modulates the voltage-gated sodium channel gating process, alters sodium current density, and regulates sodium channel protein trafficking and expression. Many mutations in the CaM-binding IQ domain give rise to diseases including epilepsy, autism, and arrhythmias by interfering with CaM interaction with the channel. In the present review, we discuss CaM interactions with the voltage-gated sodium channel and modulators involved in CaM regulation, as well as summarize CaM-binding IQ domain mutations associated with human diseases in the voltage-gated sodium channel family.

Characterization of Synthetic Tf2 as a NaV1.3 Selective Pharmacological Probe.

NaV1.3 is a subtype of the voltage-gated sodium channel family. It has been implicated in the pathogenesis of neuropathic pain, although the contribution of this channel to neuronal excitability is not well understood. Tf2, a β-scorpion toxin previously identified from the venom of Tityus fasciolatus, has been reported to selectively activate NaV1.3. Here, we describe the activity of synthetic Tf2 and assess its suitability as a pharmacological probe for NaV1.3. As described for the native toxin, synthetic Tf2 (1 µM) caused early channel opening, decreased the peak current, and shifted the voltage dependence of NaV1.3 activation in the hyperpolarizing direction by −11.3 mV, with no activity at NaV1.1, NaV1.2, and NaV1.4-NaV1.8. Additional activity was found at NaV1.9, tested using the hNav1.9_C4 chimera, where Tf2 (1 µM) shifted the voltage dependence of activation by −6.3 mV. In an attempt to convert Tf2 into an NaV1.3 inhibitor, we synthetized the analogue Tf2[S14R], a mutation previously described to remove the excitatory activity of related β-scorpion toxins. Indeed, Tf2[S14R](10 µM) had reduced excitatory activity at NaV1.3, although it still caused a small −5.8 mV shift in the voltage dependence of activation. Intraplantar injection of Tf2 (1 µM) in mice caused spontaneous flinching and swelling, which was not reduced by the NaV1.1/1.3 inhibitor ICA-121431 nor in NaV1.9-/- mice, suggesting off-target activity. In addition, despite a loss of excitatory activity, intraplantar injection of Tf2[S14R](10 µM) still caused swelling, providing strong evidence that Tf2 has additional off-target activity at one or more non-neuronal targets. Therefore, due to activity at NaV1.9 and other yet to be identified target(s), the use of Tf2 as a selective pharmacological probe may be limited.

Sodium Channelopathies: From Molecular Physiology towards Medical Genetics

Voltage-gated sodium channels are heteromeric transmembrane proteins involved in the conduction of sodium ion currents in response to membrane depolarization. In humans, nine homologous genes, SCN1A–11A, which encode different isoforms of the voltage-gated sodium channel family, are known. Sodium channel isoforms exhibit different kinetic properties that determine different types of neurons. Mutations in different channels are described in patients with various congenital disorders, from epilepsy to congenital insensitivity to pain. This review presents an analysis of the current literature on the properties of different isoforms of voltage-gated sodium channels and associated diseases.

Calcium triggers reversal of calmodulin on nested anti-parallel sites in the IQ motif of the neuronal voltage-dependent sodium channel NaV1.2

Several members of the voltage-gated sodium channel family are regulated by calmodulin (CaM) and ionic calcium. The neuronal voltage-gated sodium channel NaV1.2 contains binding sites for both apo (calcium-depleted) and calcium-saturated CaM. We have determined equilibrium dissociation constants for rat NaV1.2 IQ motif [IQRAYRRYLLK] binding to apo CaM (~3nM) and (Ca2+)4-CaM (~85nM), showing that apo CaM binding is favored by 30-fold. For both apo and (Ca2+)4-CaM, NMR demonstrated that NaV1.2 IQ motif peptide (NaV1.2IQp) exclusively made contacts with C-domain residues of CaM (CaMC). To understand how calcium triggers conformational change at the CaM-IQ interface, we determined a solution structure (2M5E.pdb) of (Ca2+)2-CaMC bound to NaV1.2IQp. The polarity of (Ca2+)2-CaMC relative to the IQ motif was opposite to that seen in apo CaMC-Nav1.2IQp (2KXW), revealing that CaMC recognizes nested, anti-parallel sites in Nav1.2IQp. Reversal of CaM may require transient release from the IQ motif during calcium binding, and facilitate a re-orientation of CaMN allowing interactions with non-IQ NaV1.2 residues or auxiliary regulatory proteins interacting in the vicinity of the IQ motif.

NaV1.7 as a pain target - From gene to pharmacology.

Na(V)1.7, a subtype of the voltage-gated sodium channel family that is highly expressed in peripheral sensory neurons, remains one of the most promising targets for the treatment of pain. However, despite compelling genetic evidence supporting a key role for Na(V)1.7 in regulating excitability of peripheral sensory neurons, the development of truly subtype-selective inhibitors has been challenging. Here, we discuss complexities surrounding targeting Na(V)1.7 pharmacologically for treatment of pain and explore future opportunities for development of effective analgesic Na(V)1.7 inhibitors. (C) 2016 Elsevier Inc. All rights reserved.

Mechanosensitivity of the Bacterial Voltage-Gated Sodium Channel NaChBac

INTRODUCTION: The voltage-gated sodium channel family spans from ancestral prokaryotic complexes found in bacterial extremophiles to eukaryotic proteins that are essential for excitability of most cell types. The SCN5A-encoded mechanosensitive voltage-gated channel Nav1.5 is expressed in electro-mechanical organs such as the heart and gastrointestinal tract. It is well-established that Nav1.5 is mechanosensitive, but biophysical studies of NaV mechanosensitivity are limited by the size of NaV proteins and their rapid kinetics, especially inactivation. Bacterial voltage-gated sodium channels recapitulate many of the eukaryotic NaV functional properties and have allowed significant advances in structural, functional and pharmacologic understanding of NaV channels and would serve as a good mechanosensitivity model. However, NaChBac mechanosensitivity has not been examined. AIMS: To determine if the bacterial Na+ channel NaChBac and a NaChBac non-inactivating mutant are mechanosensitive. METHODS: Nav1.5, NaChBac WT or the non-inactivating mutant T220A NaChBac constructs were co-transfected with GFP into HEK-293 cells. NaChBac mechanosensitivity was compared to Nav1.5 by whole Cell voltage-clamp with shear stress and in cell-attached patches using pressure clamp. RESULTS: Shear-stress increased Nav1.5 peak current by 18±3%. Similarly, shear-stress increases peak current in both WT NaChBac (+36.9%) and the non-inactivating mutant T220A (+41.6%). Clusters of single-channel openings (∼2 pS) were obtained from T220A in absence and presence of pressure (−30 mmHg). CONCLUSIONS: Effects of shear in peak current and voltage-dependency found in WT NaChBac and T220A were comparable to those found in Nav1.5. NaChBac T220A presented long-lasting frequent single channel openings, which were affected by pressure. NaChBac may serve as a good model to understand eukaryotic NaV channel mechanosensitivity.

The global distribution of the p.R1193Q polymorphism in the SCN5A gene.

The SCN5A (sodium channel, voltage-gated, type V, alpha subunit) gene encodes the cardiac sodium channel, a member of the voltage-gated sodium channel family. The p.R1193Q (c.3578G>A) polymorphism in SCN5A is known to accelerate inactivation of the sodium channel current, and has been identified in patients with Brugada and long QT syndromes. In the present study, we investigated the frequency of the p.R1193Q substitution in more than 4000 genomic DNA samples from 34 Asian, European, and African populations using TaqMan and/or APLP (amplified product length polymorphism) assays. Allele A (p.1193Q) was detected in most Asian populations, but was sporadically observed or absent in European and African populations. These results demonstrated that the p.R1193Q substitution is characteristic of Asian populations.

Ionic selectivity and thermal adaptations within the voltage-gated sodium channel family of alkaliphilic Bacillus.

Entry and extrusion of cations are essential processes in living cells. In alkaliphilic prokaryotes, high external pH activates voltage-gated sodium channels (Nav), which allows Na(+) to enter and be used as substrate for cation/proton antiporters responsible for cytoplasmic pH homeostasis. Here, we describe a new member of the prokaryotic voltage-gated Na(+) channel family (NsvBa; <underline>N</underline>on-<underline>s</underline>elective <underline>v</underline>oltage-gated, <underline>B</underline>acillus <underline>a</underline>lcalophilus) that is nonselective among Na(+), Ca(2+) and K(+) ions. Mutations in NsvBa can convert the nonselective filter into one that discriminates for Na(+) or divalent cations. Gain-of-function experiments demonstrate the portability of ion selectivity with filter mutations to other Bacillus Nav channels. Increasing pH and temperature shifts their activation threshold towards their native resting membrane potential. Furthermore, we find drugs that target Bacillus Nav channels also block the growth of the bacteria. This work identifies some of the adaptations to achieve ion discrimination and gating in Bacillus Nav channels.

Molecular Dynamics Studies of Ion Conduction in a Prokaryotic Channel

Recently, the crystal structures of putative bacterial, voltage-gated sodium channels have been published, giving an atomic scale view of the NaChBac family of voltage-gated sodium channels. The selectivity filters of the open pore structure of NavMs (McCusker et al (2012) Nature Comm. 3, 1102) and the closed pore structure of NavAb (Payandeh et al (2011) Nature 475, 353) differ only on the order of 1-2 A in diameter. Previously published studies aimed at predicting the mechanism of ion selectivity through free energy surfaces have not provided a consistent free energy surface. Electrophysiology studies suggest that sodium selectivity in the NaChBac family is slight when compared to the potassium selectivity in potassium-selective ion channels (Shaya et al (2011) PNAS 108, 12313). Multi-microsecond equilibrium simulations of NavAb by Chakrabarti et al. suggest that conformational movements of the glutamate side chains are correlated to sodium movement through the selectivity filter (PNAS (2013) 110, 11331).We present microsecond timescale molecular dynamics simulations of ion conduction through a truncated model of the NavAb pore as a function of applied voltage, in solutions containing sodium, potassium, and both, and as a function of concentration. The model NavAb is constructed from the NavAb pore in which the S5 and S6 helices are truncated, creating an open pore, and embedded in a neon support. The S5 and S6 helices and the support are restrained, but the pore helices and selectivity filter are unrestrained. We show that conformational distribution of the glutamate side chains is voltage dependent. The dependence of selectivity on voltage, concentration, and cations present is demonstrated. Additionally, it is apparent that the rate of and predominant mechanism of conduction is not only dependent upon the voltage and cation but also upon the direction of current.

An autopsy case of sudden unexpected nocturnal death syndrome with R1193Q polymorphism in the SCN5A gene

SCN5A (sodium channel, voltage-gated, type V, alpha subunit) gene encodes the cardiac sodium channel, a member of the voltage-gated sodium channel family. SCN5A mutations have been associated with a variety of inherited arrhythmias, including long QT syndrome and Brugada syndrome. We report an autopsy case of sudden unexpected nocturnal death syndrome. A man in his thirties died at night while sleeping. At autopsy, no traumatic injury, disease or drug intake was observed as a possible cause of death. We examined mutations in the SCN5A gene and identified a heterozygous mutation causing an R1193Q amino acid substitution. It was reported that the R1193Q polymorphism in the SCN5A gene destabilizes channel inactivation and may be a risk factor for Brugada and long QT syndrome. It may be considered that the cause of death in this case was sudden cardiac death.

Identification and functional characterization of the promoter of the mouse sodium‐activated sodium channel Nax gene (Scn7a)

Na(x) is a sodium channel, thought to be a descendant of the voltage-gated sodium channel family. Nevertheless, Na(x) is not activated by voltage but rather by augmentation of extracellular sodium over 150 mM. In the brain, it is localized to the circumventricular organs, important regions for salt and water homeostasis in mammals, where it operates as a sodium-level sensor of body fluid. Na(x) channel is expressed in lung, uterus, and heart, and it is also found in trigeminal and dorsal root ganglia and in nonmyelinating Schwann cells, where its physiological role remains unclarified. Here we identified the promoter and transcription start sites of Na(x) sodium channel in dorsal root ganglia neurons from mouse. We report a characterization of the basal TATA-less promoter and the sequence requirements for promoter activity in Neuro 2A neuroblastoma cells and in dorsal root ganglia neurons, where basal promoter activity seems to require NGFI-C and Ebox DNA elements. Finally, we provide evidence that a repression mechanism that inhibits Na(x) expression may be present in certain tissues. These findings provide the basis with which to understand tissue-specific regulation of Na(x) sodium channel gene (Scn7a) expression.

Hydromineral neuroendocrinology: mechanism of sensing sodium levels in the mammalian brain

Dehydration causes an increase in the sodium (Na) concentration and osmolarity of body fluids. For Na homeostasis of the body, control of Na and water intake and excretion are of prime importance. Although the circumventricular organs (CVOs) were suggested to be involved in body-fluid homeostasis, the system for sensing Na levels within the brain, which is responsible for the control of Na- and water-intake behaviour, has long been an enigma. Na(x) is an atypical sodium channel that is assumed to be a descendant of the voltage-gated sodium channel family. Our studies on the Na(x)-gene-targeting (Na(x)(-/-)) mouse revealed that Na(x) channels are localized to the CVOs and serve as a sodium-level sensor of body fluids. As the first step to understand the cellular mechanism by which the information sensed by Na(x) channels is reflected in the activity of the organs, we dissected the subcellular distribution of Na(x). Double-immunostaining and immuno-electron microscopic analyses revealed that Na(x) is exclusively localized to perineuronal lamellate processes extending from ependymal cells and astrocytes in the organs. In addition, glial cells isolated from the subfornical organ were sensitive to an increase in the extracellular sodium level, as analysed by an ion-imaging method. These results suggest that glial cells bearing Na(x) channels are the first to sense a physiological increase in the level of sodium in body fluids, and regulate the neural activity of the CVOs by enveloping neurons. Close communication between inexcitable glial cells and excitable neural cells thus appears to be the basis of the central control of salt homeostasis.

2P290 原核生物由来膜電位依存性ナトリウムチャネルのチャネル特性の多様性(生体膜・人工膜,口頭発表,第45回日本生物物理学会年会)

2P290 原核生物由来膜電位依存性ナトリウムチャネルのチャネル特性の多様性(生体膜・人工膜,口頭発表,第45回日本生物物理学会年会)

Sodium-level-sensitive sodium channel Nax is expressed in glial laminate processes in the sensory circumventricular organs

Na(x) is an atypical sodium channel that is assumed to be a descendant of the voltage-gated sodium channel family. Our recent studies on the Na(x)-gene-targeting mouse revealed that Na(x) channel is localized to the circumventricular organs (CVOs), the central loci for the salt and water homeostasis in mammals, where the Na(x) channel serves as a sodium-level sensor of the body fluid. To understand the cellular mechanism by which the information sensed by Na(x) channels is transferred to the activity of the organs, we dissected the subcellular localization of Na(x) in the present study. Double-immunostaining and immunoelectron microscopic analyses revealed that Na(x) is exclusively localized to perineuronal lamellate processes extended from ependymal cells and astrocytes in the organs. In addition, glial cells isolated from the subfornical organ, one of the CVOs, were sensitive to an increase in the extracellular sodium level, as analyzed by an ion-imaging method. These results suggest that glial cells bearing the Na(x) channel are the first to sense a physiological increase in the level of sodium in the body fluid, and they regulate the neural activity of the CVOs by enveloping neurons. Close communication between inexcitable glial cells and excitable neural cells thus appears to be the basis of the central control of the salt homeostasis.

Sodium-level-sensitive Sodium Channel and Salt-Intake Behavior

Mammals feel thirsty or develop an appetite for salt when the correct balance between water and sodium in the body fluid has been disrupted, but little is known about the mechanism in the brain that controls salt homeostasis. It has been postulated that the existence of both an osmoreceptor and a specific sodium receptor is required to accommodate the experimental data (Johnson and Edwards, 1990; Denton et al., 1996). Several candidate osmoreceptors have been reported (Oliet and Bourque, 1993; Wells, 1998; Liedtke et al., 2000); however, a specific sodium receptor has not been identified. The Nax channel—formerly called NaG/SCL11 (in rats), Nav2.3 (in mice) and Nav2.1 (in humans)—has been classified as a subfamily of voltage-gated sodium channels (Goldin et al., 2000). The primary structure of Nax, however, markedly differs from that of other voltage-gated sodium channel family members and includes differences in the key regions for voltage sensing and inactivation. The functional properties of the channel are poorly understood, as all attempts to induce functional expression of Nax in heterologous systems have failed. Several years ago, we generated mice in which the Nax gene was knocked-out by insertion of the lacZ gene in-frame and found that the Nax channel is expressed in cells in the circumventricular organs (CVOs) (Watanabe et al., 2000), in particular the subfornical organ (SFO) and organum vasculosum lamina terminalis (OVLT), which are important regions for the control of body fluid ionic balance; for the expression other than the CNS (see Watanabe et al., 2002). Under thirst conditions, Nax-deficient mice showed hyperactivity of the neurons in these two areas and ingested excessive salt: Wild-type mice take water and stop salt ingestion under dehydrated condition. Infusion of a hypertonic Na solution into the cerebral ventricle also induced extensive water intake and aversion to saline (0.3 M

Overview of the voltage-gated sodium channel family.

Selective permeation of sodium ions through voltage-dependent sodium channels is fundamental to the generation of action potentials in excitable cells such as neurons. These channels are large integral membrane proteins and are encoded by at least ten genes in mammals. The different sodium channels have remarkably similar functional properties, but small changes in sodium-channel function are biologically relevant, as underscored by mutations that cause several human diseases of hyperexcitability.

Genomic structures of SCN2A and SCN3A – candidate genes for deafness at the DFNA16 locus

DFNA16 is a form of autosomal dominant non-syndromic hearing loss (ADNSHL) characterized by fluctuating progressive hearing impairment. Earlier, we mapped the deafness-causing gene to chromosome 2q23-24.3. In this paper, we describe fine mapping results using additional markers tightly linked to the DFNA16 candidate region. Critical recombinants at markers D2S354 and D2S124 define a 3.5-cM interval that contains the DFNA16 gene. Positional candidate genes include two members of the voltage-gated sodium channel family, the type 2 α subunit ( SCN2A) and the type 3 α subunit ( SCN3A). After showing that SCN2A is expressed in human fetal cochlea, we determined its genomic structure to facilitate mutation screening in our DFNA16 kindred. We also determined the genomic structure of SCN3A. These two genes are oriented head-to-head, with their 5′ ends separated by approximately 40 kb; their homology is 82% at the nucleotide level, and 85% for identities and 90% for positives at the amino acid level. They share similar genomic structures and have alternative splice isoforms that are developmentally regulated and highly conserved between species. Although no DFNA16-causing mutations were found in either gene, haplotype analysis with polymorphic markers in SCN2A introns further narrowed the candidate gene interval to the region flanked by D2S354 and STS SHGC-82894.