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Sample records for eukaryotic sodium channel

  1. Sodium channels and pain.

    Science.gov (United States)

    Habib, Abdella M; Wood, John N; Cox, James J

    2015-01-01

    Human and mouse genetic studies have led to significant advances in our understanding of the role of voltage-gated sodium channels in pain pathways. In this chapter, we focus on Nav1.7, Nav1.8, Nav1.9 and Nav1.3 and describe the insights gained from the detailed analyses of global and conditional transgenic Nav knockout mice in terms of pain behaviour. The spectrum of human disorders caused by mutations in these channels is also outlined, concluding with a summary of recent progress in the development of selective Nav1.7 inhibitors for the treatment of pain.

  2. Targeting sodium channels in cardiac arrhythmia

    NARCIS (Netherlands)

    Remme, Carol Ann; Wilde, Arthur A. M.

    2014-01-01

    Cardiac voltage-gated sodium channels are responsible for proper electrical conduction in the heart. During acquired pathological conditions and inherited sodium channelopathies, altered sodium channel function causes conduction disturbances and ventricular arrhythmias. Although the clinical,

  3. Insect sodium channels and insecticide resistance

    OpenAIRE

    Dong, Ke

    2007-01-01

    Voltage-gated sodium channels are essential for the generation and propagation of action potentials (i.e., electrical impulses) in excitable cells. Although most of our knowledge about sodium channels is derived from decades of studies of mammalian isoforms, research on insect sodium channels is revealing both common and unique aspects of sodium channel biology. In particular, our understanding of the molecular dynamics and pharmacology of insect sodium channels has advanced greatly in recent...

  4. Biophysical Adaptations of Prokaryotic Voltage-Gated Sodium Channels.

    Science.gov (United States)

    Vien, T N; DeCaen, P G

    2016-01-01

    This chapter describes the adaptive features found in voltage-gated sodium channels (NaVs) of prokaryotes and eukaryotes. These two families are distinct, having diverged early in evolutionary history but maintain a surprising degree of convergence in function. While prokaryotic NaVs are required for growth and motility, eukaryotic NaVs selectively conduct fast electrical currents for short- and long-range signaling across cell membranes in mammalian organs. Current interest in prokaryotic NaVs is stoked by their resolved high-resolution structures and functional features which are reminiscent of eukaryotic NaVs. In this chapter, comparisons between eukaryotic and prokaryotic NaVs are made to highlight the shared and unique aspects of ion selectivity, voltage sensitivity, and pharmacology. Examples of prokaryotic and eukaryotic NaV convergent evolution will be discussed within the context of their structural features. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Functional Expression of Drosophila para Sodium Channels

    Science.gov (United States)

    Warmke, Jeffrey W.; Reenan, Robert A.G.; Wang, Peiyi; Qian, Su; Arena, Joseph P.; Wang, Jixin; Wunderler, Denise; Liu, Ken; Kaczorowski, Gregory J.; Ploeg, Lex H.T. Van der; Ganetzky, Barry; Cohen, Charles J.

    1997-01-01

    The Drosophila para sodium channel α subunit was expressed in Xenopus oocytes alone and in combination with tipE, a putative Drosophila sodium channel accessory subunit. Coexpression of tipE with para results in elevated levels of sodium currents and accelerated current decay. Para/TipE sodium channels have biophysical and pharmacological properties similar to those of native channels. However, the pharmacology of these channels differs from that of vertebrate sodium channels: (a) toxin II from Anemonia sulcata, which slows inactivation, binds to Para and some mammalian sodium channels with similar affinity (Kd ≅ 10 nM), but this toxin causes a 100-fold greater decrease in the rate of inactivation of Para/TipE than of mammalian channels; (b) Para sodium channels are >10-fold more sensitive to block by tetrodotoxin; and (c) modification by the pyrethroid insecticide permethrin is >100-fold more potent for Para than for rat brain type IIA sodium channels. Our results suggest that the selective toxicity of pyrethroid insecticides is due at least in part to the greater affinity of pyrethroids for insect sodium channels than for mammalian sodium channels. PMID:9236205

  6. Sodium channels and mammalian sensory mechanotransduction.

    OpenAIRE

    Raouf, R.; Rugiero, F.; Kiesewetter, H.; Hatch, R.; Hummler, E; Nassar, M. A.; Wang, F.; Wood, J.N.

    2012-01-01

    Abstract 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 u...

  7. Posttranslational Modification of Sodium Channels.

    Science.gov (United States)

    Pei, Zifan; Pan, Yanling; Cummins, Theodore R

    2017-10-26

    Voltage-gated sodium channels (VGSCs) are critical determinants of excitability. The properties of VGSCs are thought to be tightly controlled. However, VGSCs are also subjected to extensive modifications. Multiple posttranslational modifications that covalently modify VGSCs in neurons and muscle have been identified. These include, but are not limited to, phosphorylation, ubiquitination, palmitoylation, nitrosylation, glycosylation, and SUMOylation. Posttranslational modifications of VGSCs can have profound impact on cellular excitability, contributing to normal and abnormal physiology. Despite four decades of research, the complexity of VGSC modulation is still being determined. While some modifications have similar effects on the various VGSC isoforms, others have isoform-specific interactions. In addition, while much has been learned about how individual modifications can impact VGSC function, there is still more to be learned about how different modifications can interact. Here we review what is known about VGSC posttranslational modifications with a focus on the breadth and complexity of the regulatory mechanisms that impact VGSC properties.

  8. Mechanism of sodium channel block by local anesthetics, antiarrhythmics, and anticonvulsants

    OpenAIRE

    Tikhonov, Denis B.; Zhorov, Boris S.

    2017-01-01

    Local anesthetics, antiarrhythmics, and anticonvulsants include both charged and electroneutral compounds that block voltage-gated sodium channels. Prior studies have revealed a common drug-binding region within the pore, but details about the binding sites and mechanism of block remain unclear. Here, we use the x-ray structure of a prokaryotic sodium channel, NavMs, to model a eukaryotic channel and dock representative ligands. These include lidocaine, QX-314, cocaine, quinidine, lamotrigine...

  9. Sodium channels and mammalian sensory mechanotransduction.

    Science.gov (United States)

    Raouf, Ramin; Rugiero, Francois; Kiesewetter, Hannes; Hatch, Rachel; Hummler, Edith; Nassar, Mohammed A; Wang, Fan; Wood, John N

    2012-03-26

    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. 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. 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.

  10. Genetic control of sodium channel function

    NARCIS (Netherlands)

    Tan, Hanno L.; Bezzina, Connie R.; Smits, Jeroen P. P.; Verkerk, Arie O.; Wilde, Arthur A. M.

    2003-01-01

    Sodium ion (Na) influx through cardiac Na channels triggers the action potential in cells of the working myocardium and the specialized conduction system. Na channels thus act as key molecular determinants of cardiac excitability and impulse propagation. Na channel dysfunction may cause

  11. Analysis of the action of lidocaine on insect sodium channels

    OpenAIRE

    Song, Weizhong; Silver, Kristopher S.; Du, Yuzhe; Liu, Zhiqi; Dong, Ke

    2010-01-01

    A new class of sodium channel blocker insecticides (SCBIs), which include indoxacarb, its active metabolite, DCJW, and metaflumizone, preferably block inactivated states of both insect and mammalian sodium channels in a manner similar to that by which local anesthetic (LA) drugs block mammalian sodium channels. A recent study showed that two residues in the cockroach sodium channel, F1817 and Y1824, corresponding to two key LA-interacting residues identified in mammalian sodium channels are n...

  12. Painful peripheral neuropathy and sodium channel mutations.

    Science.gov (United States)

    Hoeijmakers, Janneke G J; Faber, Catharina G; Merkies, Ingemar S J; Waxman, Stephen G

    2015-06-02

    Peripheral neuropathy can lead to neuropathic pain in a subset of patients. Painful peripheral neuropathy is a debilitating disorder, reflected by a reduced quality of life. Therapeutic strategies are limited and often disappointing, as in most cases targeted treatment is not available. Elucidating pathogenetic factors for pain might provide a target for optimal treatment. Voltage-gated sodium channels NaV1.7-NaV1.9 are expressed in the small-diameter dorsal root ganglion neurons and their axons. By a targeted gene approach, missense gain-of-function mutations of NaV1.7-NaV1.9 have been demonstrated in painful peripheral neuropathy. Functional analyses have shown that these mutations produce a spectrum of pro-excitatory changes in channel biophysics, with the shared outcome at the cellular level of dorsal root ganglion hyperexcitability. Reduced neurite outgrowth may be another consequence of sodium channel mutations, and possible therapeutic strategies include blockade of sodium channels or block of reverse operation of the sodium-calcium exchanger. Increased understanding of the pathophysiology of painful peripheral neuropathy offers new targets that may provide a basis for more effective treatment. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  13. Sodium channels in normal and pathological pain.

    Science.gov (United States)

    Dib-Hajj, Sulayman D; Cummins, Theodore R; Black, Joel A; Waxman, Stephen G

    2010-01-01

    Nociception is essential for survival whereas pathological pain is maladaptive and often unresponsive to pharmacotherapy. Voltage-gated sodium channels, Na(v)1.1-Na(v)1.9, are essential for generation and conduction of electrical impulses in excitable cells. Human and animal studies have identified several channels as pivotal for signal transmission along the pain axis, including Na(v)1.3, Na(v)1.7, Na(v)1.8, and Na(v)1.9, with the latter three preferentially expressed in peripheral sensory neurons and Na(v)1.3 being upregulated along pain-signaling pathways after nervous system injuries. Na(v)1.7 is of special interest because it has been linked to a spectrum of inherited human pain disorders. Here we review the contribution of these sodium channel isoforms to pain.

  14. Mechanism of action of sodium channel blocker insecticides (SCBIs) on insect sodium channels

    OpenAIRE

    Silver, Kristopher S.; Song, Weizhong; NOMURA, Yoshiko; Salgado, Vincent L.; Dong, Ke

    2009-01-01

    Sodium channel blocker insecticides (SCBIs) are a relatively new class of insecticides, with a mechanism of action different from those of other classes of insecticides that target voltage-gated sodium channels. These compounds have no effect at hyperpolarized membrane potentials, but cause a voltage-dependent, nearly irreversible block as the membrane potential is depolarized. The mechanism of action of SCBIs is similar to that of local anesthetics (LAs), class I anticonvulsants and class I ...

  15. Marine Toxins That Target Voltage-gated Sodium Channels

    Directory of Open Access Journals (Sweden)

    Robert J. French

    2006-04-01

    Full Text Available Abstract: Eukaryotic, voltage-gated sodium (NaV channels are large membrane proteins which underlie generation and propagation of rapid electrical signals in nerve, muscle and heart. Nine different NaV receptor sites, for natural ligands and/or drugs, have been identified, based on functional analyses and site-directed mutagenesis. In the marine ecosystem, numerous toxins have evolved to disrupt NaV channel function, either by inhibition of current flow through the channels, or by modifying the activation and inactivation gating processes by which the channels open and close. These toxins function in their native environment as offensive or defensive weapons in prey capture or deterrence of predators. In composition, they range from organic molecules of varying size and complexity to peptides consisting of ~10-70 amino acids. We review the variety of known NaV-targeted marine toxins, outlining, where known, their sites of interaction with the channel protein and their functional effects. In a number of cases, these natural ligands have the potential applications as drugs in clinical settings, or as models for drug development.

  16. Excitability Constraints on Voltage-Gated Sodium Channels

    OpenAIRE

    Brenner, Michael; Angelino, Elaine

    2007-01-01

    We study how functional constraints bound and shape evolution through an analysis of mammalian voltage-gated sodium channels. The primary function of sodium channels is to allow the propagation of action potentials. Since Hodgkin and Huxley, mathematical models have suggested that sodium channel properties need to be tightly constrained for an action potential to propagate. There are nine mammalian genes encoding voltage-gated sodium channels, many of which are more than approximately 90% ide...

  17. Distribution of eukaryotic plankton in the English Channel and the North Sea in summer

    NARCIS (Netherlands)

    Masquelier, S.; Foulon, E.; Jouenne, F.; Ferréol, M.; Brussaard, C.P.D.; Vaulot, D.

    2011-01-01

    The distribution of eukaryotic plankton was investigated in the English Channel and the North Sea during the MICROVIR cruise in summer 2007. The size distribution of autotrophic, heterotrophic eukaryotes and species composition was analyzed with a focus on two major divisions, Haptophyta and

  18. Lidocaine block of cardiac sodium channels

    OpenAIRE

    Bean, BP; Cohen, CJ; Tsien, RW

    1983-01-01

    Lidocaine block of cardiac sodium channels was studied in voltage-clamped rabbit purkinje fibers at drug concentrations ranging from 1 mM down to effective antiarrhythmic doses (5-20 ?M). Dose-response curves indicated that lidocaine blocks the channel by binding one-to-one, with a voltage-dependent K(d). The half-blocking concentration varied from more than 300 ?M, at a negative holding potential where inactivation was completely removed, to approximately 10 ?M, at a depolarized holding pote...

  19. Shellfish Toxins Targeting Voltage-Gated Sodium Channels

    Directory of Open Access Journals (Sweden)

    Fan Zhang

    2013-11-01

    Full Text Available Voltage-gated sodium channels (VGSCs play a central role in the generation and propagation of action potentials in excitable neurons and other cells and are targeted by commonly used local anesthetics, antiarrhythmics, and anticonvulsants. They are also common targets of neurotoxins including shellfish toxins. Shellfish toxins are a variety of toxic secondary metabolites produced by prokaryotic cyanobacteria and eukaryotic dinoflagellates in both marine and fresh water systems, which can accumulate in marine animals via the food chain. Consumption of shellfish toxin-contaminated seafood may result in potentially fatal human shellfish poisoning. This article provides an overview of the structure, bioactivity, and pharmacology of shellfish toxins that act on VGSCs, along with a brief discussion on their pharmaceutical potential for pain management.

  20. Molecular biology of insect sodium channels and pyrethroid resistance.

    Science.gov (United States)

    Dong, Ke; Du, Yuzhe; Rinkevich, Frank; Nomura, Yoshiko; Xu, Peng; Wang, Lingxin; Silver, Kristopher; Zhorov, Boris S

    2014-07-01

    Voltage-gated sodium channels are essential for the initiation and propagation of the action potential in neurons and other excitable cells. Because of their critical roles in electrical signaling, sodium channels are targets of a variety of naturally occurring and synthetic neurotoxins, including several classes of insecticides. This review is intended to provide an update on the molecular biology of insect sodium channels and the molecular mechanism of pyrethroid resistance. Although mammalian and insect sodium channels share fundamental topological and functional properties, most insect species carry only one sodium channel gene, compared to multiple sodium channel genes found in each mammalian species. Recent studies showed that two posttranscriptional mechanisms, alternative splicing and RNA editing, are involved in generating functional diversity of sodium channels in insects. More than 50 sodium channel mutations have been identified to be responsible for or associated with knockdown resistance (kdr) to pyrethroids in various arthropod pests and disease vectors. Elucidation of molecular mechanism of kdr led to the identification of dual receptor sites of pyrethroids on insect sodium channels. Many of the kdr mutations appear to be located within or close to the two receptor sites. The accumulating knowledge of insect sodium channels and their interactions with insecticides provides a foundation for understanding the neurophysiology of sodium channels in vivo and the development of new and safer insecticides for effective control of arthropod pests and human disease vectors. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Molecular Biology of Insect Sodium Channels and Pyrethroid Resistance

    Science.gov (United States)

    Dong, Ke; Du, Yuzhe; Rinkevich, Frank; Nomura, Yoshiko; Xu, Peng; Wang, Lingxin; Silver, Kristopher; Zhorov, Boris S.

    2015-01-01

    Voltage-gated sodium channels are essential for the initiation and propagation of the action potential in neurons and other excitable cells. Because of their critical roles in electrical signaling, sodium channels are targets of a variety of naturally occurring and synthetic neurotoxins, including several classes of insecticides. This review is intended to provide an update on the molecular biology of insect sodium channels and the molecular mechanism of pyrethroid resistance. Although mammalian and insect sodium channels share fundamental topological and functional properties, most insect species carry only one sodium channel gene, compared to multiple sodium channel genes found in each mammalian species. Recent studies showed that two posttranscriptional mechanisms, alternative splicing and RNA editing, are involved in generating functional diversity of sodium channels in insects. More than 50 sodium channel mutations have been identified to be responsible for or associated with knockdown resistance (kdr) to pyrethroids in various arthropod pests and disease vectors. Elucidation of molecular mechanism of kdr led to the identification of dual receptor sites of pyrethroids on insect sodium channels. Most of the kdr mutations appear to be located within or close to the two receptor sites. The accumulating knowledge of insect sodium channels and their interactions with insecticides provides a foundation for understanding the neurophysiology of sodium channels in vivo and the development of new and safer insecticides for effective control of arthropod pests and human disease vectors. PMID:24704279

  2. On conduction in a bacterial sodium channel.

    Directory of Open Access Journals (Sweden)

    Simone Furini

    Full Text Available Voltage-gated Na⁺-channels are transmembrane proteins that are responsible for the fast depolarizing phase of the action potential in nerve and muscular cells. Selective permeability of Na⁺ over Ca²⁺ or K⁺ ions is essential for the biological function of Na⁺-channels. After the emergence of the first high-resolution structure of a Na⁺-channel, an anionic coordination site was proposed to confer Na⁺ selectivity through partial dehydration of Na⁺ via its direct interaction with conserved glutamate side chains. By combining molecular dynamics simulations and free-energy calculations, a low-energy permeation pathway for Na⁺ ion translocation through the selectivity filter of the recently determined crystal structure of a prokaryotic sodium channel from Arcobacter butzleri is characterised. The picture that emerges is that of a pore preferentially occupied by two ions, which can switch between different configurations by crossing low free-energy barriers. In contrast to K⁺-channels, the movements of the ions appear to be weakly coupled in Na⁺-channels. When the free-energy maps for Na⁺ and K⁺ ions are compared, a selective site is characterised in the narrowest region of the filter, where a hydrated Na⁺ ion, and not a hydrated K⁺ ion, is energetically stable.

  3. The hitchhiker’s guide to the voltage-gated sodium channel galaxy

    Science.gov (United States)

    2016-01-01

    Eukaryotic voltage-gated sodium (Nav) channels contribute to the rising phase of action potentials and served as an early muse for biophysicists laying the foundation for our current understanding of electrical signaling. Given their central role in electrical excitability, it is not surprising that (a) inherited mutations in genes encoding for Nav channels and their accessory subunits have been linked to excitability disorders in brain, muscle, and heart; and (b) Nav channels are targeted by various drugs and naturally occurring toxins. Although the overall architecture and behavior of these channels are likely to be similar to the more well-studied voltage-gated potassium channels, eukaryotic Nav channels lack structural and functional symmetry, a notable difference that has implications for gating and selectivity. Activation of voltage-sensing modules of the first three domains in Nav channels is sufficient to open the channel pore, whereas movement of the domain IV voltage sensor is correlated with inactivation. Also, structure–function studies of eukaryotic Nav channels show that a set of amino acids in the selectivity filter, referred to as DEKA locus, is essential for Na+ selectivity. Structures of prokaryotic Nav channels have also shed new light on mechanisms of drug block. These structures exhibit lateral fenestrations that are large enough to allow drugs or lipophilic molecules to gain access into the inner vestibule, suggesting that this might be the passage for drug entry into a closed channel. In this Review, we will synthesize our current understanding of Nav channel gating mechanisms, ion selectivity and permeation, and modulation by therapeutics and toxins in light of the new structures of the prokaryotic Nav channels that, for the time being, serve as structural models of their eukaryotic counterparts. PMID:26712848

  4. Excitability constraints on voltage-gated sodium channels.

    Directory of Open Access Journals (Sweden)

    Elaine Angelino

    2007-09-01

    Full Text Available We study how functional constraints bound and shape evolution through an analysis of mammalian voltage-gated sodium channels. The primary function of sodium channels is to allow the propagation of action potentials. Since Hodgkin and Huxley, mathematical models have suggested that sodium channel properties need to be tightly constrained for an action potential to propagate. There are nine mammalian genes encoding voltage-gated sodium channels, many of which are more than approximately 90% identical by sequence. This sequence similarity presumably corresponds to similarity of function, consistent with the idea that these properties must be tightly constrained. However, the multiplicity of genes encoding sodium channels raises the question: why are there so many? We demonstrate that the simplest theoretical constraints bounding sodium channel diversity--the requirements of membrane excitability and the uniqueness of the resting potential--act directly on constraining sodium channel properties. We compare the predicted constraints with functional data on mammalian sodium channel properties collected from the literature, including 172 different sets of measurements from 40 publications, wild-type and mutant, under a variety of conditions. The data from all channel types, including mutants, obeys the excitability constraint; on the other hand, channels expressed in muscle tend to obey the constraint of a unique resting potential, while channels expressed in neuronal tissue do not. The excitability properties alone distinguish the nine sodium channels into four different groups that are consistent with phylogenetic analysis. Our calculations suggest interpretations for the functional differences between these groups.

  5. Sodium Channel (Dys)Function and Cardiac Arrhythmias

    NARCIS (Netherlands)

    Remme, Carol Ann; Bezzina, Connie R.

    2010-01-01

    P>Cardiac voltage-gated sodium channels are transmembrane proteins located in the cell membrane of cardiomyocytes. Influx of sodium ions through these ion channels is responsible for the initial fast upstroke of the cardiac action potential. This inward sodium current thus triggers the initiation

  6. Evolution of the epithelial sodium channel and the sodium pump as limiting factors of aldosterone action on sodium transport.

    Science.gov (United States)

    Studer, Romain A; Person, Emilie; Robinson-Rechavi, Marc; Rossier, Bernard C

    2011-07-14

    Despite large changes in salt intake, the mammalian kidney is able to maintain the extracellular sodium concentration and osmolarity within very narrow margins, thereby controlling blood volume and blood pressure. In the aldosterone-sensitive distal nephron (ASDN), aldosterone tightly controls the activities of epithelial sodium channel (ENaC) and Na,K-ATPase, the two limiting factors in establishing transepithelial sodium transport. It has been proposed that the ENaC/degenerin gene family is restricted to Metazoans, whereas the α- and β-subunits of Na,K-ATPase have homologous genes in prokaryotes. This raises the question of the emergence of osmolarity control. By exploring recent genomic data of diverse organisms, we found that: 1) ENaC/degenerin exists in all of the Metazoans screened, including nonbilaterians and, by extension, was already present in ancestors of Metazoa; 2) ENaC/degenerin is also present in Naegleria gruberi, an eukaryotic microbe, consistent with either a vertical inheritance from the last common ancestor of Eukaryotes or a lateral transfer between Naegleria and Metazoan ancestors; and 3) The Na,K-ATPase β-subunit is restricted to Holozoa, the taxon that includes animals and their closest single-cell relatives. Since the β-subunit of Na,K-ATPase plays a key role in targeting the α-subunit to the plasma membrane and has an additional function in the formation of cell junctions, we propose that the emergence of Na,K-ATPase, together with ENaC/degenerin, is linked to the development of multicellularity in the Metazoan kingdom. The establishment of multicellularity and the associated extracellular compartment ("internal milieu") precedes the emergence of other key elements of the aldosterone signaling pathway.

  7. Phylogeny unites animal sodium leak channels with fungal calcium channels in an ancient, voltage-insensitive clade.

    Science.gov (United States)

    Liebeskind, Benjamin J; Hillis, David M; Zakon, Harold H

    2012-12-01

    Proteins in the superfamily of voltage-gated ion channels mediate behavior across the tree of life. These proteins regulate the movement of ions across cell membranes by opening and closing a central pore that controls ion flow. The best-known members of this superfamily are the voltage-gated potassium, calcium (Ca(v)), and sodium (Na(v)) channels, which underlie impulse conduction in nerve and muscle. Not all members of this family are opened by changes in voltage, however. NALCN (NA(+) leak channel nonselective) channels, which encode a voltage-insensitive "sodium leak" channel, have garnered a growing interest. This study examines the phylogenetic relationship among Na(v)/Ca(v) voltage-gated and voltage-insensitive channels in the eukaryotic group Opisthokonta, which includes animals, fungi, and their unicellular relatives. We show that NALCN channels diverged from voltage-gated channels before the divergence of fungi and animals and that the closest relatives of NALCN channels are fungal calcium channels, which they functionally resemble.

  8. Lidocaine block of cardiac sodium channels.

    Science.gov (United States)

    Bean, B P; Cohen, C J; Tsien, R W

    1983-05-01

    Lidocaine block of cardiac sodium channels was studied in voltage-clamped rabbit purkinje fibers at drug concentrations ranging from 1 mM down to effective antiarrhythmic doses (5-20 muM). Dose-response curves indicated that lidocaine blocks the channel by binding one-to-one, with a voltage-dependent K(d). The half-blocking concentration varied from more than 300 muM, at a negative holding potential where inactivation was completely removed, to approximately 10 muM, at a depolarized holding potential where inactivation was nearly complete. Lidocaine block showed prominent use dependence with trains of depolarizing pulses from a negative holding potential. During the interval between pulses, repriming of I (Na) displayed two exponential components, a normally recovering component (tauless than 0.2 s), and a lidocaine-induced, slowly recovering fraction (tau approximately 1-2 s at pH 7.0). Raising the lidocaine concentration magnified the slowly recovering fraction without changing its time course; after a long depolarization, this fraction was one-half at approximately 10 muM lidocaine, just as expected if it corresponded to drug-bound, inactivated channels. At less than or equal to 20 muM lidocaine, the slowly recovering fraction grew exponentially to a steady level as the preceding depolarization was prolonged; the time course was the same for strong or weak depolarizations, that is, with or without significant activation of I(Na). This argues that use dependence at therapeutic levels reflects block of inactivated channels, rather than block of open channels. Overall, these results provide direct evidence for the "modulated-receptor hypothesis" of Hille (1977) and Hondeghem and Katzung (1977). Unlike tetrodotoxin, lidocaine shows similar interactions with Na channels of heart, nerve, and skeletal muscle.

  9. DDESC: Dragon database for exploration of sodium channels in human

    Directory of Open Access Journals (Sweden)

    Radovanovic Aleksandar

    2008-12-01

    Full Text Available Abstract Background Sodium channels are heteromultimeric, integral membrane proteins that belong to a superfamily of ion channels. The mutations in genes encoding for sodium channel proteins have been linked with several inherited genetic disorders such as febrile epilepsy, Brugada syndrome, ventricular fibrillation, long QT syndrome, or channelopathy associated insensitivity to pain. In spite of these significant effects that sodium channel proteins/genes could have on human health, there is no publicly available resource focused on sodium channels that would support exploration of the sodium channel related information. Results We report here Dragon Database for Exploration of Sodium Channels in Human (DDESC, which provides comprehensive information related to sodium channels regarding different entities, such as "genes and proteins", "metabolites and enzymes", "toxins", "chemicals with pharmacological effects", "disease concepts", "human anatomy", "pathways and pathway reactions" and their potential links. DDESC is compiled based on text- and data-mining. It allows users to explore potential associations between different entities related to sodium channels in human, as well as to automatically generate novel hypotheses. Conclusion DDESC is first publicly available resource where the information related to sodium channels in human can be explored at different levels. This database is freely accessible for academic and non-profit users via the worldwide web http://apps.sanbi.ac.za/ddesc.

  10. Macroscopic kinetics of pentameric ligand gated ion channels: comparisons between two prokaryotic channels and one eukaryotic channel.

    Science.gov (United States)

    Laha, Kurt T; Ghosh, Borna; Czajkowski, Cynthia

    2013-01-01

    Electrochemical signaling in the brain depends on pentameric ligand-gated ion channels (pLGICs). Recently, crystal structures of prokaryotic pLGIC homologues from Erwinia chrysanthemi (ELIC) and Gloeobacter violaceus (GLIC) in presumed closed and open channel states have been solved, which provide insight into the structural mechanisms underlying channel activation. Although structural studies involving both ELIC and GLIC have become numerous, thorough functional characterizations of these channels are still needed to establish a reliable foundation for comparing kinetic properties. Here, we examined the kinetics of ELIC and GLIC current activation, desensitization, and deactivation and compared them to the GABAA receptor, a prototypic eukaryotic pLGIC. Outside-out patch-clamp recordings were performed with HEK-293T cells expressing ELIC, GLIC, or α1β2γ2L GABAA receptors, and ultra-fast ligand application was used. In response to saturating agonist concentrations, we found both ELIC and GLIC current activation were two to three orders of magnitude slower than GABAA receptor current activation. The prokaryotic channels also had slower current desensitization on a timescale of seconds. ELIC and GLIC current deactivation following 25 s pulses of agonist (cysteamine and pH 4.0 buffer, respectively) were relatively fast with time constants of 24.9 ± 5.1 ms and 1.2 ± 0.2 ms, respectively. Surprisingly, ELIC currents evoked by GABA activated very slowly with a time constant of 1.3 ± 0.3 s and deactivated even slower with a time constant of 4.6 ± 1.2 s. We conclude that the prokaryotic pLGICs undergo similar agonist-mediated gating transitions to open and desensitized states as eukaryotic pLGICs, supporting their use as experimental models. Their uncharacteristic slow activation, slow desensitization and rapid deactivation time courses are likely due to differences in specific structural elements, whose future identification may help uncover mechanisms underlying p

  11. Bioinspired Artificial Sodium and Potassium Ion Channels.

    Science.gov (United States)

    Rodríguez-Vázquez, Nuria; Fuertes, Alberto; Amorín, Manuel; Granja, Juan R

    2016-01-01

    In Nature, all biological systems present a high level of compartmentalization in order to carry out a wide variety of functions in a very specific way. Hence, they need ways to be connected with the environment for communication, homeostasis equilibrium, nutrition, waste elimination, etc. The biological membranes carry out these functions; they consist of physical insulating barriers constituted mainly by phospholipids. These amphipathic molecules spontaneously aggregate in water to form bilayers in which the polar groups are exposed to the aqueous media while the non-polar chains self-organize by aggregating to each other to stay away from the aqueous media. The insulating properties of membranes are due to the formation of a hydrophobic bilayer covered at both sides by the hydrophilic phosphate groups. Thus, lipophilic molecules can permeate the membrane freely, while the small charged or very hydrophilic molecules require the assistance of other membrane components in order to overcome the energetic cost implied in crossing the non-polar region of the bilayer. Most of the large polar species (such as oligosaccharides, polypeptides or nucleic acids) cross into and out of the cell via endocytosis and exocytosis, respectively. Nature has created a series of systems (carriers and pores) in order to control the balance of small hydrophilic molecules and ions. The most important structures to achieve these goals are the ionophoric proteins that include the channel proteins, such as the sodium and potassium channels, and ionic transporters, including the sodium/potassium pumps or calcium/sodium exchangers among others. Inspired by these, scientists have created non-natural synthetic transporting structures to mimic the natural systems. The progress in the last years has been remarkable regarding the efficient transport of Na(+) and K(+) ions, despite the fact that the selectivity and the ON/OFF state of the non-natural systems remain a present and future challenge.

  12. Voltage-gated sodium channels: action players with many faces

    NARCIS (Netherlands)

    Koopmann, Tamara T.; Bezzina, Connie R.; Wilde, Arthur A. M.

    2006-01-01

    Voltage-gated sodium channels are responsible for the upstroke of the action potential and thereby play an important role in propagation of the electrical impulse in excitable tissues like muscle, nerve and the heart. Duplication of the sodium channels encoding genes during evolution generated the

  13. Sodium Channel Mutations and Pyrethroid Resistance in Aedes aegypti

    Directory of Open Access Journals (Sweden)

    Yuzhe Du

    2016-10-01

    Full Text Available Pyrethroid insecticides are widely used to control insect pests and human disease vectors. Voltage-gated sodium channels are the primary targets of pyrethroid insecticides. Mutations in the sodium channel have been shown to be responsible for pyrethroid resistance, known as knockdown resistance (kdr, in various insects including mosquitoes. In Aedes aegypti mosquitoes, the principal urban vectors of dengue, zika, and yellow fever viruses, multiple single nucleotide polymorphisms in the sodium channel gene have been found in pyrethroid-resistant populations and some of them have been functionally confirmed to be responsible for kdr in an in vitro expression system, Xenopus oocytes. This mini-review aims to provide an update on the identification and functional characterization of pyrethroid resistance-associated sodium channel mutations from Aedes aegypti. The collection of kdr mutations not only helped us develop molecular markers for resistance monitoring, but also provided valuable information for computational molecular modeling of pyrethroid receptor sites on the sodium channel.

  14. Macroscopic kinetics of pentameric ligand gated ion channels: comparisons between two prokaryotic channels and one eukaryotic channel.

    Directory of Open Access Journals (Sweden)

    Kurt T Laha

    Full Text Available Electrochemical signaling in the brain depends on pentameric ligand-gated ion channels (pLGICs. Recently, crystal structures of prokaryotic pLGIC homologues from Erwinia chrysanthemi (ELIC and Gloeobacter violaceus (GLIC in presumed closed and open channel states have been solved, which provide insight into the structural mechanisms underlying channel activation. Although structural studies involving both ELIC and GLIC have become numerous, thorough functional characterizations of these channels are still needed to establish a reliable foundation for comparing kinetic properties. Here, we examined the kinetics of ELIC and GLIC current activation, desensitization, and deactivation and compared them to the GABAA receptor, a prototypic eukaryotic pLGIC. Outside-out patch-clamp recordings were performed with HEK-293T cells expressing ELIC, GLIC, or α1β2γ2L GABAA receptors, and ultra-fast ligand application was used. In response to saturating agonist concentrations, we found both ELIC and GLIC current activation were two to three orders of magnitude slower than GABAA receptor current activation. The prokaryotic channels also had slower current desensitization on a timescale of seconds. ELIC and GLIC current deactivation following 25 s pulses of agonist (cysteamine and pH 4.0 buffer, respectively were relatively fast with time constants of 24.9 ± 5.1 ms and 1.2 ± 0.2 ms, respectively. Surprisingly, ELIC currents evoked by GABA activated very slowly with a time constant of 1.3 ± 0.3 s and deactivated even slower with a time constant of 4.6 ± 1.2 s. We conclude that the prokaryotic pLGICs undergo similar agonist-mediated gating transitions to open and desensitized states as eukaryotic pLGICs, supporting their use as experimental models. Their uncharacteristic slow activation, slow desensitization and rapid deactivation time courses are likely due to differences in specific structural elements, whose future identification may help uncover

  15. Inhibition of voltage-gated sodium channels by sumatriptan bioisosteres

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    Roberta eCarbonara

    2015-07-01

    Full Text Available Voltage-gated sodium channels are known to play a pivotal role in perception and transmission of pain sensations. Gain-of-function mutations in the genes encoding the peripheral neuronal sodium channels, hNav1.7-1.9, cause human painful diseases. Thus while treatment of chronic pain remains an unmet clinical need, sodium channel blockers are considered as promising druggable targets. In a previous study, we evaluated the analgesic activity of sumatriptan, an agonist of serotonin 5HT1B/D receptors, and some new chiral bioisosteres, using the hot plate test in the mouse. Interestingly, we observed that the analgesic effectiveness was not necessarily correlated to serotonin agonism. In this study, we evaluated whether sumatriptan and its congeners may inhibit heterologously-expressed hNav1.7 sodium channels using the patch-clamp method. We show that sumatriptan blocks hNav1.7 channels only at very high, supratherapeutic concentrations. In contrast, its three analogues, namely 20b, (R-31b, and (S-22b, exert a dose and use-dependent sodium channel block. At 0.1 and 10 Hz stimulation frequencies, the most potent compound, (S-22b, was 4.4 and 1.7 fold more potent than the well-known sodium channel blocker mexiletine. The compound induces a negative shift of voltage dependence of fast inactivation, suggesting higher affinity to the inactivated channel. Accordingly, we show that (S-22b likely binds the conserved local anesthetic receptor within voltage-gated sodium channels. Combining these results with the previous ones, we hypothesize that use-dependent sodium channel blockade contributes to the analgesic activity of (R-31b and (S-22b. These later compounds represent promising lead compounds for the development of efficient analgesics, the mechanism of action of which may include a dual action on sodium channels and 5HT1D receptors.

  16. Inhibition of voltage-gated sodium channels by sumatriptan bioisosteres.

    Science.gov (United States)

    Carbonara, Roberta; Carocci, Alessia; Roussel, Julien; Crescenzo, Giuseppe; Buonavoglia, Canio; Franchini, Carlo; Lentini, Giovanni; Camerino, Diana Conte; Desaphy, Jean-François

    2015-01-01

    Voltage-gated sodium channels are known to play a pivotal role in perception and transmission of pain sensations. Gain-of-function mutations in the genes encoding the peripheral neuronal sodium channels, hNav1.7-1.9, cause human painful diseases. Thus while treatment of chronic pain remains an unmet clinical need, sodium channel blockers are considered as promising druggable targets. In a previous study, we evaluated the analgesic activity of sumatriptan, an agonist of serotonin 5HT1B/D receptors, and some new chiral bioisosteres, using the hot plate test in the mouse. Interestingly, we observed that the analgesic effectiveness was not necessarily correlated to serotonin agonism. In this study, we evaluated whether sumatriptan and its congeners may inhibit heterologously expressed hNav1.7 sodium channels using the patch-clamp method. We show that sumatriptan blocks hNav1.7 channels only at very high, supratherapeutic concentrations. In contrast, its three analogs, namely 20b, (R)-31b, and (S)-22b, exert a dose and use-dependent sodium channel block. At 0.1 and 10 Hz stimulation frequencies, the most potent compound, (S)-22b, was 4.4 and 1.7 fold more potent than the well-known sodium channel blocker mexiletine. The compound induces a negative shift of voltage dependence of fast inactivation, suggesting higher affinity to the inactivated channel. Accordingly, we show that (S)-22b likely binds the conserved local anesthetic receptor within voltage-gated sodium channels. Combining these results with the previous ones, we hypothesize that use-dependent sodium channel blockade contributes to the analgesic activity of (R)-31b and (S)-22b. These later compounds represent promising lead compounds for the development of efficient analgesics, the mechanism of action of which may include a dual action on sodium channels and 5HT1D receptors.

  17. Mechanism of sodium channel block by local anesthetics, antiarrhythmics, and anticonvulsants.

    Science.gov (United States)

    Tikhonov, Denis B; Zhorov, Boris S

    2017-04-03

    Local anesthetics, antiarrhythmics, and anticonvulsants include both charged and electroneutral compounds that block voltage-gated sodium channels. Prior studies have revealed a common drug-binding region within the pore, but details about the binding sites and mechanism of block remain unclear. Here, we use the x-ray structure of a prokaryotic sodium channel, NavMs, to model a eukaryotic channel and dock representative ligands. These include lidocaine, QX-314, cocaine, quinidine, lamotrigine, carbamazepine (CMZ), phenytoin, lacosamide, sipatrigine, and bisphenol A. Preliminary calculations demonstrated that a sodium ion near the selectivity filter attracts electroneutral CMZ but repels cationic lidocaine. Therefore, we further docked electroneutral and cationic drugs with and without a sodium ion, respectively. In our models, all the drugs interact with a phenylalanine in helix IVS6. Electroneutral drugs trap a sodium ion in the proximity of the selectivity filter, and this same site attracts the charged group of cationic ligands. At this position, even small drugs can block the permeation pathway by an electrostatic or steric mechanism. Our study proposes a common pharmacophore for these diverse drugs. It includes a cationic moiety and an aromatic moiety, which are usually linked by four bonds. © 2017 Tikhonov and Zhorov.

  18. Changes of sodium channel expression in experimental painful diabetic neuropathy.

    Science.gov (United States)

    Craner, Matthew J; Klein, Joshua P; Renganathan, Muthukrishnan; Black, Joel A; Waxman, Stephen G

    2002-12-01

    Although pain is experienced by many patients with diabetic neuropathy, the pathophysiology of painful diabetic neuropathy is not understood. Substantial evidence indicates that dysregulated sodium channel gene transcription contributes to hyperexcitability of dorsal root ganglion neurons, which may produce neuropathic pain after axonal transection. In this study, we examined sodium channel mRNA and protein expression in dorsal root ganglion neurons in rats with streptozotocin-induced diabetes and tactile allodynia, using in situ hybridization and immunocytochemistry for sodium channels Na(v)1.1, Na(v)1.3, Na(v)1.6, Na(v)1.7, Na(v)1.8, and Na(v)1.9. Our results show that, in rats with experimental diabetes, there is a significant upregulation of mRNA for the Na(v)1.3, Na(v)1.6, and Na(v)1.9 sodium channels and a downregulation of Na(v)1.8 mRNA 1 and 8 weeks after onset of allodynia. Channel protein levels display parallel changes. Our results demonstrate dysregulated expression of the genes for sodium channels Na(v)1.3, Na(v)1.6, Na(v)1.8, and Na(v)1.9 in dorsal root ganglion neurons in experimental diabetes and suggest that misexpression of sodium channels contributes to neuropathic pain associated with diabetic neuropathy.

  19. Sodium channels and nociception: recent concepts and therapeutic opportunities.

    Science.gov (United States)

    Krafte, Douglas S; Bannon, Anthony W

    2008-02-01

    Recent scientific advances have enhanced our understanding of the role voltage-gated sodium channels play in pain sensation. Human data on Nav1.7 show that gain-of-function mutations lead to enhanced pain while loss-of-function mutations lead to Congenital Indifference to Pain. Pre-clinical data from knockouts, anti-sense oligonucleotides, and siRNA for Nav1.3, 1.7, 1.8, and 1.9 have also demonstrated that specific subtypes of voltage-gated sodium channels play a role in different types of pain signaling. In addition, recent reports show that CNS penetration by voltage-gated sodium channel blockers is not required for efficacy in pre-clinical pain models while others have reported that identification of subtype-selective small molecules is possible. All of these data are converging to suggest next generation sodium channel blockers may offer the potential for novel pain therapies in the future.

  20. Sodium Channels in Pain and Cancer: New Therapeutic Opportunities.

    Science.gov (United States)

    Luiz, Ana Paula; Wood, John N

    2016-01-01

    Voltage-gated sodium channels (VGSCs) underpin electrical activity in the nervous system through action potential propagation. First predicted by the modeling studies of Hodgkin and Huxley, they were subsequently identified at the molecular level by groups led by Catterall and Numa. VGSC dysfunction has long been linked to neuronal and cardiac disorders with some nonselective sodium channel blockers in current use in the clinic. The lack of selectivity means that side effect issues are a major impediment to the use of broad spectrum sodium channel blockers. Nine different sodium channels are known to exist, and selective blockers are now being developed. The potential utility of these drugs to target diseases ranging from migraine, multiple sclerosis, muscle, and immune system disorders, to cancer and pain is being explored. Four channels are potential targets for pain disorders. This conclusion comes from mouse knockout studies and human mutations that prove the involvement of Nav1.3, Nav1.7, Nav1.8, and Nav1.9 in the development and maintenance of acute and chronic pain. In this chapter, we present a short overview of the possible role of Nav1.3, Nav1.7, Nav1.8, and Nav1.9 in human pain and the emerging and unexpected role of sodium channels in cancer pathogenesis. © 2016 Elsevier Inc. All rights reserved.

  1. The Epithelial Sodium Channel and the Processes of Wound Healing

    Directory of Open Access Journals (Sweden)

    Silvia Chifflet

    2016-01-01

    Full Text Available The epithelial sodium channel (ENaC mediates passive sodium transport across the apical membranes of sodium absorbing epithelia, like the distal nephron, the intestine, and the lung airways. Additionally, the channel has been involved in the transduction of mechanical stimuli, such as hydrostatic pressure, membrane stretch, and shear stress from fluid flow. Thus, in vascular endothelium, it participates in the control of the vascular tone via its activity both as a sodium channel and as a shear stress transducer. Rather recently, ENaC has been shown to participate in the processes of wound healing, a role that may also involve its activities as sodium transporter and as mechanotransducer. Its presence as the sole channel mediating sodium transport in many tissues and the diversity of its functions probably underlie the complexity of its regulation. This brief review describes some aspects of ENaC regulation, comments on evidence about ENaC participation in wound healing, and suggests possible regulatory mechanisms involved in this participation.

  2. Voltage-gated sodium channels: therapeutic targets for pain.

    Science.gov (United States)

    Dib-Hajj, Sulayman D; Black, Joel A; Waxman, Stephen G

    2009-10-01

    To provide an overview of the role of voltage-gated sodium channels in pathophysiology of acquired and inherited pain states, and of recent developments that validate these channels as therapeutic targets for treating chronic pain. Neuropathic and inflammatory pain conditions are major medical needs worldwide with only partial or low efficacy treatment options currently available. An important role of voltage-gated sodium channels in many different pain states has been established in animal models and, empirically, in humans, where sodium channel blockers partially ameliorate pain. Animal studies have causally linked changes in sodium channel expression and modulation that alter channel gating properties or current density in nociceptor neurons to different pain states. Biophysical and pharmacological studies have identified the sodium channel isoforms Na(v)1.3, Na(v)1.7, Na(v)1.8, and Na(v)1.9 as particularly important in the pathophysiology of different pain syndromes. Recently, gain-of-function mutations in SCN9A, the gene which encodes Na(v)1.7, have been linked to two human-inherited pain syndromes, inherited erythromelalgia and paroxysmal extreme pain disorder, while loss-of-function mutations in SCN9A have been linked to complete insensitivity to pain. Studies on firing properties of sensory neurons of dorsal root ganglia demonstrate that the effects of gain-of-function mutations in Na(v)1.7 on the excitability of these neurons depend on the presence of Na(v)1.8, which suggests a similar physiological interaction of these two channels in humans carrying the Na(v)1.7 pain mutation. These studies suggest that isoform-specific blockers of these channels or targeting of their modulators may provide novel approaches to treatment of pain.

  3. Solution structure of the sodium channel antagonist conotoxin GS: a new molecular caliper for probing sodium channel geometry.

    Science.gov (United States)

    Hill, J M; Alewood, P F; Craik, D J

    1997-04-15

    The venoms of Conus snails contain small, disulfide-rich inhibitors of voltage-dependent sodium channels. Conotoxin GS is a 34-residue polypeptide isolated from Conus geographus that interacts with the extracellular entrance of skeletal muscle sodium channels to prevent sodium ion conduction. Although conotoxin GS binds competitively with mu conotoxin GIIIA to the sodium channel surface, the two toxin types have little sequence identity with one another, and conotoxin GS has a four-loop structural framework rather than the characteristic three-loop mu-conotoxin framework. The structural study of conotoxin GS will form the basis for establishing a structure-activity relationship and understanding its interaction with the pore region of sodium channels. The three-dimensional structure of conotoxin GS was determined using two-dimensional NMR spectroscopy. The protein exhibits a compact fold incorporating a beta hairpin and several turns. An unusual feature of conotoxin GS is the exceptionally high proportion (100%) of cis-imide bond geometry for the three proline or hydroxyproline residues. The structure of conotoxin GS bears little resemblance to the three-loop mu conotoxins, consistent with the low sequence identity between the two toxin types and their different structural framework. However, the tertiary structure and cystine-knot motif formed by the three disulfide bonds is similar to that present in several other polypeptide ion channel inhibitors. This is the first three-dimensional structure of a 'four-loop' sodium channel inhibitor, and it represents a valuable new structural probe for the pore region of voltage-dependent sodium channels. The distribution of amino acid sidechains in the structure creates several polar and charged patches, and comparison with the mu conotoxins provides a basis for determining the binding surface of the conotoxin GS polypeptide.

  4. Sodium channels as targets for volatile anesthetics

    Directory of Open Access Journals (Sweden)

    Karl F. Herold

    2012-03-01

    Full Text Available The molecular mechanisms of modern inhaled anesthetics although widely used in clinical settings are still poorly understood. Considerable evidence supports effects on membrane proteins such as ligand- and voltage-gated ion channels of excitable cells. Na+ channels are crucial to action potential initiation and propagation, and represent potential targets for volatile anesthetics. Inhibition of presynaptic Na+ channels leads to reduced neurotransmitter release at the synapse and could therefore contribute to the mechanisms by which volatile anesthetics produce their characteristic effects: amnesia, unconsciousness, and immobility. Early studies on crayfish and squid giant axon showed inhibition of Na+ currents by volatile anesthetics. Subsequent studies using native neuronal preparations and heterologous expression systems with various mammalian Na+ channel isoforms implicated inhibition of presynaptic Na+ channels in anesthetic actions. Volatile anesthetics reduce peak Na+ current and shift the voltage of half-maximal steady-state inactivation towards more negative potentials, thus stabilizing the fast-inactivated state. Furthermore recovery from fast-inactivation is slowed together with an enhanced use-dependent block during pulse train protocols. These effects can reduce neurotransmitter release by depressing presynaptic excitability, depolarization and Ca entry, and ultimately transmitter release. This reduction in transmitter release is more portent for glutamatergic vs. GABAergic terminals. Involvement of Na+ channel inhibition in mediating the immobility caused by volatile anesthetics has been demonstrated in animal studies, in which intrathecal infusion of the Na+ channel blocker tetrodotoxin increases volatile anesthetic potency, whereas infusion of the Na+ channels agonist veratridine reduces anesthetic potency. These studies indicate that inhibition of presynaptic Na+ channels by volatile anesthetics is involved in mediating some of

  5. Physiological regulation of epithelial sodium channel by proteolysis

    DEFF Research Database (Denmark)

    Svenningsen, Per; Friis, Ulla G; Bistrup, Claus

    2011-01-01

    PURPOSE OF REVIEW: Activation of epithelial sodium channel (ENaC) by proteolysis appears to be relevant for day-to-day physiological regulation of channel activity in kidney and other epithelial tissues. Pathophysiogical, proteolytic activation of ENaC in kidney has been demonstrated in proteinuric...... disease. RECENT FINDINGS: A variation in sodium and potassium intake or plasma aldosterone changes the number of cleaved α and γ-ENaC subunits and is associated with changes in ENaC currents. The protease furin mediates intracellular cleavage, whereas the channel-activating protease prostasin (CAP-1...... opens the way for new understanding of the pathogenesis of proteinuric sodium retention, which may involve plasmin and present several potential new drug targets....

  6. Modulation of L-type calcium channels by sodium ions.

    OpenAIRE

    Balke, C W; Wier, W G

    1992-01-01

    It is universally believed that the removal of external sodium ions is without effect on calcium current. We now report that in enzymatically isolated guinea pig ventricular cells, the replacement of external sodium ions with certain other cations causes a 3- to 6-fold increase in peak L-type calcium current. The increase in current is reversibly blocked by L-type calcium-channel antagonists, not mediated by changes in internal calcium, and is inhibited by intracellular 5'-adenylyl imidodipho...

  7. Sodium-calcium exchanger and multiple sodium channel isoforms in intra-epidermal nerve terminals

    Directory of Open Access Journals (Sweden)

    Gasser Andreas

    2010-11-01

    Full Text Available Abstract Background Nociception requires transduction and impulse electrogenesis in nerve fibers which innervate the body surface, including the skin. However, the molecular substrates for transduction and action potential initiation in nociceptors are incompletely understood. In this study, we examined the expression and distribution of Na+/Ca2+ exchanger (NCX and voltage-gated sodium channel isoforms in intra-epidermal free nerve terminals. Results Small diameter DRG neurons exhibited robust NCX2, but not NCX1 or NCX3 immunolabeling, and virtually all PGP 9.5-positive intra-epidermal free nerve terminals displayed NCX2 immunoreactivity. Sodium channel NaV1.1 was not detectable in free nerve endings. In contrast, the majority of nerve terminals displayed detectable levels of expression of NaV1.6, NaV1.7, NaV1.8 and NaV1.9. Sodium channel immunoreactivity in the free nerve endings extended from the dermal boundary to the terminal tip. A similar pattern of NCX and sodium channel immunolabeling was observed in DRG neurons in vitro. Conclusions NCX2, as well as NaV1.6, NaV1.7, NaV1.8 and NaV1.9, are present in most intra-epidermal free nerve endings. The presence of NCX2, together with multiple sodium channel isoforms, in free nerve endings may have important functional implications.

  8. Sodium-calcium exchanger and multiple sodium channel isoforms in intra-epidermal nerve terminals.

    Science.gov (United States)

    Persson, Anna-Karin; Black, Joel A; Gasser, Andreas; Cheng, Xiaoyang; Fischer, Tanya Z; Waxman, Stephen G

    2010-11-30

    Nociception requires transduction and impulse electrogenesis in nerve fibers which innervate the body surface, including the skin. However, the molecular substrates for transduction and action potential initiation in nociceptors are incompletely understood. In this study, we examined the expression and distribution of Na+/Ca2+ exchanger (NCX) and voltage-gated sodium channel isoforms in intra-epidermal free nerve terminals. Small diameter DRG neurons exhibited robust NCX2, but not NCX1 or NCX3 immunolabeling, and virtually all PGP 9.5-positive intra-epidermal free nerve terminals displayed NCX2 immunoreactivity. Sodium channel NaV1.1 was not detectable in free nerve endings. In contrast, the majority of nerve terminals displayed detectable levels of expression of NaV1.6, NaV1.7, NaV1.8 and NaV1.9. Sodium channel immunoreactivity in the free nerve endings extended from the dermal boundary to the terminal tip. A similar pattern of NCX and sodium channel immunolabeling was observed in DRG neurons in vitro. NCX2, as well as NaV1.6, NaV1.7, NaV1.8 and NaV1.9, are present in most intra-epidermal free nerve endings. The presence of NCX2, together with multiple sodium channel isoforms, in free nerve endings may have important functional implications.

  9. Plasmin in nephrotic urine activates the epithelial sodium channel

    DEFF Research Database (Denmark)

    Svenningsen, Per; Bistrup, Claus; Friis, Ulla G

    2008-01-01

    Proteinuria and increased renal reabsorption of NaCl characterize the nephrotic syndrome. Here, we show that protein-rich urine from nephrotic rats and from patients with nephrotic syndrome activate the epithelial sodium channel (ENaC) in cultured M-1 mouse collecting duct cells and in Xenopus...

  10. Sodium channel variants in heart disease: expanding horizons

    NARCIS (Netherlands)

    Tan, Hanno L.

    2006-01-01

    Inherited arrhythmia syndromes have advanced our understanding of cardiac sodium (Na) channel function in health and disease. Long QT syndrome (LQT3) is consistently caused by increased net Na current secondary to inactivation defects, which give rise to persistent Na current. Conversely, various

  11. Modulation of epithelial sodium channel in human alveolar epithelial ...

    African Journals Online (AJOL)

    Purpose: To investigate the effect of lipoxin A4 (LXA4) on the expressions of protein and mRNA of alveolar epithelial sodium channel (ENaC) in normal and lipopolysaccharide (LPS)-stimulated A549 cells. Methods: A549 cell-lines were randomized into 11 groups (N = 8) and treated. EnaC level was evaluated by Western ...

  12. Patterned electrical activity modulates sodium channel expression in sensory neurons.

    Science.gov (United States)

    Klein, Joshua P; Tendi, Elisabetta A; Dib-Hajj, Sulayman D; Fields, R Douglas; Waxman, Stephen G

    2003-10-15

    Peripheral nerve injury induces changes in the level of gene expression for sodium channels Nav1.3, Nav1.8, and Nav1.9 within dorsal root ganglion (DRG) neurons, which may contribute to the development of hyperexcitability, ectopic neuronal discharge, and neuropathic pain. The mechanism of this change in sodium channel expression is unclear. Decreased availability of neurotrophic factors following axotomy contributes to these changes in gene transcription, but the question of whether changes in intrinsic neuronal activity levels alone can trigger changes in the expression of these sodium channels has not been addressed. We examined the effect of electrical stimulation on the expression of Nav1.3, Nav1.8, and Nav1.9 by using cultured embryonic mouse sensory neurons under conditions in which nerve growth factor (NGF) was not limiting. Expression of Nav1.3 was not significantly changed following stimulation. In contrast, we observed activity-dependent down-regulation of Nav1.8 and Nav1.9 mRNA and protein levels after stimulation, as demonstrated by quantitative polymerase chain reaction and immunocytochemistry. These results show that a change in neuronal activity can alter the expression of sodium channel genes in a subtype-specific manner, via a mechanism independent of NGF withdrawal. Copyright 2003 Wiley-Liss, Inc.

  13. Regulation of sodium channel function by bilayer elasticity

    DEFF Research Database (Denmark)

    Lundbaek, Jens A; Birn, Pia; Hansen, Anker J

    2004-01-01

    and kinetics of the protein conformational changes therefore will be regulated by the bilayer elasticity, which is determined by the lipid composition. This hydrophobic coupling mechanism has been studied extensively in gramicidin channels, where the channel-bilayer hydrophobic interactions link...... a "conformational" change (the monomerdimer transition) to an elastic bilayer deformation. Gramicidin channels thus are regulated by the lipid bilayer elastic properties (thickness, monolayer equilibrium curvature, and compression and bending moduli). To investigate whether this hydrophobic coupling mechanism could...... be a general mechanism regulating membrane protein function, we examined whether voltage-dependent skeletal-muscle sodium channels, expressed in HEK293 cells, are regulated by bilayer elasticity, as monitored using gramicidin A (gA) channels. Nonphysiological amphiphiles (beta-octyl-glucoside, Genapol X-100...

  14. Oxidation of multiple methionine residues impairs rapid sodium channel inactivation

    Science.gov (United States)

    Kassmann, Mario; Hansel, Alfred; Leipold, Enrico; Birkenbeil, Jan; Lu, Song-Qing; Hoshi, Toshinori; Heinemann, Stefan H.

    2010-01-01

    Reactive oxygen species (ROS) readily oxidize the sulfur-containing amino acids cysteine and methionine (Met). The impact of Met oxidation on the fast inactivation of the skeletal muscle sodium channel NaV1.4 expressed in human embryonic kidney cells was studied by applying the Met-preferring oxidant chloramine-T (ChT) or by irradiating the ROS-producing dye Lucifer Yellow in the patch pipettes. Both interventions dramatically slowed down inactivation of the sodium channels. Replacement of Met in the Ile-Phe-Met inactivation motif with Leu (M1305L) strongly attenuated the oxidizing effect on inactivation but did not eliminate it completely. Mutagenesis of conserved Met residues in the intracellular linkers connecting the membrane-spanning segments of the channel (M1469L and M1470L) also markedly diminished the oxidation sensitivity of the channel, while that of other conserved Met residues (442, 1139, 1154, 1316) were without any noticeable effect. The results of mutagenesis of results, assays of other NaV channel isoforms (NaV1.2, NaV1.5, NaV1.7) and the kinetics of the oxidation-induced removal of inactivation collectively indicate that multiple Met target residues need to be oxidized to completely impair inactivation. This arrangement using multiple Met residues confers a finely graded oxidative modulation of NaV channels and allows organisms to adapt to a variety of oxidative stress conditions, such as ischemic reperfusion. PMID:18369661

  15. Voltage-gated sodium channels in taste bud cells

    Directory of Open Access Journals (Sweden)

    Williams Mark E

    2009-03-01

    Full Text Available Abstract Background Taste bud cells transmit information regarding the contents of food from taste receptors embedded in apical microvilli to gustatory nerve fibers innervating basolateral membranes. In particular, taste cells depolarize, activate voltage-gated sodium channels, and fire action potentials in response to tastants. Initial cell depolarization is attributable to sodium influx through TRPM5 in sweet, bitter, and umami cells and an undetermined cation influx through an ion channel in sour cells expressing PKD2L1, a candidate sour taste receptor. The molecular identity of the voltage-gated sodium channels that sense depolarizing signals and subsequently initiate action potentials coding taste information to gustatory nerve fibers is unknown. Results We describe the molecular and histological expression profiles of cation channels involved in electrical signal transmission from apical to basolateral membrane domains. TRPM5 was positioned immediately beneath tight junctions to receive calcium signals originating from sweet, bitter, and umami receptor activation, while PKD2L1 was positioned at the taste pore. Using mouse taste bud and lingual epithelial cells collected by laser capture microdissection, SCN2A, SCN3A, and SCN9A voltage-gated sodium channel transcripts were expressed in taste tissue. SCN2A, SCN3A, and SCN9A were expressed beneath tight junctions in subsets of taste cells. SCN3A and SCN9A were expressed in TRPM5 cells, while SCN2A was expressed in TRPM5 and PKD2L1 cells. HCN4, a gene previously implicated in sour taste, was expressed in PKD2L1 cells and localized to cell processes beneath the taste pore. Conclusion SCN2A, SCN3A and SCN9A voltage-gated sodium channels are positioned to sense initial depolarizing signals stemming from taste receptor activation and initiate taste cell action potentials. SCN2A, SCN3A and SCN9A gene products likely account for the tetrodotoxin-sensitive sodium currents in taste receptor cells.

  16. S1-S3 counter charges in the voltage sensor module of a mammalian sodium channel regulate fast inactivation.

    Science.gov (United States)

    Groome, James R; Winston, Vern

    2013-05-01

    The movement of positively charged S4 segments through the electric field drives the voltage-dependent gating of ion channels. Studies of prokaryotic sodium channels provide a mechanistic view of activation facilitated by electrostatic interactions of negatively charged residues in S1 and S2 segments, with positive counterparts in the S4 segment. In mammalian sodium channels, S4 segments promote domain-specific functions that include activation and several forms of inactivation. We tested the idea that S1-S3 countercharges regulate eukaryotic sodium channel functions, including fast inactivation. Using structural data provided by bacterial channels, we constructed homology models of the S1-S4 voltage sensor module (VSM) for each domain of the mammalian skeletal muscle sodium channel hNaV1.4. These show that side chains of putative countercharges in hNaV1.4 are oriented toward the positive charge complement of S4. We used mutagenesis to define the roles of conserved residues in the extracellular negative charge cluster (ENC), hydrophobic charge region (HCR), and intracellular negative charge cluster (INC). Activation was inhibited with charge-reversing VSM mutations in domains I-III. Charge reversal of ENC residues in domains III (E1051R, D1069K) and IV (E1373K, N1389K) destabilized fast inactivation by decreasing its probability, slowing entry, and accelerating recovery. Several INC mutations increased inactivation from closed states and slowed recovery. Our results extend the functional characterization of VSM countercharges to fast inactivation, and support the premise that these residues play a critical role in domain-specific gating transitions for a mammalian sodium channel.

  17. Sodium Channel Blockers in the Treatment of Epilepsy.

    Science.gov (United States)

    Brodie, Martin J

    2017-07-01

    Sodium channel blockers have been the mainstay of the pharmacological management of focal and generalised tonic-clonic seizures for more than 70 years. The focus of this paper will be on phenytoin, carbamazepine, lamotrigine, oxcarbazepine, rufinamide, lacosamide and eslicarbazepine acetate. All these antiepileptic drugs have similar efficacy and share similar dose-dependent, adverse effect profiles, although phenytoin, carbamazepine and oxcarbazepine are more likely to cause idiosyncratic reactions than the others. With the exception of lamotrigine, rufinamide and lacosamide, all are enzyme inducers and most are minor teratogens; although data on teratogenicity are sparse with lacosamide and eslicarbazepine acetate. There is increasing evidence that these drugs differ mechanistically, with the newer agents, lacosamide and eslicarbazepine acetate, having their major pharmacological effect on the slow inactivation state of the sodium channel, which may be associated with better tolerability at higher dosage, although hard evidence in support of this observation is currently not available. Rufinamide is licensed only for Lennox-Gastaut syndrome in children aged 4 years and above. There is a move away from using enzyme inducers, particularly phenytoin and carbamazepine, in everyday clinical practice. There seems little doubt, however, that some sodium channel blockers will have an enduring place in the management of epilepsy well into the 21st century.

  18. Sodium permeable and "hypersensitive" TREK-1 channels cause ventricular tachycardia.

    Science.gov (United States)

    Decher, Niels; Ortiz-Bonnin, Beatriz; Friedrich, Corinna; Schewe, Marcus; Kiper, Aytug K; Rinné, Susanne; Seemann, Gunnar; Peyronnet, Rémi; Zumhagen, Sven; Bustos, Daniel; Kockskämper, Jens; Kohl, Peter; Just, Steffen; González, Wendy; Baukrowitz, Thomas; Stallmeyer, Birgit; Schulze-Bahr, Eric

    2017-04-01

    In a patient with right ventricular outflow tract (RVOT) tachycardia, we identified a heterozygous point mutation in the selectivity filter of the stretch-activated K2P potassium channel TREK-1 (KCNK2 or K2P2.1). This mutation introduces abnormal sodium permeability to TREK-1. In addition, mutant channels exhibit a hypersensitivity to stretch-activation, suggesting that the selectivity filter is directly involved in stretch-induced activation and desensitization. Increased sodium permeability and stretch-sensitivity of mutant TREK-1 channels may trigger arrhythmias in areas of the heart with high physical strain such as the RVOT We present a pharmacological strategy to rescue the selectivity defect of the TREK-1 pore. Our findings provide important insights for future studies of K2P channel stretch-activation and the role of TREK-1 in mechano-electrical feedback in the heart. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  19. Three in a row?how sodium ions cross the channel

    OpenAIRE

    K?hlbrandt, Werner

    2016-01-01

    Sodium channels are central to a host of fundamental cellular processes, including sensory perception, pain, and muscle contraction. In order to understand any of these processes in detail, it is necessary to know the atomic structure of the channel proteins both with and without bound sodium ions. In this issue, Naylor et?al (2016) present the structure of a bacterial sodium channel tetramer. The three bound, partially hydrated sodium ions line up neatly in a row inside the selectivity filte...

  20. Sodium channels gone wild: resurgent current from neuronal and muscle channelopathies

    OpenAIRE

    Cannon, Stephen C.; Bean, Bruce P.

    2009-01-01

    Voltage-dependent sodium channels are the central players in the excitability of neurons, cardiac muscle, and skeletal muscle. Hundreds of mutations in sodium channels have been associated with human disease, particularly genetic forms of epilepsy, arrhythmias, myotonia, and periodic paralysis. In this issue of the JCI, Jarecki and colleagues present evidence suggesting that many such mutations alter the gating of sodium channels to produce resurgent sodium current, an unusual form of gating ...

  1. Docking Studies of Phthalimide Pharmacophore as a Sodium Channel Blocker

    Directory of Open Access Journals (Sweden)

    Maryam Iman

    2013-09-01

    Full Text Available   Objective(s: Recently, phthalimide derivatives were designed based on ameltolide and thalidomide as they possess a similar degree of anticonvulsant potency due to their phenytoin-like profile. The ability of phthalimide pharmacophore to interact with neuronal voltage-dependent sodium channels was studied in the batrachotoxin affinity assay. Therefore, in the present study, a series of 19 compounds of phthalimide pharmacophore possessing a variety of substituents (NO2, NH2 , Me, Cl, COOH, MeO at 2-, 3-, and 4- position of the N-phenyl ring and N-(3-amino-2-methylphenyl succinimide, were subjected to docking studies in order to inhibit voltage-gated sodium channels.   Materials and Methods : Chemical structures of all compounds were designed using HYPERCHEM program and Conformational studies were performed through semi-empirical molecular orbital calculations method followed by PM3 force field. Total energy gradient calculated as a root mean square (RMS value, until the RMS gradient was 0.01 kcal mol-1. Among all energy minima conformers, the global minimum of compounds was used in docking calculations. Using a model of the open pore of Na channels, docking study was performed by AUTODOCK4.2 program. Results : Docking studies have revealed that these types of ligands interacted mainly with II-S6 residues of NaV1.2 through making hydrogen bonds and have additional hydrophobic interactions with domain I, II, III and IV in the channel's inner pore. Conclusion   : These computational studies have displayed that these compounds are capable of inhibiting Na channel, efficiently.

  2. Adaptive evolution of the vertebrate skeletal muscle sodium channel

    Directory of Open Access Journals (Sweden)

    Jian Lu

    2011-01-01

    Full Text Available Tetrodotoxin (TTX is a highly potent neurotoxin that blocks the action potential by selectively binding to voltage-gated sodium channels (Na v. The skeletal muscle Na v (Na v1.4 channels in most pufferfish species and certain North American garter snakes are resistant to TTX, whereas in most mammals they are TTX-sensitive. It still remains unclear as to whether the difference in this sensitivity among the various vertebrate species can be associated with adaptive evolution. In this study, we investigated the adaptive evolution of the vertebrate Na v1.4 channels. By means of the CODEML program of the PAML 4.3 package, the lineages of both garter snakes and pufferfishes were denoted to be under positive selection. The positively selected sites identified in the p-loop regions indicated their involvement in Na v1.4 channel sensitivity to TTX. Most of these sites were located in the intracellular regions of the Na v1.4 channel, thereby implying the possible association of these regions with the regulation of voltage-sensor movement.

  3. Epithelial Sodium and Acid-Sensing Ion Channels

    Science.gov (United States)

    Kellenberger, Stephan

    The epithelial Na+ channel (ENaC) and acid-sensing ion channels (ASICs) are non-voltage-gated Na+ channels that form their own subfamilies within the ENaC/degenerin ion channel family. ASICs are sensors of extracellular pH, and ENaC, whose main function is trans-epithelial Na+ transport, can sense extra- and intra-cellular Na+. In aldosterone-responsive epithelial cells of the kidney, ENaC plays a critical role in the control of sodium balance, blood volume and blood pressure. In airway epithelia, ENaC has a distinct role in controlling fluid reabsorption at the air-liquid interface, thereby determining the rate of mucociliary transport. In taste receptor cells of the tongue, ENaC is involved in salt taste sensation. ASICs have emerged as key sensors for extracellular protons in central and peripheral neurons. Although not all of their physiological and pathological functions are firmly established yet, there is good evidence for a role of ASICs in the brain in learning, expression of fear, and in neurodegeneration after ischaemic stroke. In sensory neurons, ASICs are involved in nociception and mechanosensation. ENaC and ASIC subunits share substantial sequence homology and the conservation of several functional domains. This chapter summarises our current understanding of the physiological functions and of the mechanisms of ion permeation, gating and regulation of ENaC and ASICs.

  4. Role of the terminal domains in sodium channel localization.

    Science.gov (United States)

    Lee, Annie; Goldin, Alan L

    2009-01-01

    Voltage-gated sodium channels are membrane proteins that initiate action potentials in neurons following membrane depolarization. Members of this family show differential distribution at the subcellular level. The mechanisms underlying the targeting of these isoforms are not understood. However, their specificity is important because the isoforms can change the excitability of the membrane due to differences in their electrophysiological properties. In this study, chimeras generated between Na(V)1.2 and Na(V)1.6 were used to test channel domains for sequence that would allow Na(V)1.2 to localize to unmyelinated axons when Na(V)1.6 could not. We show that the N-terminal 202 amino acids of the Na(V)1.2 channel can mediate membrane domain-specific sorting in polarized epithelial cells and are necessary but not sufficient for localizing the isoform to the axons of cultured neurons. The domain-sorting signal is in the region between amino acids 110-202 of the Na(V)1.2 channel. The C-terminal 451 amino acids of Na(V)1.2 likely contain determinants that interact with neuron-specific factors to direct Na(V)1.2 to the axon.

  5. Design of a Nested Eight-Channel Sodium and Four-Channel Proton Coil for 7 Tesla Knee Imaging

    Science.gov (United States)

    Brown, Ryan; Madelin, Guillaume; Lattanzi, Riccardo; Chang, Gregory; Regatte, Ravinder R.; Sodickson, Daniel K.; Wiggins, Graham C.

    2012-01-01

    The critical design aim for a dual-tuned sodium/proton coil is to maximize sodium sensitivity and transmit field (B1+) homogeneity while simultaneously providing adequate proton sensitivity and homogeneity. While most dual-frequency coils utilize lossy high-impedance trap circuits or PIN diodes to allow dual-resonance, we explored a nested-coil design for sodium/proton knee imaging at 7T. A stand-alone eight-channel sodium receive array was implemented without standard dual-resonance circuitry to provide improved sodium signal-to-noise ratio (SNR) over a volume coil. A detunable sodium birdcage was added for homogeneous sodium excitation and a four-channel proton transmit-receive array was added to provide anatomical reference imaging and B0 shimming capability. Both modules were implemented with minimal disturbance to the eight-channel sodium array by managing their respective resonances and geometrical arrangement. In vivo sodium SNR was 1.2 to 1.7 times greater in the developed eight-channel array than in a mono-nuclear sodium birdcage coil, while the developed four-channel proton array provided SNR similar to that of a commercial mono-nuclear proton birdcage coil. PMID:22887123

  6. Fast sodium channel gating supports localized and efficient axonal action potential initiation.

    Science.gov (United States)

    Schmidt-Hieber, Christoph; Bischofberger, Josef

    2010-07-28

    Action potentials (APs) are initiated in the proximal axon of most neurons. In myelinated axons, a 50-times higher sodium channel density in the initial segment compared to the soma may account for this phenomenon. However, little is known about sodium channel density and gating in proximal unmyelinated axons. To study the mechanisms underlying AP initiation in unmyelinated hippocampal mossy fibers of adult mice, we recorded sodium currents in axonal and somatic membrane patches. We demonstrate that sodium channel density in the proximal axon is approximately 5 times higher than in the soma. Furthermore, sodium channel activation and inactivation are approximately 2 times faster. Modeling revealed that the fast activation localized the initiation site to the proximal axon even upon strong synaptic stimulation, while fast inactivation contributed to energy-efficient membrane charging during APs. Thus, sodium channel gating and density in unmyelinated mossy fiber axons appear to be specialized for robust AP initiation and propagation with minimal current flow.

  7. Cardiac sodium channel palmitoylation regulates channel availability and myocyte excitability with implications for arrhythmia generation

    Science.gov (United States)

    Pei, Zifan; Xiao, Yucheng; Meng, Jingwei; Hudmon, Andy; Cummins, Theodore R.

    2016-01-01

    Cardiac voltage-gated sodium channels (Nav1.5) play an essential role in regulating cardiac electric activity by initiating and propagating action potentials in the heart. Altered Nav1.5 function is associated with multiple cardiac diseases including long-QT3 and Brugada syndrome. Here, we show that Nav1.5 is subject to palmitoylation, a reversible post-translational lipid modification. Palmitoylation increases channel availability and late sodium current activity, leading to enhanced cardiac excitability and prolonged action potential duration. In contrast, blocking palmitoylation increases closed-state channel inactivation and reduces myocyte excitability. We identify four cysteines as possible Nav1.5 palmitoylation substrates. A mutation of one of these is associated with cardiac arrhythmia (C981F), induces a significant enhancement of channel closed-state inactivation and ablates sensitivity to depalmitoylation. Our data indicate that alterations in palmitoylation can substantially control Nav1.5 function and cardiac excitability and this form of post-translational modification is likely an important contributor to acquired and congenital arrhythmias. PMID:27337590

  8. [Pain and analgesia : Mutations of voltage-gated sodium channels].

    Science.gov (United States)

    Eberhardt, M J; Leffler, A

    2017-02-01

    Voltage-gated sodium channels (Navs) are crucial for the generation and propagation of action potentials in all excitable cells, and therefore for the function of sensory neurons as well. Preclinical research over the past 20 years identified three Nav-isoforms in sensory neurons, namely Nav1.7, Nav1.8 and Nav1.9. A specific role for the function of nociceptive neurons was postulated for each. Whereas no selective sodium channel inhibitors have been established in the clinic so far, the relevance of all three isoforms regarding the pain sensitivity in humans is currently undergoing a remarkable verification through the translation of preclinical data into clinically manifest pictures. For the last ten years, Nav1.7 has been the main focus of clinical interest, as a large number of hereditary mutants were identified. The so-called "gain-of-function" mutations of Nav1.7 cause the pain syndromes hereditary erythromelalgia and paroxysmal extreme pain disorder. In addition, several Nav1.7 mutants were shown to be associated with small-fiber neuropathies. On the contrary, "loss-of-function" Nav1.7 mutants lead to a congenital insensitivity to pain. Recently, several gain-of-function mutations in Nav1.8 and Nav1.9 have been identified in patients suffering from painful peripheral neuropathies. However, another gain-of-function Nav1.9 mutation is associated with congenital insensitivity to pain. This review offers an overview of published work on painful Nav mutations with clinical relevance, and proposes possible consequences for the therapy of different pain symptoms resulting from these findings.

  9. Single sodium channels from human skeletal muscle in planar lipid bilayers: characterization and response to pentobarbital

    NARCIS (Netherlands)

    Wartenberg, Hans C.; Urban, Bernd W.

    2004-01-01

    PURPOSE: To investigate the response to general anesthetics of different sodium-channel subtypes, we examined the effects of pentobarbital, a close thiopental analogue, on single sodium channels from human skeletal muscle and compared them to existing data from human brain and human ventricular

  10. The sodium leak channel, NALCN, in health and disease

    Science.gov (United States)

    Cochet-Bissuel, Maud; Lory, Philippe; Monteil, Arnaud

    2014-01-01

    Ion channels are crucial components of cellular excitability and are involved in many neurological diseases. This review focuses on the sodium leak, G protein-coupled receptors (GPCRs)-activated NALCN channel that is predominantly expressed in neurons where it regulates the resting membrane potential and neuronal excitability. NALCN is part of a complex that includes not only GPCRs, but also UNC-79, UNC-80, NLF-1 and src family of Tyrosine kinases (SFKs). There is growing evidence that the NALCN channelosome critically regulates its ion conduction. Both in mammals and invertebrates, animal models revealed an involvement in many processes such as locomotor behaviors, sensitivity to volatile anesthetics, and respiratory rhythms. There is also evidence that alteration in this NALCN channelosome can cause a wide variety of diseases. Indeed, mutations in the NALCN gene were identified in Infantile Neuroaxonal Dystrophy (INAD) patients, as well as in patients with an Autosomal Recessive Syndrome with severe hypotonia, speech impairment, and cognitive delay. Deletions in NALCN gene were also reported in diseases such as 13q syndrome. In addition, genes encoding NALCN, NLF- 1, UNC-79, and UNC-80 proteins may be susceptibility loci for several diseases including bipolar disorder, schizophrenia, Alzheimer's disease, autism, epilepsy, alcoholism, cardiac diseases and cancer. Although the physiological role of the NALCN channelosome is poorly understood, its involvement in human diseases should foster interest for drug development in the near future. Toward this goal, we review here the current knowledge on the NALCN channelosome in physiology and diseases. PMID:24904279

  11. The crystal structure of a voltage-gated sodium channel.

    Science.gov (United States)

    Payandeh, Jian; Scheuer, Todd; Zheng, Ning; Catterall, William A

    2011-07-10

    Voltage-gated sodium (Na(V)) channels initiate electrical signalling in excitable cells and are the molecular targets for drugs and disease mutations, but the structural basis for their voltage-dependent activation, ion selectivity and drug block is unknown. Here we report the crystal structure of a voltage-gated Na(+) channel from Arcobacter butzleri (NavAb) captured in a closed-pore conformation with four activated voltage sensors at 2.7 Å resolution. The arginine gating charges make multiple hydrophilic interactions within the voltage sensor, including unanticipated hydrogen bonds to the protein backbone. Comparisons to previous open-pore potassium channel structures indicate that the voltage-sensor domains and the S4-S5 linkers dilate the central pore by pivoting together around a hinge at the base of the pore module. The NavAb selectivity filter is short, ∼4.6 Å wide, and water filled, with four acidic side chains surrounding the narrowest part of the ion conduction pathway. This unique structure presents a high-field-strength anionic coordination site, which confers Na(+) selectivity through partial dehydration via direct interaction with glutamate side chains. Fenestrations in the sides of the pore module are unexpectedly penetrated by fatty acyl chains that extend into the central cavity, and these portals are large enough for the entry of small, hydrophobic pore-blocking drugs. This structure provides the template for understanding electrical signalling in excitable cells and the actions of drugs used for pain, epilepsy and cardiac arrhythmia at the atomic level.

  12. Tetrapentylammonium block of chloramine-T and veratridine modified rat brain type IIA sodium channels.

    Science.gov (United States)

    Ghatpande, A S; Rao, S; Sikdar, S K

    2001-04-01

    Tetrapentylammonium (TPeA) block of rat brain type IIA sodium channel alpha subunit was studied using whole cell patch clamp. Results indicate that TPeA blocks the inactivating brain sodium channel in a potential and use-dependent manner similar to that of the cardiac sodium channel. Removal of inactivation using chloramine-T (CT) unmasks a time-dependent block by TPeA consistent with slow blocking kinetics. On the other hand, no time dependence is observed when inactivation is abolished by modification with veratridine. TPeA does not bind in a potential-dependent fashion to veratridine-modified channels and does not significantly affect gating of veratridine-modified channels suggesting that high affinity binding of TPeA to the brain sodium channel is lost after veratridine modification.

  13. Localization of Sodium Channels in Intercalated Disks Modulates Cardiac Conduction

    Science.gov (United States)

    Kucera, Jan P.; Rohr, Stephan; Rudy, Yoram

    2007-01-01

    It is well known that the sodium current (INa) and the degree of gap-junctional electrical coupling are the key determinants of action potential (AP) conduction in cardiac tissue. Immunohistochemical studies have shown that sodium channels (NaChs) are preferentially located in intercalated disks (IDs). Using dual immunocytochemical staining, we confirmed the colocalization of NaChs with connexin43 in cultures of neonatal rat ventricular myocytes. In mathematical simulations of conduction using the Luo-Rudy dynamic model of the ventricular AP, we assessed the hypothesis that conduction could be modulated by the preferential localization of NaChs in IDs. Localization of INa at the ID caused a large negative potential in the intercellular cleft, which influenced conduction in two opposing ways, depending on the degree of electrical coupling: (1) for normal and moderately reduced coupling, the negative cleft potential led to a large overshoot of the transmembrane potential resulting in a decreased driving force for INa itself (self-attenuation), which slowed conduction; (2) for greatly reduced coupling (<10%), the negative cleft potential induced by INa in the prejunctional membrane led to suprathreshold depolarization of the postjunctional membrane, which facilitated and accelerated conduction. When cleft potential effects were not incorporated, conduction was not significantly affected by the ID localization of INa. By enhancing conduction through the establishment of cleft potentials, the localization of NaChs in IDs might protect the myocardium from conduction block, very slow conduction, and microreentry under conditions of greatly reduced coupling. Conversely, by supporting moderately slow conduction, this mechanism could also promote arrhythmias PMID:12480819

  14. Distinct molecular sites of anaesthetic action: pentobarbital block of human brain sodium channels is alleviated by removal of fast inactivation

    NARCIS (Netherlands)

    Wartenberg, H. C.; Urban, B. W.; Duch, D. S.

    1999-01-01

    Fast inactivation of sodium channel function is modified by anaesthetics. Its quantitative contribution to the overall anaesthetic effect is assessed by removing the fast inactivation mechanism enzymatically. Sodium channels from human brain cortex were incorporated into planar lipid bilayers. After

  15. Mechanisms of action of ligands of potential-dependent sodium channels.

    Science.gov (United States)

    Tikhonov, D B

    2008-06-01

    Potential-dependent sodium channels play a leading role in generating action potentials in excitable cells. Sodium channels are the site of action of a variety of modulator ligands. Despite numerous studies, the mechanisms of action of many modulators remain incompletely understood. The main reason that many important questions cannot be resolved is that there is a lack of precise data on the structures of the channels themselves. Structurally, potential-dependent sodium channels are members of the P-loop channel superfamily, which also include potassium and calcium channels and glutamate receptor channels. Crystallization of a series of potassium channels showed that it was possible to analyze the structures of different members of the superfamily using the "homologous modeling" method. The present study addresses model investigations of the actions of ligands of sodium channels, including tetrodotoxin and batrachotoxin, as well as local anesthetics. Comparison of experimental data on sodium channel ligands with x-ray analysis data allowed us to reach a new level of understanding of the mechanisms of channel modulation and to propose a series of experimentally verifiable hypotheses.

  16. Changes in sodium channel expression following trigeminal nerve injury.

    Science.gov (United States)

    Davies, Shelley L; Loescher, Alison R; Clayton, Nick M; Bountra, Chas; Robinson, Peter P; Boissonade, Fiona M

    2006-11-01

    We have investigated the expression of TTX-sensitive (TTXs) and TTX-resistant (TTXr) sodium channel subtypes following injury to the inferior alveolar nerve (IAN), in order to determine their potential role in the development of trigeminal neuropathic pain. In seven anaesthetised ferrets, fluorogold (2%) was injected into the left IAN to identify cell bodies with axons in this nerve. In four animals, the nerve was sectioned distal to the injection site and the remaining three served as controls. After 3 days, the animals were perfused with 4% paraformaldehyde. The left and right IANs and trigeminal ganglia were processed using indirect immunofluorescence with specific primary antibodies to TTXs subtypes Na(v)1.3 and Na(v)1.7 and TTXr subtypes Na(v)1.8 and Na(v)1.9. Image analysis was used to quantify the percentage area of staining (PAS) in the nerves. In the ganglia, counts were made of positively labelled cells in the fluorogold population. PAS for Na(v)1.8 and Na(v)1.9 was significantly greater in injured nerves than in either contralateral or control nerves. After injury, significantly fewer cells in the ganglia expressed Na(v)1.3 (controls 36.9%; injured 13.1%), Na(v)1.7 (controls 17.0%; injured 8.1%) and Na(v)1.9 (controls 60.3%; injured 29.0%) (psodium channel expression may play a role in nerve injury-induced trigeminal pain.

  17. The Sodium-Activated Potassium Channel Slack Is Required for Optimal Cognitive Flexibility in Mice

    Science.gov (United States)

    Bausch, Anne E.; Dieter, Rebekka; Nann, Yvette; Hausmann, Mario; Meyerdierks, Nora; Kaczmarek, Leonard K.; Ruth, Peter; Lukowski, Robert

    2015-01-01

    "Kcnt1" encoded sodium-activated potassium channels (Slack channels) are highly expressed throughout the brain where they modulate the firing patterns and general excitability of many types of neurons. Increasing evidence suggests that Slack channels may be important for higher brain functions such as cognition and normal intellectual…

  18. The cardiac sodium channel displays differential distribution in the conduction system and transmural heterogeneity in the murine ventricular myocardium

    NARCIS (Netherlands)

    Remme, C.A.; Verkerk, A.O.; Hoogaars, W.M.H.; Aanhaanen, W.T.J.; Scicluna, B.P.; Annink, C.; van den Hoff, M.J.B.; Wilde, A.A.M.; van Veen, T.A.B.; Veldkamp, M.W.; de Bakker, J.M.T.; Christoffels, V.M.; Bezzina, C.R.

    2009-01-01

    Cardiac sodium channels are responsible for conduction in the normal and diseased heart. We aimed to investigate regional and transmural distribution of sodium channel expression and function in the myocardium. Sodium channel Scn5a mRNA and Na(v)1.5 protein distribution was investigated in adult and

  19. Involvement of voltage-gated sodium channels blockade in the analgesic effects of orphenadrine.

    Science.gov (United States)

    Desaphy, Jean-François; Dipalma, Antonella; De Bellis, Michela; Costanza, Teresa; Gaudioso, Christelle; Delmas, Patrick; George, Alfred L; Camerino, Diana Conte

    2009-04-01

    Orphenadrine is a drug acting on multiple targets, including muscarinic, histaminic, and NMDA receptors. It is used in the treatment of Parkinson's disease and in musculoskeletal disorders. It is also used as an analgesic, although its mechanism of action is still unknown. Both physiological and pharmacological results have demonstrated a critical role for voltage-gated sodium channels in many types of chronic pain syndromes. We tested the hypothesis that orphenadrine may block voltage-gated sodium channels. By using patch-clamp experiments, we evaluated the effects of the drug on whole-cell sodium currents in HEK293 cells expressing the skeletal muscle (Nav1.4), cardiac (Nav1.5) and neuronal (Nav1.1 and Nav1.7) subtypes of human sodium channels, as well as on whole-cell tetrodotoxin (TTX)-resistant sodium currents likely conducted by Nav1.8 and Nav1.9 channel subtypes in primary culture of rat DRG sensory neurons. The results indicate that orphenadrine inhibits sodium channels in a concentration-, voltage- and frequency-dependent manner. By using site-directed mutagenesis, we further show that orphenadrine binds to the same receptor as the local anesthetics. Orphenadrine affinities for resting and inactivated sodium channels were higher compared to those of known sodium channels blockers, such as mexiletine and flecainide. Low, clinically relevant orphenadrine concentration produces a significant block of Nav1.7, Nav1.8, and Nav1.9 channels, which are critical for experiencing pain sensations, indicating a role for sodium channel blockade in the clinical efficacy of orphenadrine as analgesic compound. On the other hand, block of Nav1.1 and Nav1.5 may contribute to the proconvulsive and proarrhythmic adverse reactions, especially observed during overdose.

  20. Removal of sodium inactivation and block of sodium channels by chloramine-T in crayfish and squid giant axons.

    OpenAIRE

    Huang, J. M.; Tanguy, J.; Yeh, J. Z.

    1987-01-01

    Modification of sodium channels by chloramine-T was examined in voltage clamped internally perfused crayfish and squid giant axons using the double sucrose gap and axial wire technique, respectively. Freshly prepared chloramine-T solution exerted two major actions on sodium channels: (a) an irreversible removal of the fast Na inactivation, and (b) a reversible block of the Na current. Both effects were observed when chloramine-T was applied internally or externally (5-10 mM) to axons. The fir...

  1. Sodium Channel and Sodium Pump in Normal and Pathological Muscles from Patients with Myotonic Muscular Dystrophy and Lower Motor Neuron Impairment

    OpenAIRE

    Desnuelle, C.; Lombet, A.; SERRATRICE, G.; Lazdunski, M

    1982-01-01

    Two sodium transport systems have been analyzed in this work: the voltage-sensitive sodium channel and the (Na+, K+) ATPase pump. The sodium channel has been studied using a tritiated derivative of tetrodotoxin; the sodium pump has been studied using tritiated ouabain. Properties of interaction of tritiated tetrodotoxin and of tritiated ouabain with their respective receptors were observed in normal human skeletal muscle and in muscles of patients with myotonic muscular dystrophy and with low...

  2. Use dependence of peripheral nociceptive conduction in the absence of tetrodotoxin‐resistant sodium channel subtypes

    National Research Council Canada - National Science Library

    Hoffmann, Tal; Kistner, Katrin; Nassar, Mohammed; Reeh, Peter W; Weidner, Christian

    2016-01-01

    ...‐resistant (TTXr) sodium channel (Na v 1.8, Na v 1.9) knockout and wildtype animals. We observed use‐dependent decreases of single fibre and compound action potential amplitude in peripheral mouse C...

  3. A small fraction of strongly cooperative sodium channels boosts neuronal encoding of high frequencies.

    Directory of Open Access Journals (Sweden)

    Min Huang

    Full Text Available Generation of action potentials (APs is a crucial step in neuronal information processing. Existing biophysical models for AP generation almost universally assume that individual voltage-gated sodium channels operate statistically independently, and their avalanche-like opening that underlies AP generation is coordinated only through the transmembrane potential. However, biological ion channels of various types can exhibit strongly cooperative gating when clustered. Cooperative gating of sodium channels has been suggested to explain rapid onset dynamics and large threshold variability of APs in cortical neurons. It remains however unknown whether these characteristic properties of cortical APs can be reproduced if only a fraction of channels express cooperativity, and whether the presence of cooperative channels has an impact on encoding properties of neuronal populations. To address these questions we have constructed a conductance-based neuron model in which we continuously varied the size of a fraction [Formula: see text] of sodium channels expressing cooperativity and the strength of coupling between cooperative channels [Formula: see text]. We show that starting at a critical value of the coupling strength [Formula: see text], the activation curve of sodium channels develops a discontinuity at which opening of all coupled channels becomes an all-or-none event, leading to very rapid AP onsets. Models with a small fraction, [Formula: see text], of strongly cooperative channels generate APs with the most rapid onset dynamics. In this regime APs are triggered by simultaneous opening of the cooperative channel fraction and exhibit a pronounced biphasic waveform often observed in cortical neurons. We further show that presence of a small fraction of cooperative Na+ channels significantly improves the ability of neuronal populations to phase-lock their firing to high frequency input fluctuation. We conclude that presence of a small fraction of

  4. Annotation of functional impact of voltage?gated sodium channel mutations

    OpenAIRE

    Hinard, Valérie; Britan, Aurore; Schaeffer, Mathieu; Zahn, Monique; Thomet, Urs; Rougier, Jean-Sébastien; Bairoch, Amos Marc; Abriel, Hugues; Gaudet, Pascale

    2017-01-01

    Abstract Voltage?gated sodium channels are pore?forming transmembrane proteins that selectively allow sodium ions to flow across the plasma membrane according to the electro?chemical gradient thus mediating the rising phase of action potentials in excitable cells and playing key roles in physiological processes such as neurotransmission, skeletal muscle contraction, heart rhythm, and pain sensation. Genetic variations in the nine human genes encoding these channels are known to cause a large ...

  5. Antiepileptic drugs targeting sodium channels: subunit and neuron-type specific interactions

    NARCIS (Netherlands)

    Qiao, X.

    2013-01-01

    Certain antiepileptic drugs (e.g. carbamazepine and lamotrigine) block sodium channels in an use-dependent manner and this mechanism contributes to the anti-convulsant properties of these drugs. There are, however, subtle differences in sodium current blocking properties of the antiepileptic drugs

  6. Sensory neuron voltage-gated sodium channels as analgesic drug targets.

    Science.gov (United States)

    Momin, Aliakmal; Wood, John N

    2008-08-01

    Voltage-gated sodium channels are crucial determinants of neuronal excitability and signalling; some specific channel subtypes have been implicated in a number of chronic pain conditions. Human genetic studies show gain-of-function or loss-of-function mutations in Na(V)1.7 lead to an enhancement or lack of pain, respectively, whilst transgenic mouse and knockdown studies have implicated Na(V)1.3, Na(V)1.8 and Na(V)1.9 in peripheral pain pathways. The development of subtype-specific sodium channel blockers, though clearly desirable, has been technically challenging. Recent advances exploiting both natural products and small molecule selective channel blockers have demonstrated that this approach to pain control is feasible. These observations provide a rationale for the development of new analgesics without the side effect profile of broad spectrum sodium channel blockers.

  7. Voltage-gated sodium channels in pain states: role in pathophysiology and targets for treatment.

    Science.gov (United States)

    Dib-Hajj, Sulayman D; Binshtok, Alexander M; Cummins, Theodore R; Jarvis, Michael F; Samad, Tarek; Zimmermann, Katharina

    2009-04-01

    Pain is a major unmet medical need which has been causally linked to changes in sodium channel expression, modulation, or mutations that alter channel gating properties or current density in nociceptor neurons. Voltage-gated sodium channels activate (open) then rapidly inactivate in response to a depolarization of the plasma membrane of excitable cells allowing the transient flow of sodium ions thus generating an inward current which underlies the generation and conduction of action potentials (AP) in these cells. Activation and inactivation, as well as other gating properties, of sodium channel isoforms have different kinetics and voltage-dependent properties, so that the ensemble of channels that are present determine the electrogenic properties of specific neurons. Biophysical and pharmacological studies have identified the peripheral-specific sodium channels Na(v)1.7, Na(v)1.8 and Na(v)1.9 as particularly important in the pathophysiology of different pain syndromes, and isoform-specific blockers of these channels or targeting their modulators hold the promise of a future effective therapy for treatment of pain.

  8. Distribution and function of sodium channel subtypes in human atrial myocardium.

    Science.gov (United States)

    Kaufmann, Susann G; Westenbroek, Ruth E; Maass, Alexander H; Lange, Volkmar; Renner, Andre; Wischmeyer, Erhard; Bonz, Andreas; Muck, Jenny; Ertl, Georg; Catterall, William A; Scheuer, Todd; Maier, Sebastian K G

    2013-08-01

    Voltage-gated sodium channels composed of a pore-forming α subunit and auxiliary β subunits are responsible for the upstroke of the action potential in cardiac muscle. However, their localization and expression patterns in human myocardium have not yet been clearly defined. We used immunohistochemical methods to define the level of expression and the subcellular localization of sodium channel α and β subunits in human atrial myocytes. Nav1.2 channels are located in highest density at intercalated disks where β1 and β3 subunits are also expressed. Nav1.4 and the predominant Nav1.5 channels are located in a striated pattern on the cell surface at the z-lines together with β2 subunits. Nav1.1, Nav1.3, and Nav1.6 channels are located in scattered puncta on the cell surface in a pattern similar to β3 and β4 subunits. Nav1.5 comprised approximately 88% of the total sodium channel staining, as assessed by quantitative immunohistochemistry. Functional studies using whole cell patch-clamp recording and measurements of contractility in human atrial cells and tissue showed that TTX-sensitive (non-Nav1.5) α subunit isoforms account for up to 27% of total sodium current in human atrium and are required for maximal contractility. Overall, our results show that multiple sodium channel α and β subunits are differentially localized in subcellular compartments in human atrial myocytes, suggesting that they play distinct roles in initiation and conduction of the action potential and in excitation-contraction coupling. TTX-sensitive sodium channel isoforms, even though expressed at low levels relative to TTX-sensitive Nav1.5, contribute substantially to total cardiac sodium current and are required for normal contractility. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes". Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Identification of sodium channel isoforms that mediate action potential firing in lamina I/II spinal cord neurons.

    Science.gov (United States)

    Hildebrand, Michael E; Mezeyova, Janette; Smith, Paula L; Salter, Michael W; Tringham, Elizabeth; Snutch, Terrance P

    2011-09-12

    Voltage-gated sodium channels play key roles in acute and chronic pain processing. The molecular, biophysical, and pharmacological properties of sodium channel currents have been extensively studied for peripheral nociceptors while the properties of sodium channel currents in dorsal horn spinal cord neurons remain incompletely understood. Thus far, investigations into the roles of sodium channel function in nociceptive signaling have primarily focused on recombinant channels or peripheral nociceptors. Here, we utilize recordings from lamina I/II neurons withdrawn from the surface of spinal cord slices to systematically determine the functional properties of sodium channels expressed within the superficial dorsal horn. Sodium channel currents within lamina I/II neurons exhibited relatively hyperpolarized voltage-dependent properties and fast kinetics of both inactivation and recovery from inactivation, enabling small changes in neuronal membrane potentials to have large effects on intrinsic excitability. By combining biophysical and pharmacological channel properties with quantitative real-time PCR results, we demonstrate that functional sodium channel currents within lamina I/II neurons are predominantly composed of the NaV1.2 and NaV1.3 isoforms. Overall, lamina I/II neurons express a unique combination of functional sodium channels that are highly divergent from the sodium channel isoforms found within peripheral nociceptors, creating potentially complementary or distinct ion channel targets for future pain therapeutics.

  10. Identification of sodium channel isoforms that mediate action potential firing in lamina I/II spinal cord neurons

    Directory of Open Access Journals (Sweden)

    Smith Paula L

    2011-09-01

    Full Text Available Abstract Background Voltage-gated sodium channels play key roles in acute and chronic pain processing. The molecular, biophysical, and pharmacological properties of sodium channel currents have been extensively studied for peripheral nociceptors while the properties of sodium channel currents in dorsal horn spinal cord neurons remain incompletely understood. Thus far, investigations into the roles of sodium channel function in nociceptive signaling have primarily focused on recombinant channels or peripheral nociceptors. Here, we utilize recordings from lamina I/II neurons withdrawn from the surface of spinal cord slices to systematically determine the functional properties of sodium channels expressed within the superficial dorsal horn. Results Sodium channel currents within lamina I/II neurons exhibited relatively hyperpolarized voltage-dependent properties and fast kinetics of both inactivation and recovery from inactivation, enabling small changes in neuronal membrane potentials to have large effects on intrinsic excitability. By combining biophysical and pharmacological channel properties with quantitative real-time PCR results, we demonstrate that functional sodium channel currents within lamina I/II neurons are predominantly composed of the NaV1.2 and NaV1.3 isoforms. Conclusions Overall, lamina I/II neurons express a unique combination of functional sodium channels that are highly divergent from the sodium channel isoforms found within peripheral nociceptors, creating potentially complementary or distinct ion channel targets for future pain therapeutics.

  11. Neurotoxins and their binding areas on voltage-gated sodium channels

    Directory of Open Access Journals (Sweden)

    Marijke eStevens

    2011-11-01

    Full Text Available Voltage-gated Sodium Channels (VGSCs are large transmembrane proteins that conduct sodium ions across the membrane and by doing so they generate signals of communication between many kinds of tissues. They are responsible for the generation and propagation of action potentials in excitable cells, in close collaboration with other channels like potassium channels. Genetic defects in sodium channel genes therefore can cause a wide variety of diseases, generally called ‘channelopathies’.The first insights into the mechanism of action potentials and the involvement of sodium channels originated from Hodgkin and Huxley for which they were awarded the Nobel Prize in 1963. Until now, these concepts still form the basis for understanding the functioning of VGSCs. When VGSCs sense a sufficient change in membrane potential, they are activated and will generate a massive influx of sodium ions. Immediately after, channels will start inactivating and currents decrease. In the inactivated state channels stay refractory for any new stimulus and they must return to the closed state before being susceptible to any new depolarization. On the other hand, studies with neurotoxins like tetrodotoxin (TTX and saxitoxin (STX also contributed largely to our today’s understanding of the structure and function of ion channels and specifically of VGSCs. Moreover, neurotoxins acting on ion channels turned out to be valuable tools in the development of new drugs for the enormous range of diseases in which ion channels are involved. A recent example of a synthetic neurotoxin that made it to the market is ziconotide (Prialt®, Elan. The original peptide, -MVIIA, is derived from the cone snail Conus magus and now FDA/EMEA-approved for the management of severe chronic pain by blocking the N-type voltage-gated calcium channels in neurons.This review focuses on the current status of research on neurotoxins acting on VGSC, their contribution to further unravel the

  12. Modification of sodium and potassium channel kinetics by diethyl ether and studies on sodium channel inactivation in the crayfish giant axon membrane

    Energy Technology Data Exchange (ETDEWEB)

    Bean, Bruce Palmer [Univ. of Rochester, NY (United States)

    1979-01-01

    The effects of ether and halothane on membrane currents in the voltage clamped crayfish giant axon membrane were investigated. Concentrations of ether up to 300 mM and of halothane up to 32 mM had no effect on resting potential or leakage conductance. Ether and halothane reduced the size of sodium currents without changing the voltage dependence of the peak currents or their reversal potential. Ether and halothane also produced a reversible, dose-dependent speeding of sodium current decay at all membrane potentials. Ether reduced the time constants for inactivation, and also shifted the midpoint of the steady-state inactivation curve in the hyperpolarizing direction. Potassium currents were smaller with ether present, with no change in the voltage dependence of steady-state currents. The activation of potassium channels was faster with ether present. There was no apparent change in the capacitance of the crayfish giant axon membrane with ether concentrations of up to 100 mM. Experiments on sodium channel inactivation kinetics were performed using 4-aminopyridine to block potassium currents. Sodium currents decayed with a time course generally fit well by a single exponential. The time constant of decay was a steep function of voltage, especially in the negative resistance region of the peak current vs voltage relation.The time course of inactivation was very similar to that of the decay of the current at the same potential. The measurement of steady-state inactivation curves with different test pulses showed no shifts along the voltage asix. The voltage-dependence of the integral of sodium conductance was measured to test models of sodium channel inactivation in which channels must open before inactivating; the results appear inconsistent with some of the simplest cases of such models.

  13. Action potential generation requires a high sodium channel density in the axon initial segment

    NARCIS (Netherlands)

    Kole, Maarten H. P.; Ilschner, Susanne U.; Kampa, Björn M.; Williams, Stephen R.; Ruben, Peter C.; Stuart, Greg J.

    2008-01-01

    The axon initial segment ( AIS) is a specialized region in neurons where action potentials are initiated. It is commonly assumed that this process requires a high density of voltage-gated sodium ( Na(+)) channels. Paradoxically, the results of patch-clamp studies suggest that the Na(+) channel

  14. Atrial-selective block of sodium channels by acehytisine in rabbit myocardium

    Directory of Open Access Journals (Sweden)

    Xinrong Fan

    2016-12-01

    Full Text Available Acehytisine, a multi-ion channel blocker, can markedly inhibit INa, ICa, IKur, If at various concentrations and effectively terminate and prevent atrial fibrillation (AF in patients and animal models, but the molecular mechanism underlying its blockage remains elusive. In this study, we investigated the effects of acehytisine on action potentials and sodium channels of atrial and ventricular myocytes isolated from rabbit, using whole-cell recording system. We found that acehytisine exerted stronger blocking effects on sodium channels in atria than in ventricles, especially at depolarization (IC50: 48.48 ± 7.75 μmol/L in atria vs. 560.17 ± 63.98 μmol/L in ventricles. It also significantly shifted steady state inactivation curves toward negative potentials in atrial myocytes, without affecting the recovery kinetics from inactivation of sodium channels in the same cells. In addition, acehytisine inhibited INa in a use-dependent manner and regulated slow inactivation kinetics by different gating configurations. These findings indicate that acehytisine selectively blocks atrial sodium channels and possesses affinity to sodium channel in certain states, which provides additional evidence for the anti-AF of acehytisine.

  15. The roles of sodium channels in nociception: implications for mechanisms of neuropathic pain.

    Science.gov (United States)

    Liu, Min; Wood, John N

    2011-07-01

    Animal models have provided useful insights into the development and treatment of neuropathic pain. New genetic data from both human studies and transgenic mouse models suggest that specific voltage-gated sodium channel subtypes are associated with specific types of pain and, as such, may be useful analgesic drug targets for a variety of pain types including neuropathic pain. Global voltage-gated sodium channel blockers such as lidocaine have proven efficacy in treating pain but can be limited by adverse effects when administered systemically. Selective sodium channel blockers targeting channels at the periphery (Nav1.7, Nav1.8, and Nav1.9) could potentially reduce the side effect profile. Individual isoforms of voltage-gated sodium channels have been linked to particular types of pain. Nav1.7 is a useful target for ameliorating acute mechanical pain and inflammatory pain, and strong evidence also suggests that Nav1.9 could be targeted for treating inflammatory pain. Selective blockers of Nav1.8 could also have clinical benefit for visceral pain. Although there is no association between a single sodium channel isoform and neuropathic pain, combined blockade of peripherally expressed isoforms Nav1.7, Nav1.8, and Nav1.9 may prove useful. Wiley Periodicals, Inc.

  16. Distribution and function of voltage-gated sodium channels in the nervous system.

    Science.gov (United States)

    Wang, Jun; Ou, Shao-Wu; Wang, Yun-Jie

    2017-09-18

    Voltage-gated sodium channels (VGSCs) are the basic ion channels for neuronal excitability, which are crucial for the resting potential and the generation and propagation of action potentials in neurons. To date, at least nine distinct sodium channel isoforms have been detected in the nervous system. Recent studies have identified that voltage-gated sodium channels not only play an essential role in the normal electrophysiological activities of neurons but also have a close relationship with neurological diseases. In this study, the latest research findings regarding the structure, type, distribution, and function of VGSCs in the nervous system and their relationship to neurological diseases, such as epilepsy, neuropathic pain, brain tumors, neural trauma, and multiple sclerosis, are reviewed in detail.

  17. Role of Sodium Channel on Cardiac Action Potential

    Directory of Open Access Journals (Sweden)

    S. H. Sabzpoushan

    2012-06-01

    Full Text Available Sudden cardiac death is a major cause of death worldwide. In most cases, it's caused by abnormal action potential propagation that leads to cardiac arrhythmia. The aim of this article is to study the abnormal action potential propagation through sodium ion concentration variations. We use a new electrophysiological model that is both detailed and computationally efficient. This efficient model is based on the partial differential equation method. The central finite difference method is used for numerical solving of the two-dimensional (2D wave propagation equation. Simulations are implemented in two stages, as a single cardiac cell and as a two-dimensional grid of cells. In both stages, the normal action potential formation in case of a single cell and it's normal propagation in case of a two-dimensional grid of cells were simulated with nominal sodium ion conductance. Then, the effect of sodium ion concentration on the action potential signal was studied by reducing the sodium ion conductance. It is concluded that reducing the sodium ion conductance, decreases both passing ability and conduction velocity of the action potential wave front.

  18. Activation of Drosophila Sodium Channels Promotes Modification by Deltamethrin

    Science.gov (United States)

    Vais, Horia; Williamson, Martin S.; Goodson, Susannah J.; Devonshire, Alan L.; Warmke, Jeffrey W.; Usherwood, Peter N.R.; Cohen, Charles J.

    2000-01-01

    kdr and super-kdr are mutations in houseflies and other insects that confer 30- and 500-fold resistance to the pyrethroid deltamethrin. They correspond to single (L1014F) and double (L1014F+M918T) mutations in segment IIS6 and linker II(S4–S5) of Na channels. We expressed Drosophila para Na channels with and without these mutations and characterized their modification by deltamethrin. All wild-type channels can be modified by Anemonia sulcata, which slows inactivation. The mutations reduce channel opening by enhancing closed-state inactivation. In addition, these mutations reduce the affinity for open channels by 20- and 100-fold, respectively. Deltamethrin inhibits channel closing and the mutations reduce the time that channels remain open once drug has bound. The super-kdr mutations effectively reduce the number of deltamethrin binding sites per channel from two to one. Thus, the mutations reduce both the potency and efficacy of insecticide action. PMID:10694259

  19. Significant natriuretic and antihypertensive action of the epithelial sodium channel blocker amiloride in diabetic patients with and without nephropathy

    DEFF Research Database (Denmark)

    Andersen, Henrik; Hansen, Pernille B L; Bistrup, Claus

    2016-01-01

    OBJECTIVE: Diabetic nephropathy is associated with aberrant glomerular filtration of serine proteases. The study was designed to test the hypothesis that the epithelial sodium channel is activated proteolytically by urine plasmin in diabetic nephropathy and mediates renal sodium retention. METHOD...

  20. A highly polarized excitable cell separates sodium channels from sodium-activated potassium channels by more than a millimeter.

    Science.gov (United States)

    Ban, Yue; Smith, Benjamin E; Markham, Michael R

    2015-07-01

    The bioelectrical properties and resulting metabolic demands of electrogenic cells are determined by their morphology and the subcellular localization of ion channels. The electric organ cells (electrocytes) of the electric fish Eigenmannia virescens generate action potentials (APs) with Na(+) currents >10 μA and repolarize the AP with Na(+)-activated K(+) (KNa) channels. To better understand the role of morphology and ion channel localization in determining the metabolic cost of electrocyte APs, we used two-photon three-dimensional imaging to determine the fine cellular morphology and immunohistochemistry to localize the electrocytes' ion channels, ionotropic receptors, and Na(+)-K(+)-ATPases. We found that electrocytes are highly polarized cells ∼ 1.5 mm in anterior-posterior length and ∼ 0.6 mm in diameter, containing ∼ 30,000 nuclei along the cell periphery. The cell's innervated posterior region is deeply invaginated and vascularized with complex ultrastructural features, whereas the anterior region is relatively smooth. Cholinergic receptors and Na(+) channels are restricted to the innervated posterior region, whereas inward rectifier K(+) channels and the KNa channels that terminate the electrocyte AP are localized to the anterior region, separated by >1 mm from the only sources of Na(+) influx. In other systems, submicrometer spatial coupling of Na(+) and KNa channels is necessary for KNa channel activation. However, our computational simulations showed that KNa channels at a great distance from Na(+) influx can still terminate the AP, suggesting that KNa channels can be activated by distant sources of Na(+) influx and overturning a long-standing assumption that AP-generating ion channels are restricted to the electrocyte's posterior face. Copyright © 2015 the American Physiological Society.

  1. Regulation/modulation of sensory neuron sodium channels.

    Science.gov (United States)

    Chahine, Mohamed; O'Leary, Michael E

    2014-01-01

    The pseudounipolar sensory neurons of the dorsal root ganglia (DRG) give rise to peripheral branches that convert thermal, mechanical, and chemical stimuli into electrical signals that are transmitted via central branches to the spinal cord. These neurons express unique combinations of tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Na(+) channels that contribute to the resting membrane potential, action potential threshold, and regulate neuronal firing frequency. The small-diameter neurons (35 μm) are typically low-threshold A-fibers that predominately express TTX-S Na(+) currents. Peripheral nerve damage, inflammation, and metabolic diseases alter the expression and function of these Na(+) channels leading to increases in neuronal excitability and pain. The Na(+) channels expressed in these neurons are the target of intracellular signaling cascades that regulate the trafficking, cell surface expression, and gating properties of these channels. Post-translational regulation of Na(+) channels by protein kinases (PKA, PKC, MAPK) alter the expression and function of the channels. Injury-induced changes in these signaling pathways have been linked to sensory neuron hyperexcitability and pain. This review examines the signaling pathways and regulatory mechanisms that modulate the voltage-gated Na(+) channels of sensory neurons.

  2. Voltage-Gated Sodium Channels: Evolutionary History and Distinctive Sequence Features.

    Science.gov (United States)

    Kasimova, M A; Granata, D; Carnevale, V

    2016-01-01

    Voltage-gated sodium channels (Nav) are responsible for the rising phase of the action potential. Their role in electrical signal transmission is so relevant that their emergence is believed to be one of the crucial factors enabling development of nervous system. The presence of voltage-gated sodium-selective channels in bacteria (BacNav) has raised questions concerning the evolutionary history of the ones in animals. Here we review some of the milestones in the field of Nav phylogenetic analysis and discuss some of the most important sequence features that distinguish these channels from voltage-gated potassium channels and transient receptor potential channels. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Neurotrophin-evoked depolarization requires the sodium channel Na(V)1.9.

    Science.gov (United States)

    Blum, Robert; Kafitz, Karl W; Konnerth, Arthur

    2002-10-17

    Brain-derived neurotrophic factor (BDNF) and other neurotrophins are essential for normal brain function. Many types of neurons in the central nervous system are excited by BDNF or neurotrophin-4/5, an action that has recently been implicated in synaptic plasticity. The mechanisms involved in this transmitter-like action of neurotrophins remains unclear. Here, by screening candidate genes with an antisense messenger RNA expression approach and by co-expressing the receptor tyrosine kinase TrkB and various sodium channels, we demonstrate that the tetrodotoxin-insensitive sodium channel Na(V)1.9 underlies the neurotrophin-evoked excitation. These results establish the molecular basis of neurotrophin-evoked depolarization and reveal a mechanism of ligand-mediated sodium channel activation.

  4. Down-regulation of voltage-dependent sodium channels initiated by sodium influx in developing neurons

    Energy Technology Data Exchange (ETDEWEB)

    Dargent, B.; Couraud, F. (Centre National de la Recherche Scientifique, Marseille (France))

    1990-08-01

    To address the issue of whether regulatory feedback exists between the electrical activity of a neuron and ion-channel density, the authors investigated the effect of Na{sup +}-channel activators (scorpion {alpha} toxin, batrachotoxin, and veratridine) on the density of Na{sup +} channels in fetal rat brain neurons in vitro. A partial but rapid (t{sub 1/2}, 15 min) disappearance of surface Na{sup +} channels was observed as measured by a decrease in the specific binding of ({sup 3}H)saxitoxin and {sup 125}I-labeled scorpion {beta} toxin and a decrease in specific {sup 22}Na{sup +} uptake. Moreover, the increase in the number of Na{sup +} channels that normally occurs during neuronal maturation in vitro was inhibited by chronic channel activator treatment. The induced disappearance of Na{sup +} channels was abolished by tetrodotoxin, was found to be dependent on the external Na{sup +} concentration, and was prevented when either choline (a nonpermeant ion) or Li{sup +} (a permeant ion) was substituted for Na{sup +}. Amphotericin B, a Na{sup +} ionophore, and monensin were able to mimick the effect of Na{sup +}-channel activators, while a KCl depolarization failed to do this. This feedback regulation seems to be a neuronal property since Na{sup +}-channel density in cultured astrocytes was not affected by channel activator treatment or by amphotericin B. The present evidence suggests that an increase in intracellular Na{sup +} concentration, whether elicited by Na{sup +}-channel activators or mediated by a Na{sup +} ionophore, can induce a decrease in surface Na{sup +} channels and therefore is involved in down-regulation of Na{sup +}-channel density in fetal rat brain neurons in vitro.

  5. Use-dependent block of cardiac sodium channels by quaternary derivatives of lidocaine.

    Science.gov (United States)

    Gintant, G A; Hoffman, B F

    1984-02-01

    Modulation of the reduction of fast inward sodium current by local anesthetics due to changes in electrical activity has been termed use-dependent block ( Courtney 1975). To determine the mechanisms responsible for use-dependent block of cardiac sodium channels and to compare use-dependent block in cardiac and nerve preparations, we investigated use-dependent block of cardiac sodium channels by the quaternary lidocaine analogues QX -314 and QX -222 (two agents previously studied in nerve). We used canine cardiac Purkinje fibers, and assessed changes in the fast inward sodium current using changes in the maximum rate of rise of the action potential upstroke (Vmax). Two microelectrode voltage clamp and current clamp techniques were used to control membrane potential prior to stimulated upstrokes . Use-dependent block was not affected by shortening the action potential duration during rapid stimulation. Partial recovery from use-dependent block was observed during rapid stimulation with brief depolarizing prepulses terminating immediately prior to the upstroke. Similar prepulses also prevented the development of use-dependent block following an abrupt increase in the stimulation rate. Hyperpolarizing prepulses during rapid stimulation caused recovery from use-dependent block; recovery was greater and more rapid with increasingly negative prepulses . Hyperpolarization during periods of electrical quiescence also caused greater recovery. These results, interpreted using the modulated receptor hypothesis ( Hille 1977; Hondeghem and Katzung 1977), suggest that use-dependent block of cardiac sodium channels by quaternary local anesthetics is due to drug association with the inactivated sodium channel receptor which occurs only after these drugs gain access to the receptor site through open sodium channels.

  6. Evolution of the epithelial sodium channel and the sodium pump as limiting factors of aldosterone action on sodium transport

    National Research Council Canada - National Science Library

    Romain A. Studer; Emilie Person; Marc Robinson-Rechavi; Bernard C. Rossier

    2011-01-01

    Despite large changes in salt intake, the mammalian kidney is able to maintain the extracellular sodium concentration and osmolarity within very narrow margins, thereby controlling blood volume and blood pressure...

  7. Axonal sodium channel distribution shapes the depolarized action potential threshold of dentate granule neurons.

    Science.gov (United States)

    Kress, Geraldine J; Dowling, Margaret J; Eisenman, Lawrence N; Mennerick, Steven

    2010-04-01

    Intrinsic excitability is a key feature dictating neuronal response to synaptic input. Here we investigate the recent observation that dentate granule neurons exhibit a more depolarized voltage threshold for action potential initiation than CA3 pyramidal neurons. We find no evidence that tonic GABA currents, leak or voltage-gated potassium conductances, or the expression of sodium channel isoform differences can explain this depolarized threshold. Axonal initial segment voltage-gated sodium channels, which are dominated by the Na(V)1.6 isoform in both cell types, distribute more proximally and exhibit lower overall density in granule neurons than in CA3 neurons. To test possible contributions of sodium channel distributions to voltage threshold and to test whether morphological differences participate, we performed simulations of dentate granule neurons and of CA3 pyramidal neurons. These simulations revealed that cell morphology and sodium channel distribution combine to yield the characteristic granule neuron action potential upswing and voltage threshold. Proximal axon sodium channel distribution strongly contributes to the higher voltage threshold of dentate granule neurons for two reasons. First, action potential initiation closer to the somatodendritic current sink causes the threshold of the initiating axon compartment to rise. Second, the proximity of the action potential initiation site to the recording site causes somatic recordings to more faithfully reflect the depolarized threshold of the axon than in cells like CA3 neurons, with distally initiating action potentials. Our results suggest that the proximal location of axon sodium channels in dentate granule neurons contributes to the intrinsic excitability differences between DG and CA3 neurons and may participate in the low-pass filtering function of dentate granule neurons. (c) 2009 Wiley-Liss, Inc.

  8. Removal of sodium channel inactivation in squid axon by the oxidant chloramine-T

    Science.gov (United States)

    1985-01-01

    We have investigated the effects of a mild oxidant, chloramine-T(CT), on the sodium and potassium currents of squid axons under voltage-clamp conditions. Sodium channel inactivation of squid giant axons can be completely removed by CT at neutral pH. Internal and external CT treatment are both effective. CT apparently removes inactivation in an irreversible, all-or-none manner. The activation process of sodium channels is little affected, as judged from the voltage dependence of peak sodium currents, the rising phase of sodium currents, and the time course of tail currents following the repolarization. The removal of inactivation by CT is pH-dependent; higher pH decreases the removal rate, whereas lower pH increases it. Internal metabisulfite, a strong reductant, does not protect inactivation from the action of external CT, nor does external metabisulfite protect from internal CT application. CT slightly depresses the peak potassium currents at comparable concentrations but has no apparent effects on their kinetics. Our results suggest that the neutral form of CT modifies an embedded methionine residue that is involved in sodium channel inactivation. PMID:2413161

  9. Role of Sodium Channel on Cardiac Action Potential

    OpenAIRE

    S. H. Sabzpoushan; A. Faghani Ghodrat

    2012-01-01

    Sudden cardiac death is a major cause of death worldwide. In most cases, it's caused by abnormal action potential propagation that leads to cardiac arrhythmia. The aim of this article is to study the abnormal action potential propagation through sodium ion concentration variations. We use a new electrophysiological model that is both detailed and computationally efficient. This efficient model is based on the partial differential equation method. The central finite difference method is used f...

  10. Comparative effects of sodium channel blockers in short term rat whole embryo culture

    Energy Technology Data Exchange (ETDEWEB)

    Nilsson, Mats F, E-mail: Mats.Nilsson@farmbio.uu.se [Department of Pharmaceutical Biosciences, Uppsala University (Sweden); Sköld, Anna-Carin; Ericson, Ann-Christin; Annas, Anita; Villar, Rodrigo Palma [AstraZeneca R and D Södertälje (Sweden); Cebers, Gvido [AstraZeneca R and D, iMed, 141 Portland Street, Cambridge, MA 02139 (United States); Hellmold, Heike; Gustafson, Anne-Lee [AstraZeneca R and D Södertälje (Sweden); Webster, William S [Department of Anatomy and Histology, University of Sydney (Australia)

    2013-10-15

    This study was undertaken to examine the effect on the rat embryonic heart of two experimental drugs (AZA and AZB) which are known to block the sodium channel Nav1.5, the hERG potassium channel and the L-type calcium channel. The sodium channel blockers bupivacaine, lidocaine, and the L-type calcium channel blocker nifedipine were used as reference substances. The experimental model was the gestational day (GD) 13 rat embryo cultured in vitro. In this model the embryonic heart activity can be directly observed, recorded and analyzed using computer assisted image analysis as it responds to the addition of test drugs. The effect on the heart was studied for a range of concentrations and for a duration up to 3 h. The results showed that AZA and AZB caused a concentration-dependent bradycardia of the embryonic heart and at high concentrations heart block. These effects were reversible on washout. In terms of potency to cause bradycardia the compounds were ranked AZB > bupivacaine > AZA > lidocaine > nifedipine. Comparison with results from previous studies with more specific ion channel blockers suggests that the primary effect of AZA and AZB was sodium channel blockage. The study shows that the short-term rat whole embryo culture (WEC) is a suitable system to detect substances hazardous to the embryonic heart. - Highlights: • Study of the effect of sodium channel blocking drugs on embryonic heart function • We used a modified method rat whole embryo culture with image analysis. • The drugs tested caused a concentration dependent bradycardia and heart block. • The effect of drugs acting on multiple ion channels is difficult to predict. • This method may be used to detect cardiotoxicity in prenatal development.

  11. Tetrodotoxin-resistant voltage-dependent sodium channels in identified muscle afferent neurons.

    Science.gov (United States)

    Ramachandra, Renuka; McGrew, Stephanie Y; Baxter, James C; Kiveric, Esad; Elmslie, Keith S

    2012-10-01

    Muscle afferents are critical regulators of motor function (Group I and II) and cardiovascular responses to exercise (Group III and IV). However, little is known regarding the expressed voltage-dependent ion channels. We identified muscle afferent neurons in dorsal root ganglia (DRGs), using retrograde labeling to examine voltage-dependent sodium (Na(V)) channels. In patch-clamp recordings, we found that the dominant Na(V) current in the majority of identified neurons was insensitive to tetrodotoxin (TTX-R), with Na(V) current in only a few (14%) neurons showing substantial (>50%) TTX sensitivity (TTX-S). The TTX-R current was sensitive to a Na(V)1.8 channel blocker, A803467. Immunocytochemistry demonstrated labeling of muscle afferent neurons by a Na(V)1.8 antibody, which further supported expression of these channels. A portion of the TTX-R Na(V) current appeared to be noninactivating during our 25-ms voltage steps, which suggested activity of Na(V)1.9 channels. The majority of the noninactivating current was insensitive to A803467 but sensitive to extracellular sodium. Immunocytochemistry showed labeling of muscle afferent neurons by a Na(V)1.9 channel antibody, which supports expression of these channels. Further examination of the muscle afferent neurons showed that functional TTX-S channels were expressed, but were largely inactivated at physiological membrane potentials. Immunocytochemistry showed expression of the TTX-S channels Na(V)1.6 and Na(V)1.7 but not Na(V)1.1. Na(V)1.8 and Na(V)1.9 appear to be the dominant functional sodium channels in small- to medium-diameter muscle afferent neurons. The expression of these channels is consistent with the identification of these neurons as Group III and IV, which mediate the exercise pressor reflex.

  12. On the multiple roles of the voltage gated sodium channel β1 subunit in genetic diseases

    Directory of Open Access Journals (Sweden)

    Debora eBaroni

    2015-05-01

    Full Text Available Voltage-gated sodium channels are intrinsic plasma membrane proteins that initiate the action potential in electrically excitable cells. They are composed of a pore-forming α-subunit and associated β-subunits. The β1-subunit was the first accessory subunit to be cloned. It can be important for controlling cell excitability and modulating multiple aspects of sodium channel physiology. Mutations of β1 are implicated in a wide variety of inherited pathologies, including epilepsy and cardiac conduction diseases. This review summarizes β1-subunit related channelopathies pointing out the current knowledge concerning their genetic background and their underlying molecular mechanisms.

  13. Molecular basis for class Ib anti-arrhythmic inhibition of cardiac sodium channels

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Galpin, Jason D; Frankel, Adam

    2011-01-01

    -Williams classification system into classes Ia-c based on their distinct effects on the electrocardiogram. How can these drugs elicit distinct effects on the cardiac action potential by binding to a common receptor? Here we use fluorinated phenylalanine derivatives to test whether the electronegative surface potential......Cardiac sodium channels are established therapeutic targets for the management of inherited and acquired arrhythmias by class I anti-arrhythmic drugs (AADs). These drugs share a common target receptor bearing two highly conserved aromatic side chains, and are subdivided by the Vaughan...... the inhibition of cardiac sodium channels by clinically relevant drugs and provide information for the directed design of AADs....

  14. Functionalized Fullerene Targeting Human Voltage-Gated Sodium Channel, hNav1.7.

    Science.gov (United States)

    Hilder, Tamsyn A; Robinson, Anna; Chung, Shin-Ho

    2017-08-16

    Mutations of hNa v 1.7 that cause its activities to be enhanced contribute to severe neuropathic pain. Only a small number of hNa v 1.7 specific inhibitors have been identified, most of which interact with the voltage-sensing domain of the voltage-activated sodium ion channel. In our previous computational study, we demonstrated that a [Lys 6 ]-C 84 fullerene binds tightly (affinity of 46 nM) to Na v Ab, the voltage-gated sodium channel from the bacterium Arcobacter butzleri. Here, we extend this work and, using molecular dynamics simulations, demonstrate that the same [Lys 6 ]-C 84 fullerene binds strongly (2.7 nM) to the pore of a modeled human sodium ion channel hNa v 1.7. In contrast, the fullerene binds only weakly to a mutated model of hNa v 1.7 (I1399D) (14.5 mM) and a model of the skeletal muscle hNa v 1.4 (3.7 mM). Comparison of one representative sequence from each of the nine human sodium channel isoforms shows that only hNa v 1.7 possesses residues that are critical for binding the fullerene derivative and blocking the channel pore.

  15. Tetrodotoxin-resistant voltage-dependent sodium channels in identified muscle afferent neurons

    OpenAIRE

    Ramachandra, Renuka; McGrew, Stephanie Y.; Baxter, James C.; Kiveric, Esad; Elmslie, Keith S.

    2012-01-01

    Muscle afferents are critical regulators of motor function (Group I and II) and cardiovascular responses to exercise (Group III and IV). However, little is known regarding the expressed voltage-dependent ion channels. We identified muscle afferent neurons in dorsal root ganglia (DRGs), using retrograde labeling to examine voltage-dependent sodium (NaV) channels. In patch-clamp recordings, we found that the dominant NaV current in the majority of identified neurons was insensitive to tetrodoto...

  16. Sodium channel blockade with QRS widening after an escitalopram overdose.

    Science.gov (United States)

    Schreffler, Susan M; Marraffa, Jeanna M; Stork, Christine M; Mackey, Jennifer

    2013-09-01

    Escitalopram is rarely associated with prolongation of the QTc interval; however, there are no reported cases of QRS complex widening associated with escitalopram overdose. We report a case of a patient who presented with both QRS complex widening and QTc interval prolongation after an escitalopram overdose. A 16-year-old girl presented to the emergency department after ingestion of escitalopram, tramadol/acetaminophen, and hydrocodone/acetaminophen. Laboratory results were significant for 4-hour acetaminophen 21.1 μg/mL. Serum electrolytes including potassium, magnesium, and calcium were all normal. Initial electrocardiogram (ECG) revealed a widened QRS with an incomplete right bundle branch pattern. After administration of 100-mEq sodium bicarbonate, a repeat ECG revealed narrowing of the QRS complex and a prolonged QTc interval. Magnesium sulfate 2 g intravenous and sodium bicarbonate drip were initiated. A repeat ECG, 1 hour after the second, revealed normalization of the QRS complex and QTc interval. Prolongation of the QTc interval is an expected effect of escitalopram. Both escitalopram and citalopram are metabolized to the cardiotoxic metabolite S-didesmethylcitalopram and didesmethylcitalopram, respectively, which have been implicated in numerous cardiac abnormalities including widening of the QRS complex. Although never previously described with escitalopram, this mechanism provides a reasonable explanation for the QRS complex widening and incomplete right bundle branch block that occurred in our patient. Both QRS complex widening and QTc interval prolongation should be monitored in cases of escitalopram and citalopram overdoses.

  17. Current-voltage curve of sodium channels and concentration dependence of sodium permeability in frog skin

    DEFF Research Database (Denmark)

    Fuchs, W; Larsen, Erik Hviid; Lindemann, B

    1977-01-01

    transport through open Na-selective channels of the outward facing membrane of the stratum granulosum cells can be described as an electrodiffusion process which as such does not saturate with increasing (Na)(o). However, when added to the outer border of the membrane Na causes a decrease of P(Na) within...... was recorded. This procedure was repeated after blocking the Na channels with amiloride to obtain the current-voltage curve of transmembrane and paracellular shunt pathways. The current-voltage curve of the Na channels was computed by subtracting the shunt current from the total current.4. The instantaneous I...... several seconds. It is considered that binding of Na results in closure of Na channels....

  18. Molecular mechanism of allosteric modification of voltage-dependent sodium channels by local anesthetics.

    Science.gov (United States)

    Arcisio-Miranda, Manoel; Muroi, Yukiko; Chowdhury, Sandipan; Chanda, Baron

    2010-11-01

    The hallmark of many intracellular pore blockers such as tetra-alkylammonium compounds and local anesthetics is their ability to allosterically modify the movement of the voltage sensors in voltage-dependent ion channels. For instance, the voltage sensor of domain III is specifically stabilized in the activated state when sodium currents are blocked by local anesthetics. The molecular mechanism underlying this long-range interaction between the blocker-binding site in the pore and voltage sensors remains poorly understood. Here, using scanning mutagenesis in combination with voltage clamp fluorimetry, we systematically evaluate the role of the internal gating interface of domain III of the sodium channel. We find that several mutations in the S4-S5 linker and S5 and S6 helices dramatically reduce the stabilizing effect of lidocaine on the activation of domain III voltage sensor without significantly altering use-dependent block at saturating drug concentrations. In the wild-type skeletal muscle sodium channel, local anesthetic block is accompanied by a 21% reduction in the total gating charge. In contrast, point mutations in this critical intracellular region reduce this charge modification by local anesthetics. Our analysis of a simple model suggests that these mutations in the gating interface are likely to disrupt the various coupling interactions between the voltage sensor and the pore of the sodium channel. These findings provide a molecular framework for understanding the mechanisms underlying allosteric interactions between a drug-binding site and voltage sensors.

  19. Molecular mechanism of allosteric modification of voltage-dependent sodium channels by local anesthetics

    Science.gov (United States)

    Arcisio-Miranda, Manoel; Muroi, Yukiko; Chowdhury, Sandipan

    2010-01-01

    The hallmark of many intracellular pore blockers such as tetra-alkylammonium compounds and local anesthetics is their ability to allosterically modify the movement of the voltage sensors in voltage-dependent ion channels. For instance, the voltage sensor of domain III is specifically stabilized in the activated state when sodium currents are blocked by local anesthetics. The molecular mechanism underlying this long-range interaction between the blocker-binding site in the pore and voltage sensors remains poorly understood. Here, using scanning mutagenesis in combination with voltage clamp fluorimetry, we systematically evaluate the role of the internal gating interface of domain III of the sodium channel. We find that several mutations in the S4–S5 linker and S5 and S6 helices dramatically reduce the stabilizing effect of lidocaine on the activation of domain III voltage sensor without significantly altering use-dependent block at saturating drug concentrations. In the wild-type skeletal muscle sodium channel, local anesthetic block is accompanied by a 21% reduction in the total gating charge. In contrast, point mutations in this critical intracellular region reduce this charge modification by local anesthetics. Our analysis of a simple model suggests that these mutations in the gating interface are likely to disrupt the various coupling interactions between the voltage sensor and the pore of the sodium channel. These findings provide a molecular framework for understanding the mechanisms underlying allosteric interactions between a drug-binding site and voltage sensors. PMID:20937693

  20. The voltage-gated sodium channel NaV 1.9 in visceral pain

    National Research Council Canada - National Science Library

    Hockley, J R F; Winchester, W J; Bulmer, D C

    2016-01-01

    ... sodium channel subtype 1.9 (NaV 1.9). Here, we address the role of NaV 1.9 in visceral pain and what known human NaV 1.9 mutants can tell us about NaV 1.9 function in gut physiology and pathophysiology.

  1. Loss-of-Function Sodium Channel Mutations in Infancy A Pattern Unfolds

    NARCIS (Netherlands)

    Chockalingam, Priya; Wilde, Arthur A. M.

    2012-01-01

    The role of channelopathies in the pathogenesis of sudden cardiac death (SCD) in patients with structurally normal hearts is a rapidly evolving story.(1) Many ion channels are involved, including loss-of-function sodium channelopathies of which the phenotypic spectrum ranges from lethal arrhythmias

  2. Cardiac sodium channel overlap syndromes: different faces of SCN5A mutations

    NARCIS (Netherlands)

    Remme, Carol Ann; Wilde, Arthur A. M.; Bezzina, Connie R.

    2008-01-01

    Cardiac sodium channel dysfunction caused by mutations in the SCN5A gene is associated with a number of relatively uncommon arrhythmia syndromes, including long-QT syndrome type 3 (LQT3), Brugada syndrome, conduction disease, sinus node dysfunction, and atrial standstill, which potentially lead to

  3. Distribution and function of sodium channel subtypes in human atrial myocardium

    NARCIS (Netherlands)

    Kaufmann, Susann G.; Westenbroek, Ruth E.; Maass, Alexander H.; Lange, Volkmar; Renner, Andre; Wischmeyer, Erhard; Bonz, Andreas; Muck, Jenny; Ertl, Georg; Catterall, William A.; Scheuer, Todd; Maier, Sebastian K. G.

    Voltage-gated sodium channels composed of a pore-forming alpha subunit and auxiliary beta subunits are responsible for the upstroke of the action potential in cardiac muscle. However, their localization and expression patterns in human myocardium have not yet been clearly defined. We used

  4. SCN9A mutations define primary erythermalgia as a neuropathic disorder of voltage gated sodium channels.

    NARCIS (Netherlands)

    Drenth, J.P.H.; Morsche, R.H.M. te; Guillet, G.; Taieb, A.; Kirby, R.L.; Jansen, J.B.M.J.

    2005-01-01

    Primary erythermalgia is a rare disorder characterized by recurrent attacks of red, warm and painful hands, and/or feet. We previously localized the gene for primary erythermalgia to a 7.94 cM region on chromosome 2q. Recently, Yang et al identified two missense mutations of the sodium channel alpha

  5. Phenotypical Manifestations of Mutations in the Genes Encoding Subunits of the Cardiac Sodium Channel

    NARCIS (Netherlands)

    Wilde, Arthur A. M.; Brugada, Ramon

    2011-01-01

    Variations in the gene encoding for the major sodium channel (Na(v)1.5) in the heart, SCN5A, has been shown to cause a number of arrhythmia syndromes (with or without structural changes in the myocardium), including the long-QT syndrome (type 3), Brugada syndrome, (progressive) cardiac conduction

  6. Slick (Kcnt2 Sodium-Activated Potassium Channels Limit Peptidergic Nociceptor Excitability and Hyperalgesia

    Directory of Open Access Journals (Sweden)

    Danielle L Tomasello

    2017-09-01

    Full Text Available The Slick (Kcnt2 sodium-activated potassium (K Na channel is a rapidly gating and weakly voltage-dependent and sodium-dependent potassium channel with no clearly defined physiological function. Within the dorsal root ganglia (DRGs, we show Slick channels are exclusively expressed in small-sized and medium-sized calcitonin gene–related peptide (CGRP-containing DRG neurons, and a pool of channels are localized to large dense-core vesicles (LDCV-containing CGRP. We stimulated DRG neurons for CGRP release and found Slick channels contained within CGRP-positive LDCV translocated to the neuronal membrane. Behavioral studies in Slick knockout (KO mice indicated increased basal heat detection and exacerbated thermal hyperalgesia compared with wild-type littermate controls during neuropathic and chronic inflammatory pain. Electrophysiologic recordings of DRG neurons from Slick KO mice revealed that Slick channels contribute to outward current, propensity to fire action potentials (APs, and to AP properties. Our data suggest that Slick channels restrain the excitability of CGRP-containing neurons, diminishing pain behavior after inflammation and injury.

  7. Slick (Kcnt2) Sodium-Activated Potassium Channels Limit Peptidergic Nociceptor Excitability and Hyperalgesia

    Science.gov (United States)

    Tomasello, Danielle L; Hurley, Edward; Wrabetz, Lawrence; Bhattacharjee, Arin

    2017-01-01

    The Slick (Kcnt2) sodium-activated potassium (KNa) channel is a rapidly gating and weakly voltage-dependent and sodium-dependent potassium channel with no clearly defined physiological function. Within the dorsal root ganglia (DRGs), we show Slick channels are exclusively expressed in small-sized and medium-sized calcitonin gene–related peptide (CGRP)-containing DRG neurons, and a pool of channels are localized to large dense-core vesicles (LDCV)-containing CGRP. We stimulated DRG neurons for CGRP release and found Slick channels contained within CGRP-positive LDCV translocated to the neuronal membrane. Behavioral studies in Slick knockout (KO) mice indicated increased basal heat detection and exacerbated thermal hyperalgesia compared with wild-type littermate controls during neuropathic and chronic inflammatory pain. Electrophysiologic recordings of DRG neurons from Slick KO mice revealed that Slick channels contribute to outward current, propensity to fire action potentials (APs), and to AP properties. Our data suggest that Slick channels restrain the excitability of CGRP-containing neurons, diminishing pain behavior after inflammation and injury. PMID:28943756

  8. Molecular and functional characterization of voltage-gated sodium channels in human sperm.

    Science.gov (United States)

    Pinto, Francisco M; Ravina, Cristina G; Fernández-Sánchez, Manuel; Gallardo-Castro, Manuel; Cejudo-Román, Antonio; Candenas, Luz

    2009-07-16

    We have investigated the expression of voltage-gated sodium channels in human spermatozoa and characterized their role in sperm motility. Freshly ejaculated semen was collected from thirty normozoospermic human donors, with each donor supplying 2 different samples. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence techniques were used to detect the mRNAs and proteins of interest. Sperm motility was measured by a computer-assisted sperm analysis system (CASA). Cytosolic free calcium was determined by fluorimetry in cells loaded with the fluorescent calcium indicator Fura-2. The mRNAs that encode the different Nav alpha subunits (Nav1.1-1.9) were all expressed in capacitated human spermatozoa. The mRNAs of the auxiliary subunits beta1, beta3 and beta4 were also present. Immunofluorescence studies showed that, with the exception of Nav1.1 and Nav1.3, the Nav channel proteins were present in sperm cells and show specific and different sites of localization. Veratridine, a voltage-gated sodium channel activator, caused time- and concentration-dependent increases in progressive sperm motility. In sperm suspensions loaded with Fura-2, veratridine did not modify intracellular free calcium levels. This research shows the presence of voltage-gated sodium channels in human sperm and supports a role for these channels in the regulation of mature sperm function.

  9. Molecular and functional characterization of voltage-gated sodium channels in human sperm

    Directory of Open Access Journals (Sweden)

    Cejudo-Román Antonio

    2009-07-01

    Full Text Available Abstract Background We have investigated the expression of voltage-gated sodium channels in human spermatozoa and characterized their role in sperm motility. Methods Freshly ejaculated semen was collected from thirty normozoospermic human donors, with each donor supplying 2 different samples. Reverse transcription-polymerase chain reaction (RT-PCR and immunofluorescence techniques were used to detect the mRNAs and proteins of interest. Sperm motility was measured by a computer-assisted sperm analysis system (CASA. Cytosolic free calcium was determined by fluorimetry in cells loaded with the fluorescent calcium indicator Fura-2. Results The mRNAs that encode the different Nav alpha subunits (Nav1.1-1.9 were all expressed in capacitated human spermatozoa. The mRNAs of the auxiliary subunits beta1, beta3 and beta4 were also present. Immunofluorescence studies showed that, with the exception of Nav1.1 and Nav1.3, the Nav channel proteins were present in sperm cells and show specific and different sites of localization. Veratridine, a voltage-gated sodium channel activator, caused time- and concentration-dependent increases in progressive sperm motility. In sperm suspensions loaded with Fura-2, veratridine did not modify intracellular free calcium levels. Conclusion This research shows the presence of voltage-gated sodium channels in human sperm and supports a role for these channels in the regulation of mature sperm function.

  10. Tetrodotoxin-sensitive α-subunits of voltage-gated sodium channels are relevant for inhibition of cardiac sodium currents by local anesthetics.

    Science.gov (United States)

    Stoetzer, C; Doll, T; Stueber, T; Herzog, C; Echtermeyer, F; Greulich, F; Rudat, C; Kispert, A; Wegner, F; Leffler, A

    2016-06-01

    The sodium channel α-subunit (Nav) Nav1.5 is regarded as the most prevalent cardiac sodium channel required for generation of action potentials in cardiomyocytes. Accordingly, Nav1.5 seems to be the main target molecule for local anesthetic (LA)-induced cardiotoxicity. However, recent reports demonstrated functional expression of several "neuronal" Nav's in cardiomyocytes being involved in cardiac contractility and rhythmogenesis. In this study, we examined the relevance of neuronal tetrodotoxin (TTX)-sensitive Nav's for inhibition of cardiac sodium channels by the cardiotoxic LAs ropivacaine and bupivacaine. Effects of LAs on recombinant Nav1.2, 1.3, 1.4, and 1.5 expressed in human embryonic kidney cell line 293 (HEK-293) cells, and on sodium currents in murine, cardiomyocytes were investigated by whole-cell patch clamp recordings. Expression analyses were performed by reverse transcription PCR (RT-PCR). Cultured cardiomyocytes from neonatal mice express messenger RNA (mRNA) for Nav1.2, 1.3, 1.5, 1.8, and 1.9 and generate TTX-sensitive sodium currents. Tonic and use-dependent block of sodium currents in cardiomyocytes by ropivacaine and bupivacaine were enhanced by 200 nM TTX. Inhibition of recombinant Nav1.5 channels was similar to that of TTX-resistant currents in cardiomyocytes but stronger as compared to inhibition of total sodium current in cardiomyocytes. Recombinant Nav1.2, 1.3, 1.4, and 1.5 channels displayed significant differences in regard to use-dependent block by ropivacaine. Finally, bupivacaine blocked sodium currents in cardiomyocytes as well as recombinant Nav1.5 currents significantly stronger in comparison to ropivacaine. Our data demonstrate for the first time that cardiac TTX-sensitive sodium channels are relevant for inhibition of cardiac sodium currents by LAs.

  11. Bacterial voltage-gated sodium channels (BacNa(V)s) from the soil, sea, and salt lakes enlighten molecular mechanisms of electrical signaling and pharmacology in the brain and heart.

    Science.gov (United States)

    Payandeh, Jian; Minor, Daniel L

    2015-01-16

    Voltage-gated sodium channels (Na(V)s) provide the initial electrical signal that drives action potential generation in many excitable cells of the brain, heart, and nervous system. For more than 60years, functional studies of Na(V)s have occupied a central place in physiological and biophysical investigation of the molecular basis of excitability. Recently, structural studies of members of a large family of bacterial voltage-gated sodium channels (BacNa(V)s) prevalent in soil, marine, and salt lake environments that bear many of the core features of eukaryotic Na(V)s have reframed ideas for voltage-gated channel function, ion selectivity, and pharmacology. Here, we analyze the recent advances, unanswered questions, and potential of BacNa(V)s as templates for drug development efforts. Copyright © 2014. Published by Elsevier Ltd.

  12. Nav1.5 cardiac sodium channels, regulation and clinical implications

    Directory of Open Access Journals (Sweden)

    Henry Humberto León-Ariza

    2014-10-01

    Full Text Available Voltage-gated sodium channels constitute a group of membrane proteins widely distributed thought the body. In the heart, there are at least six different isoforms, being the Nav1.5 the most abundant. The channel is composed of an α subunit that is formed by four domains of six segments each, and four much smaller β subunits that provide stability and integrate other channels into the α subunit. The function of the Nav1.5 channel is modulated by intracellular cytoskeleton proteins, extracellular proteins, calcium concentration, free radicals, and medications, among other things. The study of the channel and its alterations has grown thanks to its association with pathogenic conditions such as Long QT syndrome, Brugada syndrome, atrial fibrillation, arrhythmogenic ventricular dysplasia and complications during ischemic processes.

  13. Sea anemone toxins affecting voltage-gated sodium channels--molecular and evolutionary features.

    Science.gov (United States)

    Moran, Yehu; Gordon, Dalia; Gurevitz, Michael

    2009-12-15

    The venom of sea anemones is rich in low molecular weight proteinaceous neurotoxins that vary greatly in structure, site of action, and phyletic (insect, crustacean or vertebrate) preference. This toxic versatility likely contributes to the ability of these sessile animals to inhabit marine environments co-habited by a variety of mobile predators. Among these toxins, those that show prominent activity at voltage-gated sodium channels and are critical in predation and defense, have been extensively studied for more than three decades. These studies initially focused on the discovery of new toxins, determination of their covalent and folded structures, understanding of their mechanisms of action on different sodium channels, and identification of the primary sites of interaction of the toxins with their channel receptors. The channel binding site for Type I and the structurally unrelated Type III sea anemone toxins was identified as neurotoxin receptor site 3, a site previously shown to be targeted by scorpion alpha-toxins. The bioactive surfaces of toxin representatives from these two sea anemone types have been characterized by mutagenesis. These analyses pointed to heterogeneity of receptor site 3 at various sodium channels. A turning point in evolutionary studies of sea anemone toxins was the recent release of the genome sequence of Nematostella vectensis, which enabled analysis of the genomic organization of the corresponding genes. This analysis demonstrated that Type I toxins in Nematostella and other species are encoded by gene families and suggested that these genes developed by concerted evolution. The current review provides a brief historical description of the discovery and characterization of sea anemone toxins that affect voltage-gated sodium channels and delineates recent advances in the study of their structure-activity relationship and evolution.

  14. Post-repolarization block of cardiac sodium channels by saxitoxin.

    Science.gov (United States)

    Makielski, J C; Satin, J; Fan, Z

    1993-08-01

    Phasic block of rat cardiac Na+ current by saxitoxin was assessed using pulse trains and two-pulse voltage clamp protocols, and the results were fit to several kinetic models. For brief depolarizations (5 to 50 ms) the depolarization duration did not affect the rate of development or the amplitude of phasic block for pulse trains. The pulse train data were well described by a recurrence relation based upon the guarded receptor model, and it provided rate constants that accurately predicted first-pulse block as well as recovery time constants in response to two-pulse protocols. However, the amplitudes and rates of phasic block development at rapid rates (> 5 Hz) were less than the model predicted. For two pulse protocols with a short (10 ms) conditioning step to -30 mV, block developed only after repolarization to -150 mV and then recovered as the interpulse interval was increased. This suggested that phasic block under these conditions was caused by binding with increased affinity to a state that exists transiently after repolarization to -150 mV. This "post-repolarization block" was fit to a three-state model consisting of a transient state with high affinity for the toxin, the toxin bound state, and the ultimate resting state of the channel. This model accounted for the biphasic post-repolarization block development and recovery observed in two-pulse protocols, and it more accurately described phasic block in pulse trains. The transient state after repolarization was predicted to have a dwell time of 570 ms, an on rate for saxitoxin of 16 s-1 micro M-1, and an off rate of 0.2 s-1 (KD = 12 nM). These results and the proposed model suggest a novel variation on phasic block mechanisms and suggest a long-lived transient Na+ channel conformation during recovery.

  15. Calcium-activated SK channels control firing regularity by modulating sodium channel availability in midbrain dopamine neurons.

    Science.gov (United States)

    Iyer, Rajeshwari; Ungless, Mark A; Faisal, Aldo A

    2017-07-12

    Dopamine neurons in the substantia nigra pars compacta and ventral tegmental area regulate behaviours such as reward-related learning, and motor control. Dysfunction of these neurons is implicated in Schizophrenia, addiction to drugs, and Parkinson's disease. While some dopamine neurons fire single spikes at regular intervals, others fire irregular single spikes interspersed with bursts. Pharmacological inhibition of calcium-activated potassium (SK) channels increases the variability in their firing pattern, sometimes also increasing the number of spikes fired in bursts, indicating that SK channels play an important role in maintaining dopamine neuron firing regularity and burst firing. However, the exact mechanisms underlying these effects are still unclear. Here, we develop a biophysical model of a dopamine neuron incorporating ion channel stochasticity that enabled the analysis of availability of ion channels in multiple states during spiking. We find that decreased firing regularity is primarily due to a significant decrease in the AHP that in turn resulted in a reduction in the fraction of available voltage-gated sodium channels due to insufficient recovery from inactivation. Our model further predicts that inhibition of SK channels results in a depolarisation of action potential threshold along with an increase in its variability.

  16. Structure-based assessment of disease-related mutations in human voltage-gated sodium channels

    Directory of Open Access Journals (Sweden)

    Weiyun Huang

    2017-02-01

    Full Text Available ABSTRACT Voltage-gated sodium (Nav channels are essential for the rapid upstroke of action potentials and the propagation of electrical signals in nerves and muscles. Defects of Nav channels are associated with a variety of channelopathies. More than 1000 disease-related mutations have been identified in Nav channels, with Nav1.1 and Nav1.5 each harboring more than 400 mutations. Nav channels represent major targets for a wide array of neurotoxins and drugs. Atomic structures of Nav channels are required to understand their function and disease mechanisms. The recently determined atomic structure of the rabbit voltage-gated calcium (Cav channel Cav1.1 provides a template for homology-based structural modeling of the evolutionarily related Nav channels. In this Resource article, we summarized all the reported disease-related mutations in human Nav channels, generated a homologous model of human Nav1.7, and structurally mapped disease-associated mutations. Before the determination of structures of human Nav channels, the analysis presented here serves as the base framework for mechanistic investigation of Nav channelopathies and for potential structure-based drug discovery.

  17. Structure-based assessment of disease-related mutations in human voltage-gated sodium channels.

    Science.gov (United States)

    Huang, Weiyun; Liu, Minhao; Yan, S Frank; Yan, Nieng

    2017-06-01

    Voltage-gated sodium (Na v ) channels are essential for the rapid upstroke of action potentials and the propagation of electrical signals in nerves and muscles. Defects of Na v channels are associated with a variety of channelopathies. More than 1000 disease-related mutations have been identified in Na v channels, with Na v 1.1 and Na v 1.5 each harboring more than 400 mutations. Na v channels represent major targets for a wide array of neurotoxins and drugs. Atomic structures of Na v channels are required to understand their function and disease mechanisms. The recently determined atomic structure of the rabbit voltage-gated calcium (Ca v ) channel Ca v 1.1 provides a template for homology-based structural modeling of the evolutionarily related Na v channels. In this Resource article, we summarized all the reported disease-related mutations in human Na v channels, generated a homologous model of human Na v 1.7, and structurally mapped disease-associated mutations. Before the determination of structures of human Na v channels, the analysis presented here serves as the base framework for mechanistic investigation of Na v channelopathies and for potential structure-based drug discovery.

  18. Conduction velocity is regulated by sodium channel inactivation in unmyelinated axons innervating the rat cranial meninges

    Science.gov (United States)

    De Col, Roberto; Messlinger, Karl; Carr, Richard W

    2008-01-01

    Axonal conduction velocity varies according to the level of preceding impulse activity. In unmyelinated axons this typically results in a slowing of conduction velocity and a parallel increase in threshold. It is currently held that Na+–K+-ATPase-dependent axonal hyperpolarization is responsible for this slowing but this has long been equivocal. We therefore examined conduction velocity changes during repetitive activation of single unmyelinated axons innervating the rat cranial meninges. In direct contradiction to the currently accepted postulate, Na+–K+-ATPase blockade actually enhanced activity-induced conduction velocity slowing, while the degree of velocity slowing was curtailed in the presence of lidocaine (10–300 μm) and carbamazepine (30–500 μm) but not tetrodotoxin (TTX, 10–80 nm). This suggests that a change in the number of available sodium channels is the most prominent factor responsible for activity-induced changes in conduction velocity in unmyelinated axons. At moderate stimulus frequencies, axonal conduction velocity is determined by an interaction between residual sodium channel inactivation following each impulse and the retrieval of channels from inactivation by a concomitant Na+–K+-ATPase-mediated hyperpolarization. Since the process is primarily dependent upon sodium channel availability, tracking conduction velocity provides a means of accessing relative changes in the excitability of nociceptive neurons. PMID:18096592

  19. Use dependence of peripheral nociceptive conduction in the absence of tetrodotoxin‐resistant sodium channel subtypes

    Science.gov (United States)

    Kistner, Katrin; Nassar, Mohammed; Reeh, Peter W.; Weidner, Christian

    2016-01-01

    Key points This study examines conduction in peripheral nerves and its use dependence in tetrodotoxin‐resistant (TTXr) sodium channel (Nav1.8, Nav1.9) knockout and wildtype animals.We observed use‐dependent decreases of single fibre and compound action potential amplitude in peripheral mouse C‐fibres (wildtype). This matches the previously published hypothesis that increased Na/K‐pump activity is not the underlying mechanism for use‐dependent changes of neural conduction.Knocking out TTXr sodium channels influences use‐dependent changes of conductive properties (action potential amplitude, latency, conduction safety) in the order Nav1.8 KO > Nav1.9KO > wildtype.This is most likely explained by different subsets of presumably (relatively) Nav1.7‐rich conducting fibres in knockout animals as compared to wildtypes, in combination with reduced per‐pulse sodium influx. Abstract Use dependency of peripheral nerves, especially of nociceptors, correlates with receptive properties. Slow inactivation of voltage‐gated sodium channels has been discussed to be the underlying mechanism – pointing to a receptive class‐related difference of sodium channel equipment. Using electrophysiological recordings of single unmyelinated cutaneous fibres and their compound action potential (AP), we evaluated use‐dependent changes in mouse peripheral nerves, and the contribution of the tetrodotoxin‐resistant (TTXr) sodium channels Nav1.8 and Nav1.9 to these changes. Nerve fibres were electrically stimulated using single or double pulses at 2 Hz. Use‐dependent changes of latency, AP amplitude, and duration as well as the fibres’ ability to follow the stimulus were evaluated. AP amplitudes substantially diminished in used fibres from C57BL/6 but increased in Nav1.8 knockout (KO) mice, with Nav1.9 KO in between. Activity‐induced latency slowing was in contrast the most pronounced in Nav1.8 KOs and the least in wildtype mice. The genotype was also predictive of

  20. Channel waveguides in glass via silver-sodium field-assisted ion exchange

    Science.gov (United States)

    Forrest, K.; Pagano, S. J.; Viehmann, W.

    1986-01-01

    Multimode channel waveguides have been formed in sodium aluminosilicate glass by field-assisted diffusion of Ag(+) ions from vacuum-evaporated Ag films. The two-dimensional refractive index profiles of the waveguides were controlled by varying the diffusion time, the diffusion temperature, and the electric field strength. Estimates of the diffusion rate through a strip aperture were obtained, assuming the electric field was strong 120-240 V/mm. The maximum change in refractive index in the sodium aluminosilicate glasses was estimated near 65 percent of the change in soda-lime silicate glass. The physical properties of the glasses are given in a table.

  1. A mechanistic interpretation of the action of toxin II from Anemonia sulcata on the cardiac sodium channel.

    Science.gov (United States)

    Schreibmayer, W; Kazerani, H; Tritthart, H A

    1987-07-23

    Cardiac sodium channels, modified by Anemonia sulcata toxin II, have been analyzed by the patch-clamp method. The open state of the modified sodium channels proved to be prolonged highly significantly and reopening from a closed state denoted c*-state frequently occurred, interrupted by silent periods, denoted i*-state. Activation from the c*-state was apparently not affected by toxin action, whereas activation from the i*-state was markedly prolonged. Upon higher depolarizations toxin-induced sodium channels disappeared and this behaviour has been attributed to dissociation of the toxin from the channel by use of a special pulse-protocol. The onset of the toxin effect on the action potential proved to depend on stimulation, and it is concluded that the toxin binds preferentially to the open (o)-state. Taking together the results, a kinetic scheme is suggested for action of the toxin on the cardiac sodium channel.

  2. CC chemokine ligand 2 upregulates the current density and expression of TRPV1 channels and Nav 1.8 sodium channels in dorsal root ganglion neurons

    National Research Council Canada - National Science Library

    Der-Jang Kao; Allen H Li; Jin-Chung Chen; Ro-Sun Luo; Ying-Ling Chen; Juu-Chin Lu; Hung-Li Wang

    2012-01-01

    .... Transient receptor potential vanilloid receptor 1 (TRPV1) and tetrodotoxin (TTX)-resistant Na v 1.8 sodium channels play an essential role in regulating the excitability and pain transmission of DRG nociceptive neurons...

  3. Voltage-dependent sodium channels and calcium-activated potassium channels in human odontoblasts in vitro.

    Science.gov (United States)

    Ichikawa, Hideki; Kim, Hyong-Jung; Shuprisha, Apichai; Shikano, Tetsuo; Tsumura, Maki; Shibukawa, Yoshiyuki; Tazaki, Masakazu

    2012-10-01

    Transmembrane ionic signaling regulates many cellular processes in both physiological and pathologic settings. In this study, the biophysical properties of voltage-dependent Na(+) channels in odontoblasts derived from human dental pulp (HOB cells) were investigated together with the effect of bradykinin on intracellular Ca(2+) signaling and expression of Ca(2+)-activated K(+) channels. Ionic channel activity was characterized by using whole-cell patch-clamp recording and fura-2 fluorescence. Mean resting membrane potential in the HOB cells was -38 mV. Depolarizing steps from a holding potential of -80 mV activated transient voltage-dependent inward currents with rapid activation/inactivation properties. At a holding potential of -50 mV, no inward current was recorded. Fast-activation kinetics exhibited dependence on membrane potential, whereas fast-inactivation kinetics did not. Steady-state inactivation was described by a Boltzmann function with a half-maximal inactivation potential of -70 mV, indicating that whereas the channels were completely inactivated at physiological resting membrane potential, they could be activated when the cells were hyperpolarized. Inward currents disappeared in Na(+)-free extracellular solution. Bradykinin activated intracellular Ca(2+)-releasing and influx pathways. When the HOB cells were clamped at a holding potential of -50 mV, outward currents were recorded at positive potentials, indicating sensitivity to inhibitors of intermediate-conductance Ca(2+)-activated K(+) channels. Human odontoblasts expressed voltage-dependent Na(+) channels, bradykinin receptors, and Ca(2+)-activated K(+) channels, which play an important role in driving cellular functions by channel-receptor signal interaction and membrane potential regulation. Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  4. Molecular characterization of neurally expressing genes in the para sodium channel gene cluster of Drosophila

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Chang-Sook; Ganetzky, B. [Univ. of Wisconsin, Madison, WI (United States)

    1996-03-01

    To elucidate the mechanisms regulating expression of para, which encodes the major class of sodium channels in the Drosophila nervous system, we have tried to locate upstream cis-acting regulatory elements by mapping the transcriptional start site and analyzing the region immediately upstream of para in region 14D of the polytene chromosomes. From these studies, we have discovered that the region contains a cluster of neurally expressing genes. Here we report the molecular characterization of the genomic organization of the 14D region and the genes within this region, which are: calnexin (Cnx), actin related protein 14D (Arp14D), calcineurin A 14D (CnnA14D), and chromosome associated protein (Cap). The tight clustering of these genes, their neuronal expression patterns, and their potential functions related to expression, modulation, or regulation of sodium channels raise the possibility that these genes represent a functionally related group sharing some coordinate regulatory mechanism. 76 refs., 11 figs.

  5. Characterization of a Sodium Channel Mutation in Permethrin-Resistant Rhipicephalus sanguineus (Acari: Ixodidae).

    Science.gov (United States)

    Tucker, Nicholas S G; Kaufman, Phillip E; Weeks, Emma N I; Rowland, Jessica; Tidwell, Jason; Miller, Robert J

    2017-11-07

    The brown dog tick, Rhipicephalus sanguineus (Latrielle) sensu lato, is an important ectoparasite of dogs and occasionally humans, capable of transmitting several pathogens, such as Rickettsia and Ehrlichia, which are of veterinary and medical importance. The brown dog tick is distributed worldwide and has an affinity for human habitations in much of its range. In some populations, lack of integrated pest management plans and overuse of pyrethroid pesticides and other sodium channel inhibitors has resulted in high levels of resistance to permethrin. Recently, a highly conserved region of the R. sanguineus sodium channel was sequenced, indicating that a single nucleotide polymorphism of thymine to cytosine on domain III segment VI of the sodium channel could confer resistance. A molecular assay targeting a point mutation in the sodium channel was developed and optimized to separate ticks expressing permethrin resistance from those from a susceptible colony. Thereafter, multiple field-collected phenotypically permethrin-resistant populations were evaluated using this molecular assay to determine genotype. As confirmed by DNA sequencing, a point mutation was present at a high rate in phenotypically resistant tick populations that was not present in the susceptible strain. These data suggest an additional permethrin resistance mechanism to metabolic resistance, which has been reported for this tick species, and confirm its association with phenotypic resistance. The results of this study further emphasize the need to preserve acaricide chemistry through rotation of active ingredients used to control ectoparasites. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  6. Irreversible modification of sodium channel inactivation in toad myelinated nerve fibres by the oxidant chloramine-T.

    Science.gov (United States)

    Wang, G K

    1984-01-01

    The effects of externally applied chloramine-T on the excitability of single toad myelinated nerve fibres were studied. Chloramine-T is a mild oxidant which reacts specifically with the cysteine and methionine residues of proteins. Chloramine-T prolongs the action potential of a single myelinated fibre by more than 1000-fold. This effect is concentration- and time-dependent; higher concentrations and longer incubation times increase prolongation. Under voltage-clamp conditions, sodium channel inactivation is markedly inhibited by chloramine-T while sodium channel activation remains normal. Prolonged depolarization of the membrane leads to a maintained sodium current. The maintained sodium currents show activation kinetics, dependence on membrane potential, and reversal potentials which are similar to those of normal, inactivating sodium currents in untreated fibres. Both the maintained and the peak sodium currents are equally inhibited by tetrodotoxin. After partial removal of sodium inactivation by brief exposures to chloramine-T, the voltage dependence of the steady-state sodium current inactivation (h infinity) is shifted in the depolarized direction by about 20 mV, even after correction for the non-inactivating component contributed by the maintained current. The phenomena described here imply that cysteine or methionine residues are critical for the sodium channel inactivation processes. The two different modifications of inactivation, its removal shown by the maintained current, and the shift in the voltage-dependence of the remaining inactivatable channels, reveal that at least two separate residues are modified by chloramine-T. PMID:6321714

  7. Contactin associates with sodium channel Nav1.3 in native tissues and increases channel density at the cell surface.

    Science.gov (United States)

    Shah, Bhaval S; Rush, Anthony M; Liu, Shujun; Tyrrell, Lynda; Black, Joel A; Dib-Hajj, Sulayman D; Waxman, Stephen G

    2004-08-18

    The upregulation of voltage-gated sodium channel Na(v)1.3 has been linked to hyperexcitability of axotomized dorsal root ganglion (DRG) neurons, which underlies neuropathic pain. However, factors that regulate delivery of Na(v)1.3 to the cell surface are not known. Contactin/F3, a cell adhesion molecule, has been shown to interact with and enhance surface expression of sodium channels Na(v)1.2 and Na(v)1.9. In this study we show that contactin coimmunoprecipitates with Na(v)1.3 from postnatal day 0 rat brain where this channel is abundant, and from human embryonic kidney (HEK) 293 cells stably transfected with Na(v)1.3 (HEK-Na(v)1.3). Purified GST fusion proteins of the N and C termini of Na(v)1.3 pull down contactin from lysates of transfected HEK 293 cells. Transfection of HEK-Na(v)1.3 cells with contactin increases the amplitude of the current threefold without changing the biophysical properties of the channel. Enzymatic removal of contactin from the cell surface of cotransfected cells does not reduce the elevated levels of the Na(v)1.3 current. Finally, we show that, similar to Na(v)1.3, contactin is upregulated in axotomized DRG neurons and accumulates within the neuroma of transected sciatic nerve. We propose that the upregulation of contactin and its colocalization with Na(v)1.3 in axotomized DRG neurons may contribute to the hyper-excitablity of the injured neurons.

  8. [Genetic and molecular basis for sodium channel-mediated Brugada syndrome].

    Science.gov (United States)

    Barajas-Martínez, Héctor; Hu, Dan; Antzelevitch, Charles

    2013-01-01

    Brugada syndrome is a genetic disease that is characterized by abnormal electrocardiogram findings and an increased risk of sudden cardiac death. This syndrome is linked to mutations in the SCN5A gene in approximately 20% of Brugada syndrome probands. SCN5A encodes the α subunit of the cardiac sodium channel. Studies conducted over the past decade have identified 11 other Brugada syndrome susceptibility genes besides to SCN5A, pointing to genetic heterogeneity of the syndrome. Transmission of the disease shows an autosomal dominant inheritance pattern. This brief review focuses on a reported case of sodium channel-mediated Brugada syndrome, guiding the reader through the process of identification of the genetic variants responsible for the clinically-diagnosed syndrome, mutagenesis to clone SCN5A with and without the 2 variants identified and transfection of the 2 variants into TSA201 cells to determine the functional consequence of these genetic variants on sodium channel expression and function. Copyright © 2013 Instituto Nacional de Cardiología Ignacio Chávez. Published by Masson Doyma México S.A. All rights reserved.

  9. Voltage-gated sodium channels confer excitability to human odontoblasts: possible role in tooth pain transmission.

    Science.gov (United States)

    Allard, Bruno; Magloire, Henry; Couble, Marie Lise; Maurin, Jean Christophe; Bleicher, Françoise

    2006-09-29

    Odontoblasts are responsible for the dentin formation. They are suspected to play a role in tooth pain transmission as sensor cells because of their close relationship with nerve, but this role has never been evidenced. We demonstrate here that human odontoblasts in vitro produce voltage-gated tetrodotoxin-sensitive Na(+) currents in response to depolarization under voltage clamp conditions and are able to generate action potentials. Odontoblasts express neuronal isoforms of alpha2 and beta2 subunits of sodium channels. Co-cultures of odontoblasts with trigeminal neurons indicate a clustering of alpha2 and beta2 sodium channel subunits and, at the sites of cell-cell contact, a co-localization of odontoblasts beta2 subunits with peripherin. In vivo, sodium channels are expressed in odontoblasts. Ankyrin(G) and beta2 co-localize, suggesting a link for signal transduction between axons and odontoblasts. Evidence for excitable properties of odontoblasts and clustering of key molecules at the site of odontoblast-nerve contact strongly suggest that odontoblasts may operate as sensor cells that initiate tooth pain transmission.

  10. Tissue distribution and subcellular localization of the cardiac sodium channel during mouse heart development.

    Science.gov (United States)

    Domínguez, Jorge N; de la Rosa, Angel; Navarro, Francisco; Franco, Diego; Aránega, Amelia E

    2008-04-01

    The aim of this study was to analyse the mRNA expression levels and protein distribution of the cardiac sodium channel Scn5a/Nav1.5 during mouse cardiogenesis. Scn5a mRNA levels were determined by real-time RT-PCR using embryonic hearts ranging from E9.5 to E17.5 as well as postnatal and adult hearts. In addition, Scn5a protein (Nav1.5) distribution was analysed by immunohistochemistry and confocal microscopy. Scn5a mRNA levels displayed a peak at stage E11.5, decreased during the subsequent stages and then steadily increased from E17.5 onwards, and throughout the postnatal to the adult stages. Immunohistochemistry experiments revealed comparable distribution of Nav1.5 between the different cardiac chambers at early embryonic stages. During the foetal stages, Nav1.5 showed an enhanced expression in the trabeculated myocardium and in the bundle branches. At the subcellular level, Nav1.5 and Scn1b double-immunostaining analysis is consistent with the presence of both sodium channel subunits in the T-tubule system and the intercalated discs. Our results demonstrate that the cardiac sodium channel, Nav1.5, shows a dynamic expression pattern during mouse heart development, indicating that it could play an important role in the acquisition of a mature pattern of conduction and contraction during cardiogenesis.

  11. Site of anticonvulsant action on sodium channels: autoradiographic and electrophysiological studies in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Worley, P.F.; Baraban, J.M.

    1987-05-01

    The anticonvulsants phenytoin and carbamazepine interact allosterically with the batrachotoxin binding site of sodium channels. In the present study, we demonstrate an autoradiographic technique to localize the batrachotoxin binding site on sodium channels in rat brain using (/sup 3/H)batrachotoxinin-A 20-alpha-benzoate (BTX-B). Binding of (/sup 3/H)BTX-B to brain sections is dependent on potentiating allosteric interactions with scorpion venom and is displaced by BTX-B (Kd approximately 200 nM), aconitine, veratridine, and phenytoin with the same rank order of potencies as described in brain synaptosomes. The maximum number of (/sup 3/H)BTX-B binding sites in forebrain sections also agrees with biochemical determinations. Autoradiographic localizations indicate that (/sup 3/H)BTX-B binding sites are not restricted to cell bodies and axons but are present in synaptic zones throughout the brain. For example, a particularly dense concentration of these sites in the substantia nigra is associated with afferent terminals of the striatonigral projection. By contrast, myelinated structures possess much lower densities of binding sites. In addition, we present electrophysiological evidence that synaptic transmission, as opposed to axonal conduction, is preferentially sensitive to the action of aconitine and veratridine. Finally, the synaptic block produced by these sodium channel activators is inhibited by phenytoin and carbamazepine at therapeutic anticonvulsant concentrations.

  12. Biophysical and Pharmacological Characterization of Nav1.9 Voltage Dependent Sodium Channels Stably Expressed in HEK-293 Cells

    National Research Council Canada - National Science Library

    Lin, Zhixin; Santos, Sonia; Padilla, Karen; Printzenhoff, David; Castle, Neil A

    2016-01-01

    The voltage dependent sodium channel Nav1.9, is expressed preferentially in peripheral sensory neurons and has been linked to human genetic pain disorders, which makes it target of interest for the development of new pain therapeutics...

  13. Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET.

    Science.gov (United States)

    Kubota, Tomoya; Durek, Thomas; Dang, Bobo; Finol-Urdaneta, Rocio K; Craik, David J; Kent, Stephen B H; French, Robert J; Bezanilla, Francisco; Correa, Ana M

    2017-03-07

    Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: β-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating.

  14. Surface expression of sodium channels and transporters in rat kidney: effects of dietary sodium.

    Science.gov (United States)

    Frindt, Gustavo; Palmer, Lawrence G

    2009-11-01

    The abundance of Na transport proteins in the luminal membrane of the rat kidney was assessed using in situ biotinylation and immunoblotting. When animals were fed an Na-deficient diet for 1 wk, the amounts of epithelial Na channel (ENaC) beta-subunit (beta-ENaC) and gamma-subunit (gamma-ENaC) and Na-Cl cotransporter (NCC) protein in the surface fraction increased relative to controls by 1.9-, 3.5-, and 1.5-fold, respectively. The amounts of the luminal Na/H exchanger (NHE3) and the luminal Na-K-2Cl cotransporter (NKCC2) did not change significantly. The increases in ENaC subunits were mimicked by administration of aldosterone for 1 wk, but the increase in NCC was not. When the animals were fed a high-Na (5% NaCl) diet for 1 wk, the surface expression of beta-ENaC increased by 50%, whereas that of the other membrane proteins did not change, relative to controls. The biochemical parameter most strongly affected by dietary Na was the abundance of the 65-kDa cleaved form of gamma-ENaC at the surface. This increased by 8.5-fold with Na depletion and decreased by 40% with Na loading. The overall 14-fold change reflected regulation of the total abundance of the subunit as well as the fraction of the subunit protein in the cleaved form. We conclude that cleavage of gamma-ENaC and its expression at the apical surface play a major role in the regulation of renal Na reabsorption.

  15. Long-QT syndrome-related sodium channel mutations probed by the dynamic action potential clamp technique

    NARCIS (Netherlands)

    Berecki, Géza; Zegers, Jan G.; Bhuiyan, Zahurul A.; Verkerk, Arie O.; Wilders, Ronald; van Ginneken, Antoni C. G.

    2006-01-01

    Long-QT3 syndrome (LQT3) is linked to cardiac sodium channel gene (SCN5A) mutations. In this study, we used the 'dynamic action potential clamp' (dAPC) technique to effectively replace the native sodium current (I(Na)) of the Priebe-Beuckelmann human ventricular cell model with wild-type (WT) or

  16. Differential distribution of the sodium-activated potassium channels slick and slack in mouse brain.

    Science.gov (United States)

    Rizzi, Sandra; Knaus, Hans-Günther; Schwarzer, Christoph

    2016-07-01

    The sodium-activated potassium channels Slick (Slo2.1, KCNT2) and Slack (Slo2.2, KCNT1) are high-conductance potassium channels of the Slo family. In neurons, Slick and Slack channels are involved in the generation of slow afterhyperpolarization, in the regulation of firing patterns, and in setting and stabilizing the resting membrane potential. The distribution and subcellular localization of Slick and Slack channels in the mouse brain have not yet been established in detail. The present study addresses this issue through in situ hybridization and immunohistochemistry. Both channels were widely distributed and exhibited distinct distribution patterns. However, in some brain regions, their expression overlapped. Intense Slick channel immunoreactivity was observed in processes, varicosities, and neuronal cell bodies of the olfactory bulb, granular zones of cortical regions, hippocampus, amygdala, lateral septal nuclei, certain hypothalamic and midbrain nuclei, and several regions of the brainstem. The Slack channel showed primarily a diffuse immunostaining pattern, and labeling of cell somata and processes was observed only occasionally. The highest Slack channel expression was detected in the olfactory bulb, lateral septal nuclei, basal ganglia, and distinct areas of the midbrain, brainstem, and cerebellar cortex. In addition, comparing our data obtained from mouse brain with a previously published study on rat brain revealed some differences in the expression and distribution of Slick and Slack channels in these species. J. Comp. Neurol. 524:2093-2116, 2016. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

  17. The sialic acid component of the beta1 subunit modulates voltage-gated sodium channel function.

    Science.gov (United States)

    Johnson, Daniel; Montpetit, Marty L; Stocker, Patrick J; Bennett, Eric S

    2004-10-22

    Voltage-gated sodium channels (Nav) are responsible for initiation and propagation of nerve, skeletal muscle, and cardiac action potentials. Nav are composed of a pore-forming alpha subunit and often one to several modulating beta subunits. Previous work showed that terminal sialic acid residues attached to alpha subunits affect channel gating. Here we show that the fully sialylated beta1 subunit induces a uniform, hyperpolarizing shift in steady state and kinetic gating of the cardiac and two neuronal alpha subunit isoforms. Under conditions of reduced sialylation, the beta1-induced gating effect was eliminated. Consistent with this, mutation of beta1 N-glycosylation sites abolished all effects of beta1 on channel gating. Data also suggest an interaction between the cis effect of alpha sialic acids and the trans effect of beta1 sialic acids on channel gating. Thus, beta1 sialic acids had no effect gating on the of the heavily glycosylated skeletal muscle alpha subunit. However, when glycosylation of the skeletal muscle alpha subunit was reduced through chimeragenesis such that alpha sialic acids did not impact gating, beta1 sialic acids caused a significant hyperpolarizing shift in channel gating. Together, the data indicate that beta1 N-linked sialic acids can modulate Nav gating through an apparent saturating electrostatic mechanism. A model is proposed in which a spectrum of differentially sialylated Nav can directly modulate channel gating, thereby impacting cardiac, skeletal muscle, and neuronal excitability.

  18. Action potential generation requires a high sodium channel density in the axon initial segment.

    Science.gov (United States)

    Kole, Maarten H P; Ilschner, Susanne U; Kampa, Björn M; Williams, Stephen R; Ruben, Peter C; Stuart, Greg J

    2008-02-01

    The axon initial segment (AIS) is a specialized region in neurons where action potentials are initiated. It is commonly assumed that this process requires a high density of voltage-gated sodium (Na(+)) channels. Paradoxically, the results of patch-clamp studies suggest that the Na(+) channel density at the AIS is similar to that at the soma and proximal dendrites. Here we provide data obtained by antibody staining, whole-cell voltage-clamp and Na(+) imaging, together with modeling, which indicate that the Na(+) channel density at the AIS of cortical pyramidal neurons is approximately 50 times that in the proximal dendrites. Anchoring of Na(+) channels to the cytoskeleton can explain this discrepancy, as disruption of the actin cytoskeleton increased the Na(+) current measured in patches from the AIS. Computational models required a high Na(+) channel density (approximately 2,500 pS microm(-2)) at the AIS to account for observations on action potential generation and backpropagation. In conclusion, action potential generation requires a high Na(+) channel density at the AIS, which is maintained by tight anchoring to the actin cytoskeleton.

  19. The voltage-gated sodium channel nav1.8 is expressed in human sperm.

    Directory of Open Access Journals (Sweden)

    Antonio Cejudo-Roman

    Full Text Available The role of Na(+ fluxes through voltage-gated sodium channels in the regulation of sperm cell function remains poorly understood. Previously, we reported that several genes encoding voltage-gated Na(+ channels were expressed in human testis and mature spermatozoa. In this study, we analyzed the presence and function of the TTX-resistant VGSC α subunit Nav1.8 in human capacitated sperm cells. Using an RT-PCR assay, we found that the mRNA of the gene SCN10A, that encode Na v1.8, was abundantly and specifically expressed in human testis and ejaculated spermatozoa. The Na v1.8 protein was detected in capacitated sperm cells using three different specific antibodies against this channel. Positive immunoreactivity was mainly located in the neck and the principal piece of the flagellum. The presence of Na v1.8 in sperm cells was confirmed by Western blot. Functional studies demonstrated that the increases in progressive motility produced by veratridine, a voltage-gated sodium channel activator, were reduced in sperm cells preincubated with TTX (10 μM, the Na v1.8 antagonist A-803467, or a specific Na v1.8 antibody. Veratridine elicited similar percentage increases in progressive motility in sperm cells maintained in Ca(2+-containing or Ca(2+-free solution and did not induce hyperactivation or the acrosome reaction. Veratridine caused a rise in sperm intracellular Na(+, [Na(+]i, and the sustained phase of the response was inhibited in the presence of A-803467. These results verify that the Na(+ channel Na v1.8 is present in human sperm cells and demonstrate that this channel participates in the regulation of sperm function.

  20. The Voltage-Gated Sodium Channel Nav1.8 Is Expressed in Human Sperm

    Science.gov (United States)

    Cejudo-Roman, Antonio; Pinto, Francisco M.; Subirán, Nerea; Ravina, Cristina G.; Fernández-Sánchez, Manuel; Pérez-Hernández, Natalia; Pérez, Ricardo; Pacheco, Alberto; Irazusta, Jon; Candenas, Luz

    2013-01-01

    The role of Na+ fluxes through voltage-gated sodium channels in the regulation of sperm cell function remains poorly understood. Previously, we reported that several genes encoding voltage-gated Na+ channels were expressed in human testis and mature spermatozoa. In this study, we analyzed the presence and function of the TTX-resistant VGSC α subunit Nav1.8 in human capacitated sperm cells. Using an RT-PCR assay, we found that the mRNA of the gene SCN10A, that encode Na v1.8, was abundantly and specifically expressed in human testis and ejaculated spermatozoa. The Na v1.8 protein was detected in capacitated sperm cells using three different specific antibodies against this channel. Positive immunoreactivity was mainly located in the neck and the principal piece of the flagellum. The presence of Na v1.8 in sperm cells was confirmed by Western blot. Functional studies demonstrated that the increases in progressive motility produced by veratridine, a voltage-gated sodium channel activator, were reduced in sperm cells preincubated with TTX (10 μM), the Na v1.8 antagonist A-803467, or a specific Na v1.8 antibody. Veratridine elicited similar percentage increases in progressive motility in sperm cells maintained in Ca2+-containing or Ca2+-free solution and did not induce hyperactivation or the acrosome reaction. Veratridine caused a rise in sperm intracellular Na+, [Na+]i, and the sustained phase of the response was inhibited in the presence of A-803467. These results verify that the Na+ channel Na v1.8 is present in human sperm cells and demonstrate that this channel participates in the regulation of sperm function. PMID:24086692

  1. Recent developments regarding voltage-gated sodium channel blockers for the treatment of inherited and acquired neuropathic pain syndromes

    Directory of Open Access Journals (Sweden)

    Jonathan W. Theile

    2011-10-01

    Full Text Available Chronic and neuropathic pain constitute significant health problems affecting millions of individuals each year. Pain sensations typically originate in sensory neurons of the peripheral nervous system (PNS which relay information to the central nervous system (CNS. Pathological pain sensations can arise as result of changes in excitability of these peripheral sensory neurons. Voltage-gated sodium channels are key determinants regulating action-potential generation and propagation; thus, changes in sodium channel function can have profound effects on neuronal excitability and pain signaling. At present, most of the clinically available sodium channel blockers used to treat pain are non-selective across sodium channel isoforms and can contribute to cardio-toxicity, motor impairments and CNS side effects. Numerous strides have been made over the last decade in an effort to develop more selective and efficacious sodium channel blockers to treat pain. The purpose of this review is to highlight some of the more recent developments put forth by research universities and pharmaceutical companies alike in the pursuit of developing more targeted sodium channel therapies for the treatment of a variety of neuropathic pain conditions.

  2. Tracking voltage-dependent conformational changes in skeletal muscle sodium channel during activation.

    Science.gov (United States)

    Chanda, Baron; Bezanilla, Francisco

    2002-11-01

    The primary voltage sensor of the sodium channel is comprised of four positively charged S4 segments that mainly differ in the number of charged residues and are expected to contribute differentially to the gating process. To understand their kinetic and steady-state behavior, the fluorescence signals from the sites proximal to each of the four S4 segments of a rat skeletal muscle sodium channel were monitored simultaneously with either gating or ionic currents. At least one of the kinetic components of fluorescence from every S4 segment correlates with movement of gating charge. The fast kinetic component of fluorescence from sites S216C (S4 domain I), S660C (S4 domain II), and L1115C (S4 domain III) is comparable to the fast component of gating currents. In contrast, the fast component of fluorescence from the site S1436C (S4 domain IV) correlates with the slow component of gating. In all the cases, the slow component of fluorescence does not have any apparent correlation with charge movement. The fluorescence signals from sites reflecting the movement of S4s in the first three domains initiate simultaneously, whereas the fluorescence signals from the site S1436C exhibit a lag phase. These results suggest that the voltage-dependent movement of S4 domain IV is a later step in the activation sequence. Analysis of equilibrium and kinetic properties of fluorescence over activation voltage range indicate that S4 domain III is likely to move at most hyperpolarized potentials, whereas the S4s in domain I and domain II move at more depolarized potentials. The kinetics of fluorescence changes from sites near S4-DIV are slower than the activation time constants, suggesting that the voltage-dependent movement of S4-DIV may not be a prerequisite for channel opening. These experiments allow us to map structural features onto the kinetic landscape of a sodium channel during activation.

  3. Bisphenol A binds to the local anesthetic receptor site to block the human cardiac sodium channel.

    Directory of Open Access Journals (Sweden)

    Andrias O O'Reilly

    Full Text Available Bisphenol A (BPA has attracted considerable public attention as it leaches from plastic used in food containers, is detectable in human fluids and recent epidemiologic studies link BPA exposure with diseases including cardiovascular disorders. As heart-toxicity may derive from modified cardiac electrophysiology, we investigated the interaction between BPA and hNav1.5, the predominant voltage-gated sodium channel subtype expressed in the human heart. Electrophysiology studies of heterologously-expressed hNav1.5 determined that BPA blocks the channel with a K(d of 25.4±1.3 µM. By comparing the effects of BPA and the local anesthetic mexiletine on wild type hNav1.5 and the F1760A mutant, we demonstrate that both compounds share an overlapping binding site. With a key binding determinant thus identified, an homology model of hNav1.5 was generated based on the recently-reported crystal structure of the bacterial voltage-gated sodium channel NavAb. Docking predictions position both ligands in a cavity delimited by F1760 and contiguous with the DIII-IV pore fenestration. Steered molecular dynamics simulations used to assess routes of ligand ingress indicate that the DIII-IV pore fenestration is a viable access pathway. Therefore BPA block of the human heart sodium channel involves the local anesthetic receptor and both BPA and mexiletine may enter the closed-state pore via membrane-located side fenestrations.

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

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    Mohamed-Yassine eAMAROUCH

    2015-02-01

    Full Text Available 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 voltage-gated sodium channels. 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 voltage-gated sodium channels by shifting the voltage-dependence of steady state activation towards more negative potentials.

  5. [Inhibition of sodium channels in acutely isolated hippocampal neurons by scorpion venom heat resistant protein].

    Science.gov (United States)

    Zhang, Xiao-Yun; Wang, Yue; Zhang, Jian; Wang, Jing-Yu; Zhao, Jie; Zhang, Wan-Qin; Li, Shao

    2007-06-25

    The effects of scorpion venom heat resistant protein (SVHRP) on sodium channel were studied in freshly isolated hippocampal neurons in rat using the whole-cell patch-clamp technique. The results indicated that tetrodotoxin-sensitive voltage-dependent sodium current in hippocampal neurons was inhibited by SVHRP in a dose-dependent manner. The half-inhibition concentration (IC(50)) was (0.0034+/-0.0004) microg/mL, Hill constant (n) was 0.4361+/-0.0318. After SVHRP application, a clear shift of the activation curve of Na(+) channel was shown towards more depolarized potential, resulting in channel opening at more positive membrane potentials. In the presence of 0.1 mug/mL SVHRP, the voltage for half-activation (V(1/2)) and the slope factor of the activation curve were (-23.96+/-0.41) mV and 3.73+/-0.08 (n=8, P<0.05) compared with the control recordings of (-34.38+/-0.62) mV and 4.52+/-0.52 (n=16), respectively. Averaged and normalized curve of steady-state inactivation of Na(+) channel was shifted towards negative potential after treatment of 0.1 and 0.01 mug/mL SVHRP. In the presence of 0.1 mug/mL SVHRP, the voltage for half-inactivation (V(1/2)) and the slope factor determined by a sigmoid fit of the inactivation curve were (-50.69+/-2.55) mV (n=8, P<0.01) and 5.49+/-0.72 (n=8, P<0.05) compared with the control recordings of (-32.60+/-1.52) mV and 6.73+/-0.51 (n=16), respectively. These results suggest that SVHRP blocks the voltage-dependent sodium currents and alters the sodium channel kinetics to decrease the excitability of neurons. This might be an interpretation for the antiepileptic effects of SVHRP.

  6. Correlation of the electrophysiological profiles and sodium channel transcripts of individual rat dorsal root ganglia neurons

    Science.gov (United States)

    Thériault, Olivier; Chahine, Mohamed

    2014-01-01

    Voltage gated sodium channels (Nav channels) play an important role in nociceptive transmission. They are intimately tied to the genesis and transmission of neuronal firing. Five different isoforms (Nav1.3, Nav1.6, Nav1.7, Nav1.8, and Nav1.9) have been linked to nociceptive responses. A change in the biophysical properties of these channels or in their expression levels occurs in different pathological pain states. However, the precise involvement of the isoforms in the genesis and transmission of nociceptive responses is unknown. The aim of the present study was to investigate the synergy between the different populations of Nav channels that give individual neurons a unique electrophysical profile. We used the patch-clamp technique in the whole-cell configuration to record Nav currents and action potentials from acutely dissociated small diameter DRG neurons (<30 μm) from adult rats. We also performed single cell qPCR on the same neurons. Our results revealed that there is a strong correlation between Nav currents and mRNA transcripts in individual neurons. A cluster analysis showed that subgroups formed by Nav channel transcripts by mRNA quantification have different biophysical properties. In addition, the firing frequency of the neurons was not affected by the relative populations of Nav channel. The synergy between populations of Nav channel in individual small diameter DRG neurons gives each neuron a unique electrophysiological profile. The Nav channel remodeling that occurs in different pathological pain states may be responsible for the sensitization of the neurons. PMID:25285069

  7. Reporting sodium channel activity using calcium flux: pharmacological promiscuity of cardiac Nav1.5.

    Science.gov (United States)

    Zhang, Hongkang; Zou, Beiyan; Du, Fang; Xu, Kaiping; Li, Min

    2015-02-01

    Voltage-gated sodium (Nav) channels are essential for membrane excitability and represent therapeutic targets for treating human diseases. Recent reports suggest that these channels, e.g., Nav1.3 and Nav1.5, are inhibited by multiple structurally distinctive small molecule drugs. These studies give reason to wonder whether these drugs collectively target a single site or multiple sites in manifesting such pharmacological promiscuity. We thus investigate the pharmacological profile of Nav1.5 through systemic analysis of its sensitivity to diverse compound collections. Here, we report a dual-color fluorescent method that exploits a customized Nav1.5 [calcium permeable Nav channel, subtype 5 (SoCal5)] with engineered-enhanced calcium permeability. SoCal5 retains wild-type (WT) Nav1.5 pharmacological profiles. WT SoCal5 and SoCal5 with the local anesthetics binding site mutated (F1760A) could be expressed in separate cells, each with a different-colored genetically encoded calcium sensor, which allows a simultaneous report of compound activity and site dependence. The pharmacological profile of SoCal5 reveals a hit rate (>50% inhibition) of around 13% at 10 μM, comparable to that of hERG. The channel activity is susceptible to blockage by known drugs and structurally diverse compounds. The broad inhibition profile is highly dependent on the F1760 residue in the inner cavity, which is a residue conserved among all nine subtypes of Nav channels. Both promiscuity and dependence on F1760 seen in Nav1.5 were replicated in Nav1.4. Our evidence of a broad inhibition profile of Nav channels suggests a need to consider off-target effects on Nav channels. The site-dependent promiscuity forms a foundation to better understand Nav channels and compound interactions. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  8. Correlation of the electrophysiological profiles and sodium channel transcripts of individual rat dorsal root ganglia neurons

    Directory of Open Access Journals (Sweden)

    Olivier eTheriault

    2014-09-01

    Full Text Available Voltage gated sodium channels (Na+ channels play an important role in nociceptive transmission. They are intimately tied to the genesis and transmission of neuronal firing. Five different isoforms (Nav1.3, Nav1.6, Nav1.7, Nav1.8, and Nav1.9 have been linked to nociceptive responses. A change in the biophysical properties of these channels or in their expression levels occurs in different pathological pain states. However, the precise involvement of the isoforms in the genesis and transmission of nociceptive responses is unknown. The aim of the present study was to investigate the synergy between the different populations Na+ channels that give individual neurons a unique electrophysical profile.We used the patch-clamp technique in the whole-cell configuration to record Na+ currents and action potentials from acutely dissociated small diameter DRG neurons (<30 µM from adult rats. We also performed single cell qPCR on the same neurons. Our results revealed that there is a strong correlation between Na+ currents and mRNA transcripts in individual neurons. A cluster analysis showed that subgroups formed by Na+ channel transcripts by mRNA quantification have different biophysical properties. In addition, the firing frequency of the neurons was not affected by the relative populations of Na+ channel. The synergy between populations of Na+ channel in individual small diameter DRG neurons gives each neuron a unique electrophysiological profile. The Na+ channel remodeling that occurs in different pathological pain states may be responsible for the sensitization of the neurons.

  9. Mechanisms of regulation of epithelial sodium channel by SGK1 in A6 cells.

    Science.gov (United States)

    Alvarez de la Rosa, Diego; Paunescu, Teodor G; Els, Willem J; Helman, Sandy I; Canessa, Cecilia M

    2004-10-01

    The serum and glucocorticoid induced kinase 1 (SGK1) participates in the regulation of sodium reabsorption in the distal segment of the renal tubule, where it may modify the function of the epithelial sodium channel (ENaC). The molecular mechanism underlying SGK1 regulation of ENaC in renal epithelial cells remains controversial. We have addressed this issue in an A6 renal epithelial cell line that expresses SGK1 under the control of a tetracycline-inducible system. Expression of a constitutively active mutant of SGK1 (SGK1T(S425D)) induced a sixfold increase in amiloride-sensitive short-circuit current (Isc). Using noise analysis we demonstrate that SGK1 effect on Isc is due to a fourfold increase in the number of functional ENaCs in the membrane and a 43% increase in channel open probability. Impedance analysis indicated that SGK1T(S425D) increased the absolute value of cell equivalent capacitance by an average of 13.7%. SGK1T(S425D) also produced a 1.6-1.9-fold increase in total and plasma membrane subunit abundance, without changing the half-life of channels in the membrane. We conclude that in contrast to aldosterone, where stimulation of transport can be explained simply by an increase in channel synthesis, SGK1 effects are more complex and involve at least three actions: (1) increase of ENaC open probability; (2) increase of subunit abundance within apical membranes and intracellular compartments; and (3) activation of one or more pools of preexistent channels within the apical membranes and/or intracellular compartments.

  10. Rbfox proteins regulate alternative splicing of neuronal sodium channel SCN8A.

    Science.gov (United States)

    O'Brien, Janelle E; Drews, Valerie L; Jones, Julie M; Dugas, Jason C; Barres, Ben A; Meisler, Miriam H

    2012-02-01

    The SCN8A gene encodes the voltage-gated sodium channel Na(v)1.6, a major channel in neurons of the CNS and PNS. SCN8A contains two alternative exons,18N and 18A, that exhibit tissue specific splicing. In brain, the major SCN8A transcript contains exon 18A and encodes the full-length sodium channel. In other tissues, the major transcript contains exon 18N and encodes a truncated protein, due to the presence of an in-frame stop codon. Selection of exon 18A is therefore essential for generation of a functional channel protein, but the proteins involved in this selection have not been identified. Using a 2.6 kb Scn8a minigene containing exons 18N and 18A, we demonstrate that co-transfection with Fox-1 or Fox-2 initiates inclusion of exon 18A. This effect is dependent on the consensus Fox binding site located 28 bp downstream of exon 18A. We examined the alternative splicing of human SCN8A and found that the postnatal switch to exon 18A is completed later than 10 months of age. In purified cell populations, transcripts containing exon 18A predominate in neurons but are not present in oligodendrocytes or astrocytes. Transcripts containing exon 18N appear to be degraded by nonsense-mediated decay in HEK cells. Our data indicate that RBFOX proteins contribute to the cell-specific expression of Na(v)1.6 channels in mature neurons. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. Use dependence of peripheral nociceptive conduction in the absence of tetrodotoxin-resistant sodium channel subtypes.

    Science.gov (United States)

    Hoffmann, Tal; Kistner, Katrin; Nassar, Mohammed; Reeh, Peter W; Weidner, Christian

    2016-10-01

    This study examines conduction in peripheral nerves and its use dependence in tetrodotoxin-resistant (TTXr) sodium channel (Nav 1.8, Nav 1.9) knockout and wildtype animals. We observed use-dependent decreases of single fibre and compound action potential amplitude in peripheral mouse C-fibres (wildtype). This matches the previously published hypothesis that increased Na/K-pump activity is not the underlying mechanism for use-dependent changes of neural conduction. Knocking out TTXr sodium channels influences use-dependent changes of conductive properties (action potential amplitude, latency, conduction safety) in the order Nav 1.8 KO > Nav 1.9KO > wildtype. This is most likely explained by different subsets of presumably (relatively) Nav 1.7-rich conducting fibres in knockout animals as compared to wildtypes, in combination with reduced per-pulse sodium influx. Use dependency of peripheral nerves, especially of nociceptors, correlates with receptive properties. Slow inactivation of voltage-gated sodium channels has been discussed to be the underlying mechanism - pointing to a receptive class-related difference of sodium channel equipment. Using electrophysiological recordings of single unmyelinated cutaneous fibres and their compound action potential (AP), we evaluated use-dependent changes in mouse peripheral nerves, and the contribution of the tetrodotoxin-resistant (TTXr) sodium channels Nav 1.8 and Nav 1.9 to these changes. Nerve fibres were electrically stimulated using single or double pulses at 2 Hz. Use-dependent changes of latency, AP amplitude, and duration as well as the fibres' ability to follow the stimulus were evaluated. AP amplitudes substantially diminished in used fibres from C57BL/6 but increased in Nav 1.8 knockout (KO) mice, with Nav 1.9 KO in between. Activity-induced latency slowing was in contrast the most pronounced in Nav 1.8 KOs and the least in wildtype mice. The genotype was also predictive of how long fibres could follow the

  12. Inherited Cardiac Diseases Caused by Mutations in the Nav1.5 Sodium Channel

    DEFF Research Database (Denmark)

    Tfelt-Hansen, Jacob; Winkel, Bo Gregers; Grunnet, Morten

    2009-01-01

    Cardiac Diseases Caused by SCN5A Mutations. A prerequisite for a normal cardiac function is a proper generation and propagation of electrical impulses. Contraction of the heart is obtained through a delicate matched transmission of the electrical impulses. A pivotal element of the impulse...... propagation is the depolarizing sodium current, responsible for the initial depolarization of the cardiomyocytes. Recent research has shown that mutations in the SCN5A gene, encoding the cardiac sodium channel Nav1.5, are associated with both rare forms of ventricular arrhythmia, as well as the most frequent...... form of arrhythmia, atrial fibrillation (AF). In this comprehensive review, we describe the functional role of Nav1.5 and its associated proteins in propagation and depolarization both in a normal- and in a pathophysiological setting. Furthermore, several of the arrhythmogenic diseases, such as long...

  13. Molecular dissection of lubeluzole use-dependent block of voltage-gated sodium channels discloses new therapeutic potentials.

    Science.gov (United States)

    Desaphy, Jean-François; Carbonara, Roberta; Costanza, Teresa; Lentini, Giovanni; Cavalluzzi, Maria Maddalena; Bruno, Claudio; Franchini, Carlo; Camerino, Diana Conte

    2013-02-01

    Lubeluzole, which acts on various targets in vitro, including voltage-gated sodium channels, was initially proposed as a neuroprotectant. The lubeluzole structure contains a benzothiazole moiety [N-methyl-1,3-benzothiazole-2-amine (R-like)] related to riluzole and a phenoxy-propranol-amine moiety [(RS)-1-(3,4-difluorophenoxy)-3-(piperidin-1-yl)propan-2-ol (A-core)] recalling propranolol. Both riluzole and propranolol are efficient sodium channel blockers. We studied in detail the effects of lubeluzole (racemic mixture and single isomers), the aforementioned lubeluzole moieties, and riluzole on sodium channels to increase our knowledge of drug-channel molecular interactions. Compounds were tested on hNav1.4 sodium channels, and on F1586C or Y1593C mutants functionally expressed in human embryonic kidney 293 cells, using the patch-clamp technique. Lubeluzole blocked sodium channels with a remarkable effectiveness. No stereoselectivity was found. Compared with mexiletine, the dissociation constant for inactivated channels was ~600 times lower (~11 nM), conferring to lubeluzole a huge use-dependence of great therapeutic value. The F1586C mutation only partially impaired the use-dependent block, suggesting that additional amino acids are critically involved in high-affinity binding. Lubeluzole moieties were modest sodium channel blockers. Riluzole blocked sodium channels efficiently but lacked use dependence, similar to R-like. F1586C fully abolished A-core use dependence, suggesting that A-core binds to the local anesthetic receptor. Thus, lubeluzole likely binds to the local anesthetic receptor through its phenoxy-propranol-amine moiety, with consequent use-dependent behavior. Nevertheless, compared with other known sodium channel blockers, lubeluzole adds a third pharmacophoric point through its benzothiazole moiety, which greatly enhances high-affinity binding and use-dependent block. If sufficient isoform specificity can be attained, the huge use-dependent block

  14. Crystal Structure of the Eukaryotic Strong Inward-Rectifier K[superscript +] Channel Kir2.2 at 3.1 Å Resolution

    Energy Technology Data Exchange (ETDEWEB)

    Tao, Xiao; Avalos, Jose L.; Chen, Jiayun; MacKinnon, Roderick; (Rockefeller)

    2010-03-29

    Inward-rectifier potassium (K{sup +}) channels conduct K{sup +} ions most efficiently in one direction, into the cell. Kir2 channels control the resting membrane voltage in many electrically excitable cells, and heritable mutations cause periodic paralysis and cardiac arrhythmia. We present the crystal structure of Kir2.2 from chicken, which, excluding the unstructured amino and carboxyl termini, is 90% identical to human Kir2.2. Crystals containing rubidium (Rb{sup +}), strontium (Sr{sup 2+}), and europium (Eu{sup 3+}) reveal binding sites along the ion conduction pathway that are both conductive and inhibitory. The sites correlate with extensive electrophysiological data and provide a structural basis for understanding rectification. The channel's extracellular surface, with large structured turrets and an unusual selectivity filter entryway, might explain the relative insensitivity of eukaryotic inward rectifiers to toxins. These same surface features also suggest a possible approach to the development of inhibitory agents specific to each member of the inward-rectifier K{sup +} channel family.

  15. Behavioral changes and trigeminal ganglion sodium channel regulation in an orofacial neuropathic pain model.

    Science.gov (United States)

    Eriksson, Jonas; Jablonski, Aleksandra; Persson, Anna-Karin; Hao, Jing-Xia; Kouya, Poli Francois; Wiesenfeld-Hallin, Zsuzsanna; Xu, Xiao-Jun; Fried, Kaj

    2005-12-15

    We used a photochemical method to generate a partial ischemic injury to the infraorbital branch of the trigeminal nerve in rats. Following injury, rats developed a bilateral persistent hypersensitivity to mechanical stimulation in the territory innervated by the infraorbital nerve. In addition, spread of mechanical hypersensitivity beyond the facial region was noted. Heat hypersensitivity was also present, although to a lesser extent and of a shorter duration. In some rats, excessive facial grooming/scratching were observed. Morphological examination revealed a graded damage to the irradiated portion of the infraorbital nerve that was related to the duration of laser irradiation. Investigations of gene expression changes in injured trigeminal ganglion neurons of animals with behavioral signs of neuropathic pain demonstrated that the sodium channel alpha-subunit Na(v)1.3-absent in sham-operated animals-was expressed to a limited extent. mRNAs for Na(v)1.8 and Na(v)1.9 were reduced both with respect to proportions of expressing neurons and to intensities, whereas the beta 3 subunit was markedly upregulated. mRNA levels of p11, a regulatory factor that facilitates the surface expression of Na(v)1.8, were unchanged. Previous findings have shown that injury to the trigeminal nerve branches may elicit responses that differ from those of segmental spinal nerves. Despite this we conclude that the key sodium channel regulations that are reported as consequences of nerve damage in the dorsal root ganglia seem to appear also in the trigeminal ganglion. Thus, novel analgesic drugs designed to target the sodium channel subtypes involved could be of use for the treatment of orofacial pain.

  16. Sudden Infant Death Syndrome-Associated Mutations in the Sodium Channel Beta Subunits

    Science.gov (United States)

    Tan, Bi-Hua; Pundi, Kavitha N; Van Norstrand, David W; Valdivia, Carmen R; Tester, David J; Medeiros-Domingo, Argelia; Makielski, Jonathan C.; Ackerman, Michael J.

    2010-01-01

    Background Approximately 10% of sudden infant death syndrome (SIDS) may stem from potentially lethal cardiac channelopathies, with approximately half of channelopathic SIDS involving the NaV1.5 cardiac sodium channel. Recently, NaV beta subunits have been implicated in various cardiac arrhythmias. Thus, the four genes encoding NaV beta subunits represent plausible candidate genes for SIDS. Objective To determine the spectrum, prevalence and functional consequences of sodium channel beta subunit mutations in a SIDS cohort. Methods In this IRB-approved study, mutational analysis of the 4 beta subunit genes: SCN1B – 4B was performed using PCR, DHPLC, and direct DNA sequencing of DNA derived from 292 SIDS cases. Engineered mutations were co-expressed with SCN5A in HEK 293 cells, and whole cell patch clamped. One of the putative SIDS-associated mutations was similarly studied in adenovirally transduced adult rat ventricular myocytes. Results 3 rare (absent in 200–800 reference alleles) missense mutations (β3-V36M, β3-V54G and β4-S206L) were identified in 3/292 SIDS cases. Compared to SCN5A+β3-WT, β3-V36M significantly decreased peak INa and increased late INa while β3-V54G resulted in a marked loss-of-function. β4-S206L accentuated late INa and positively shifted the midpoint of inactivation compared to SCN5A+β4-WT. In native cardiomyocytes, β4-S206L accentuated late INa and increased the ventricular action potential duration (APD) compared to β4-WT. Conclusion This study provides the first molecular and functional evidence to implicate the NaV beta subunits in SIDS pathogenesis. Altered NaV1.5 sodium channel function due to beta subunit mutations may account for the molecular pathogenic mechanism underlying approximately 1% of SIDS. PMID:20226894

  17. Distribution of cardiac sodium channels in clusters potentiates ephaptic interactions in the intercalated disc.

    Science.gov (United States)

    Hichri, Echrak; Abriel, Hugues; Kucera, Jan P

    2017-12-06

    It has been proposed that ephaptic conduction, relying on interactions between the sodium (Na+ ) current and the extracellular potential in intercalated discs, might contribute to cardiac conduction when gap junctional coupling is reduced, but this mechanism is still controversial. In intercalated discs, Na+ channels form clusters near gap junction plaques, but the functional significance of these clusters has never been evaluated. In HEK cells expressing cardiac Na+ channels, we show that restricting the extracellular space modulates the Na+ current, as predicted by corresponding simulations accounting for ephaptic effects. In a high-resolution model of the intercalated disc, clusters of Na+ channels that face each other across the intercellular cleft facilitate ephaptic impulse transmission when gap junctional coupling is reduced. Thus, our simulations reveal a functional role for the clustering of Na+ channels in intercalated discs, and suggest that rearrangement of these clusters in disease may influence cardiac conduction. It has been proposed that ephaptic interactions in intercalated discs, mediated by extracellular potentials, contribute to cardiac impulse propagation when gap junctional coupling is reduced. However, experiments demonstrating ephaptic effects on the cardiac Na+ current (INa ) are scarce. Furthermore, Na+ channels form clusters around gap junction plaques, but the electrophysiological significance of these clusters has never been investigated. In patch clamp experiments with HEK cells stably expressing human Nav 1.5 channels, we examined how restricting the extracellular space modulates INa elicited by an activation protocol. In parallel, we developed a high-resolution computer model of the intercalated disc to investigate how the distribution of Na+ channels influences ephaptic interactions. Approaching the HEK cells to a non-conducting obstacle always increased peak INa at step potentials near the threshold of INa activation and decreased

  18. Increased renal sodium absorption by inhibition of prostaglandin synthesis during fasting in healthy man. A possible role of the epithelial sodium channels

    Directory of Open Access Journals (Sweden)

    Graffe Carolina C

    2010-10-01

    Full Text Available Abstract Background Treatment with prostaglandin inhibitors can reduce renal function and impair renal water and sodium excretion. We tested the hypotheses that a reduction in prostaglandin synthesis by ibuprofen treatment during fasting decreased renal water and sodium excretion by increased absorption of water and sodium via the aquaporin2 water channels and the epithelial sodium channels. Methods The effect of ibuprofen, 600 mg thrice daily, was measured during fasting in a randomized, placebo-controlled, double-blinded crossover study of 17 healthy humans. The subjects received a standardized diet on day 1, fasted at day 2, and received an IV infusion of 3% NaCl on day 3. The effect variables were urinary excretions of aquaporin2 (u-AQP2, the beta-fraction of the epithelial sodium channel (u-ENaCbeta, cyclic-AMP (u-cAMP, prostaglandin E2 (u-PGE2. Free water clearance (CH2O, fractional excretion of sodium (FENa, and plasma concentrations of vasopressin, angiotensin II, aldosterone, atrial-, and brain natriuretic peptide. Results Ibuprofen decreased u-AQP2, u-PGE2, and FENa at all parts of the study. During the same time, ibuprofen significantly increased u-ENaCbeta. Ibuprofen did not change the response in p-AVP, u-c-AMP, urinary output, and free water clearance during any of these periods. Atrial-and brain natriuretic peptide were higher. Conclusion During inhibition of prostaglandin synthesis, urinary sodium excretion decreased in parallel with an increase in sodium absorption and increase in u-ENaCbeta. U-AQP2 decreased indicating that water transport via AQP2 fell. The vasopressin-c-AMP-axis did not mediate this effect, but it may be a consequence of the changes in the natriuretic peptide system and/or the angiotensin-aldosterone system Trial Registration Clinical Trials Identifier: NCT00281762

  19. EXPRESS: Voltage-dependent sodium (NaV) channels in group IV sensory afferents.

    Science.gov (United States)

    Ramachandra, Renuka; Elmslie, Keith S

    2016-01-01

    Patients with intermittent claudication suffer from both muscle pain and an exacerbated exercise pressor reflex. Excitability of the group III and group IV afferent fibers mediating these functions is controlled in part by voltage-dependent sodium (NaV) channels. We previously found tetrodotoxin-resistant NaV1.8 channels to be the primary type in muscle afferent somata. However, action potentials in group III and IV afferent axons are blocked by TTX, supporting a minimal role of NaV1.8 channels. To address these apparent differences in NaV channel expression between axon and soma, we used immunohistochemistry to identify the NaV channels expressed in group IV axons within the gastrocnemius muscle and the dorsal root ganglia sections. Positive labeling by an antibody against the neurofilament protein peripherin was used to identify group IV neurons and axons. We show that >67% of group IV fibers express NaV1.8, NaV1.6, or NaV1.7. Interestingly, expression of NaV1.8 channels in group IV somata was significantly higher than in the fibers, whereas there were no significant differences for either NaV1.6 or NaV1.7. When combined with previous work, our results suggest that NaV1.8 channels are expressed in most group IV axons, but that, under normal conditions, NaV1.6 and/or NaV1.7 play a more important role in action potential generation to signal muscle pain and the exercise pressor reflex.

  20. Single-cell analysis of sodium channel expression in dorsal root ganglion neurons.

    Science.gov (United States)

    Ho, Cojen; O'Leary, Michael E

    2011-01-01

    Sensory neurons of the dorsal root ganglia (DRG) express multiple voltage-gated sodium (Na) channels that substantially differ in gating kinetics and pharmacology. Small-diameter (30 μm) predominately express fast TTX-S Na current. Na channel expression was further investigated using single-cell RT-PCR to measure the transcripts present in individually harvested DRG neurons. Consistent with cellular electrophysiology, the small neurons expressed transcripts encoding for both TTX-S (Nav1.1, Nav1.2, Nav1.6, and Nav1.7) and TTX-R (Nav1.8 and Nav1.9) Na channels. Nav1.7, Nav1.8 and Nav1.9 were the predominant Na channels expressed in the small neurons. The large neurons highly expressed TTX-S isoforms (Nav1.1, Nav1.6, and Nav1.7) while TTX-R channels were present at comparatively low levels. A unique subpopulation of the large neurons was identified that expressed TTX-R Na current and high levels of Nav1.8 transcript. DRG neurons also displayed substantial differences in the expression of neurofilaments (NF200, peripherin) and Necl-1, a neuronal adhesion molecule involved in myelination. The preferential expression of NF200 and Necl-1 suggests that large-diameter neurons give rise to thick myelinated axons. Small-diameter neurons expressed peripherin, but reduced levels of NF200 and Necl-1, a pattern more consistent with thin unmyelinated axons. Single-cell analysis of Na channel transcripts indicates that TTX-S and TTX-R Na channels are differentially expressed in large myelinated (Nav1.1, Nav1.6, and Nav1.7) and small unmyelinated (Nav1.7, Nav1.8, and Nav1.9) sensory neurons. Copyright © 2010 Elsevier Inc. All rights reserved.

  1. Functional Properties and Toxin Pharmacology of a Dorsal Root Ganglion Sodium Channel Viewed through its Voltage Sensors

    OpenAIRE

    Bosmans, Frank; Puopolo, Michelino; Martin-Eauclaire, Marie-France; Swartz, Kenton J.; Bean, Bruce Palmer

    2011-01-01

    The voltage-activated sodium (Nav) channel Nav1.9 is expressed in dorsal root ganglion (DRG) neurons where it is believed to play an important role in nociception. Progress in revealing the functional properties and pharmacological sensitivities of this non-canonical Nav channel has been slow because attempts to express this channel in a heterologous expression system have been unsuccessful. Here, we use a protein engineering approach to dissect the contributions of the four Nav1.9 voltage se...

  2. Sodium

    Science.gov (United States)

    Table salt is a combination of two minerals - sodium and chloride Your body needs some sodium to work properly. It helps with the function ... in your body. Your kidneys control how much sodium is in your body. If you have too ...

  3. NaV1.9: a sodium channel linked to human pain.

    Science.gov (United States)

    Dib-Hajj, Sulayman D; Black, Joel A; Waxman, Stephen G

    2015-09-01

    The voltage-gated sodium channel Na(V)1.9 is preferentially expressed in nociceptors and has been shown in rodent models to have a major role in inflammatory and neuropathic pain. These studies suggest that by selectively targeting Na(V)1.9, it might be possible to ameliorate pain without inducing adverse CNS side effects such as sedation, confusion and addictive potential. Three recent studies in humans--two genetic and functional studies in rare genetic disorders, and a third study showing a role for Na(V)1.9 in painful peripheral neuropathy--have demonstrated that Na(V)1.9 plays an important part both in regulating sensory neuron excitability and in pain signalling. With this human validation, attention is turning to this channel as a potential therapeutic target for pain.

  4. Molecular insights into the local anesthetic receptor within voltage-gated sodium channels using hydroxylated analogues of mexiletine

    Directory of Open Access Journals (Sweden)

    Jean-François eDesaphy

    2012-02-01

    Full Text Available We previously showed that the β-adrenoceptor modulators, clenbuterol and propranolol, directly blocked voltage-gated sodium channels, whereas salbutamol and nadolol did not (Desaphy et al., 2003, suggesting the presence of two hydroxyl groups on the aromatic moiety of the drugs as a molecular requisite for impeding sodium channel block. To verify such an hypothesis, we synthesized five new mexiletine analogues by adding one or two hydroxyl groups to the aryl moiety of the sodium channel blocker and tested these compounds on hNav1.4 channels expressed in HEK293 cells. Concentration-response relationships were constructed using an holding potential of -120 mV at 0.1 Hz (tonic block and 10 Hz (use-dependent block stimulation frequencies. The half-maximum inhibitory concentrations (IC50 were linearly correlated to drug lipophilicity: the less lipophilic the drug, minor was the block. The same compounds were also tested on F1586C and Y1593C hNav1.4 channel mutants, to gain further information on the molecular interactions of mexiletine with its receptor within the sodium channel pore. Alteration of tonic block suggests that the aryl moiety of mexiletine may interact either directly or indirectly with Phe1586 in the closed sodium channel to produce low-affinity binding block, and that this interaction depends on the electrostatic potential of the drug aromatic tail. Alteration of use-dependent block suggests that addition of hydroxyl groups to the aryl moiety may modify high-affinity binding of the drug ammine terminal to Phe1586 through cooperativity between the two pharmacophores, this effect being mainly related to drug lipophilicity. Mutation of Tyr1593 further impaired such cooperativity. In conclusion, these results confirm our former hypothesis showing that the presence of hydroxyl groups to the aryl moiety of mexiletine greatly reduced sodium channel block, and provide molecular insights into the intimate interaction of local anesthetics with

  5. T-type channels become highly permeable to sodium ions using an alternative extracellular turret region (S5-P) outside the selectivity filter.

    Science.gov (United States)

    Senatore, Adriano; Guan, Wendy; Boone, Adrienne N; Spafford, J David

    2014-04-25

    T-type (Cav3) channels are categorized as calcium channels, but invertebrate ones can be highly sodium-selective channels. We illustrate that the snail LCav3 T-type channel becomes highly sodium-permeable through exon splicing of an extracellular turret and descending helix in domain II of the four-domain Cav3 channel. Highly sodium-permeable T-type channels are generated without altering the invariant ring of charged residues in the selectivity filter that governs calcium selectivity in calcium channels. The highly sodium-permeant T-type channel expresses in the brain and is the only splice isoform expressed in the snail heart. This unique splicing of turret residues offers T-type channels a capacity to serve as a pacemaking sodium current in the primitive heart and brain in lieu of Nav1-type sodium channels and to substitute for voltage-gated sodium channels lacking in many invertebrates. T-type channels would also contribute substantially to sodium leak conductances at rest in invertebrates because of their large window currents.

  6. Actions of sea anemone type 1 neurotoxins on voltage-gated sodium channel isoforms.

    Science.gov (United States)

    Wanke, Enzo; Zaharenko, André Junqueira; Redaelli, Elisa; Schiavon, Emanuele

    2009-12-15

    most of the structural and electrophysiological studies were performed on type 1 sea anemone sodium channel toxins, we will present a comprehensive and updated review on the current understanding of the physiological actions of these Na channel modifiers.

  7. Mechanism of Ion Permeation in Mammalian Voltage-Gated Sodium Channels.

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    Somayeh Mahdavi

    Full Text Available Recent determination of the crystal structures of bacterial voltage-gated sodium (NaV channels have raised hopes that modeling of the mammalian counterparts could soon be achieved. However, there are substantial differences between the pore domains of the bacterial and mammalian NaV channels, which necessitates careful validation of mammalian homology models constructed from the bacterial NaV structures. Such a validated homology model for the NaV1.4 channel was constructed recently using the extensive mutagenesis data available for binding of μ-conotoxins. Here we use this NaV1.4 model to study the ion permeation mechanism in mammalian NaV channels. Linking of the DEKA residues in the selectivity filter with residues in the neighboring domains is found to be important for keeping the permeation pathway open. Molecular dynamics simulations and potential of mean force calculations reveal that there is a binding site for a Na+ ion just inside the DEKA locus, and 1-2 Na+ ions can occupy the vestibule near the EEDD ring. These sites are separated by a low free energy barrier, suggesting that inward conduction occurs when a Na+ ion in the vestibule goes over the free energy barrier and pushes the Na+ ion in the filter to the intracellular cavity, consistent with the classical knock-on mechanism. The NaV1.4 model also provides a good description of the observed Na+/K+ selectivity.

  8. Sensory functions for degenerin/epithelial sodium channels (DEG/ENaC).

    Science.gov (United States)

    Ben-Shahar, Yehuda

    2011-01-01

    All animals use a sophisticated array of receptor proteins to sense their external and internal environments. Major advances have been made in recent years in understanding the molecular and genetic bases for sensory transduction in diverse modalities, indicating that both metabotropic and ionotropic pathways are important in sensory functions. Here, I review the historical background and recent advances in understanding the roles of a relatively newly discovered family of receptors, the degenerin/epithelial sodium channels (DEG/ENaC). These animal-specific cation channels show a remarkable sequence and functional diversity in different species and seem to exert their functions in diverse sensory modalities. Functions for DEG/ENaC channels have been implicated in mechanosensation as well as chemosensory transduction pathways. In spite of overall sequence diversity, all family members share a unique protein topology that includes just two transmembrane domains and an unusually large and highly structured extracellular domain, that seem to be essential for both their mechanical and chemical sensory functions. This review will discuss many of the recent discoveries and controversies associated with sensory function of DEG/ENaC channels in both vertebrate and invertebrate model systems, covering the role of family members in taste, mechanosensation, and pain. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. Lysine and the Na+/K+ Selectivity in Mammalian Voltage-Gated Sodium Channels.

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    Yang Li

    Full Text Available Voltage-gated sodium (Nav channels are critical in the generation and transmission of neuronal signals in mammals. The crystal structures of several prokaryotic Nav channels determined in recent years inspire the mechanistic studies on their selection upon the permeable cations (especially between Na+ and K+ ions, a property that is proposed to be mainly determined by residues in the selectivity filter. However, the mechanism of cation selection in mammalian Nav channels lacks direct explanation at atomic level due to the difference in amino acid sequences between mammalian and prokaryotic Nav homologues, especially at the constriction site where the DEKA motif has been identified to determine the Na+/K+ selectivity in mammalian Nav channels but is completely absent in the prokaryotic counterparts. Among the DEKA residues, Lys is of the most importance since its mutation to Arg abolishes the Na+/K+ selectivity. In this work, we modeled the pore domain of mammalian Nav channels by mutating the four residues at the constriction site of a prokaryotic Nav channel (NavRh to DEKA, and then mechanistically investigated the contribution of Lys in cation selection using molecular dynamics simulations. The DERA mutant was generated as a comparison to understand the loss of ion selectivity caused by the K-to-R mutation. Simulations and free energy calculations on the mutants indicate that Lys facilitates Na+/K+ selection by electrostatically repelling the cation to a highly Na+-selective location sandwiched by the carboxylate groups of Asp and Glu at the constriction site. In contrast, the electrostatic repulsion is substantially weakened when Lys is mutated to Arg, because of two intrinsic properties of the Arg side chain: the planar geometric design and the sparse charge distribution of the guanidine group.

  10. Nav1.7 and other voltage-gated sodium channels as drug targets for pain relief.

    Science.gov (United States)

    Emery, Edward C; Luiz, Ana Paula; Wood, John N

    2016-08-01

    Chronic pain is a massive clinical problem. We discuss the potential of subtype selective sodium channel blockers that may provide analgesia with limited side effects. Sodium channel subtypes have been linked to human pain syndromes through genetic studies. Gain of function mutations in Nav1.7, 1.8 and 1.9 can cause pain, whilst loss of function Nav1.7 mutations lead to loss of pain in otherwise normal people. Intriguingly, both human and mouse Nav1.7 null mutants have increased opioid drive, because naloxone, an opioid antagonist, can reverse the analgesia associated with the loss of Nav1.7 expression. We believe there is a great future for sodium channel antagonists, particularly Nav1.7 antagonists in treating most pain syndromes. This review deals with recent attempts to develop specific sodium channel blockers, the mechanisms that underpin the Nav1.7 null pain-free phenotype and new routes to analgesia using, for example, gene therapy or combination therapy with subtype specific sodium channel blockers and opioids. The use of selective Nav1.7 antagonists together with either enkephalinase inhibitors or low dose opioids has the potential for side effect-free analgesia, as well as an important opioid sparing function that may be clinically very significant.

  11. Expression patterns, mutation detection and RNA interference of Rhopalosiphum padi voltage-gated sodium channel genes

    Science.gov (United States)

    Zuo, Yayun; Peng, Xiong; Wang, Kang; Lin, Fangfei; Li, Yuting; Chen, Maohua

    2016-07-01

    The voltage-gated sodium channel (VGSC) is the target of sodium-channel-blocking insecticides. Traditionally, animals were thought to have only one VGSC gene comprising a α-subunit with four homologous domains (DI-DIV). The present study showed that Rhopalosiphum padi, an economically important crop pest, owned a unique heterodimeric VGSC (H1 and H2 subunits) encoded by two genes (Rpvgsc1 and Rpvgsc2), which is unusual in insects and other animals. The open reading frame (ORF) of Rpvgsc1 consisted 1150 amino acids, and the ORF of Rpvgsc2 had 957 amino acids. Rpvgsc1 showed 64.1% amino acid identity to DI-DII of Drosophila melanogaster VGSC and Rpvgsc2 showed 64.0% amino acid identity to DIII-DIV of D. melanogaster VGSC. A M918L mutation previously reported in pyrethroids-resistant strains of other insects was found in the IIS4-S6 region of R. padi field sample. The two R. padi VGSC genes were expressed at all developmental stages and showed similar expression patterns after treatment with beta-cypermethrin. Knockdown of Rpvgsc1 or Rpvgsc2 caused significant reduction in mortality rate of R. padi after exposure to beta-cypermethrin. These findings suggest that the two R. padi VGSC genes are both functional genes.

  12. The cardiac sodium channel displays differential distribution in the conduction system and transmural heterogeneity in the murine ventricular myocardium.

    Science.gov (United States)

    Remme, C A; Verkerk, A O; Hoogaars, W M H; Aanhaanen, W T J; Scicluna, B P; Annink, C; van den Hoff, M J B; Wilde, A A M; van Veen, T A B; Veldkamp, M W; de Bakker, J M T; Christoffels, V M; Bezzina, C R

    2009-09-01

    Cardiac sodium channels are responsible for conduction in the normal and diseased heart. We aimed to investigate regional and transmural distribution of sodium channel expression and function in the myocardium. Sodium channel Scn5a mRNA and Na(v)1.5 protein distribution was investigated in adult and embryonic mouse heart through immunohistochemistry and in situ hybridization. Functional sodium channel availability in subepicardial and subendocardial myocytes was assessed using patch-clamp technique. Adult and embryonic (ED14.5) mouse heart sections showed low expression of Na(v)1.5 in the HCN4-positive sinoatrial and atrioventricular nodes. In contrast, high expression levels of Na(v)1.5 were observed in the HCN4-positive and Cx43-negative AV or His bundle, bundle branches and Purkinje fibers. In both ventricles, a transmural gradient was observed, with a low Na(v)1.5 labeling intensity in the subepicardium as compared to the subendocardium. Similar Scn5a mRNA expression patterns were observed on in situ hybridization of embryonic and adult tissue. Maximal action potential upstroke velocity was significantly lower in subepicardial myocytes (mean +/- SEM 309 +/- 32 V/s; n = 14) compared to subendocardial myocytes (394 +/- 32 V/s; n = 11; P channel availability in subepicardium compared to subendocardium. Scn5a and Na(v)1.5 show heterogeneous distribution patterns within the cardiac conduction system and across the ventricular wall. This differential distribution of the cardiac sodium channel may have profound consequences for conduction disease phenotypes and arrhythmogenesis in the setting of sodium channel disease.

  13. Modulation of the cardiac sodium channel Nav1.5 by fibroblast growth factor homologous factor 1B.

    Science.gov (United States)

    Liu, Chuan-ju; Dib-Hajj, Sulayman D; Renganathan, Muthukrishnan; Cummins, Theodore R; Waxman, Stephen G

    2003-01-10

    We have previously shown that fibroblast growth factor homologous factor 1B (FHF1B), a cytosolic member of the fibroblast growth factor family, associates with the sensory neuron-specific channel Na(v)1.9 but not with the other sodium channels present in adult rat dorsal root ganglia neurons. We show in this study that FHF1B binds to the C terminus of the cardiac voltage-gated sodium channel Na(v)1.5 and modulates the properties of the channel. The N-terminal 41 amino acid residues of FHF1B are essential for binding to Na(v)1.5, and the conserved acidic rich domain (amino acids 1773-1832) in the C terminus of Na(v)1.5 is sufficient for association with this factor. Binding of the growth factor to recombinant wild type human Na(v)1.5 in human embryonic kidney 293 cells produces a significant hyperpolarizing shift in the voltage dependence of channel inactivation. An aspartic acid to glycine substitution at position 1790 of the channel, which underlies one of the LQT-3 phenotypes of cardiac arrythmias, abolishes the interaction of the Na(v)1.5 channel with FHF1B. This is the first report showing that interaction with a growth factor can modulate properties of a voltage-gated sodium channel.

  14. A novel tarantula toxin stabilizes the deactivated voltage sensor of bacterial sodium channel.

    Science.gov (United States)

    Tang, Cheng; Zhou, Xi; Nguyen, Phuong Tran; Zhang, Yunxiao; Hu, Zhaotun; Zhang, Changxin; Yarov-Yarovoy, Vladimir; DeCaen, Paul G; Liang, Songping; Liu, Zhonghua

    2017-07-01

    Voltage-gated sodium channels (Na V s) are activated by transiting the voltage sensor from the deactivated to the activated state. The crystal structures of several bacterial Na V s have captured the voltage sensor module (VSM) in an activated state, but structure of the deactivated voltage sensor remains elusive. In this study, we sought to identify peptide toxins stabilizing the deactivated VSM of bacterial Na V s. We screened fractions from several venoms and characterized a cystine knot toxin called JZTx-27 from the venom of tarantula Chilobrachys jingzhao as a high-affinity antagonist of the prokaryotic Na V s Ns V Ba (nonselective voltage-gated Bacillus alcalophilus ) and NaChBac (bacterial sodium channel from Bacillus halodurans ) (IC 50 = 112 nM and 30 nM, respectively). JZTx-27 was more efficacious at weaker depolarizing voltages and significantly slowed the activation but accelerated the deactivation of Ns V Ba, whereas the local anesthetic drug lidocaine was shown to antagonize Ns V Ba without affecting channel gating. Mutation analysis confirmed that JZTx-27 bound to S3-4 linker of Ns V Ba, with F98 being the critical residue in determining toxin affinity. All electrophysiological data and in silico analysis suggested that JZTx-27 trapped VSM of Ns V Ba in one of the deactivated states. In mammalian Na V s, JZTx-27 preferably inhibited the inactivation of Na V 1.5 by targeting the fourth transmembrane domain. To our knowledge, this is the first report of peptide antagonist for prokaryotic Na V s. More important, we proposed that JZTx-27 stabilized the Ns V Ba VSM in the deactivated state and may be used as a probe to determine the structure of the deactivated VSM of Na V s.-Tang, C., Zhou, X., Nguyen, P. T., Zhang, Y., Hu, Z., Zhang, C., Yarov-Yarovoy, V., DeCaen, P. G., Liang, S., Liu, Z. A novel tarantula toxin stabilizes the deactivated voltage sensor of bacterial sodium channel. © FASEB.

  15. Epithelial Sodium Channel-Mediated Sodium Transport Is Not Dependent on the Membrane-Bound Serine Protease CAP2/Tmprss4.

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    Anna Keppner

    Full Text Available The membrane-bound serine protease CAP2/Tmprss4 has been previously identified in vitro as a positive regulator of the epithelial sodium channel (ENaC. To study its in vivo implication in ENaC-mediated sodium absorption, we generated a knockout mouse model for CAP2/Tmprss4. Mice deficient in CAP2/Tmprss4 were viable, fertile, and did not show any obvious histological abnormalities. Unexpectedly, when challenged with sodium-deficient diet, these mice did not develop any impairment in renal sodium handling as evidenced by normal plasma and urinary sodium and potassium electrolytes, as well as normal aldosterone levels. Despite minor alterations in ENaC mRNA expression, we found no evidence for altered proteolytic cleavage of ENaC subunits. In consequence, ENaC activity, as monitored by the amiloride-sensitive rectal potential difference (ΔPD, was not altered even under dietary sodium restriction. In summary, ENaC-mediated sodium balance is not affected by lack of CAP2/Tmprss4 expression and thus, does not seem to directly control ENaC expression and activity in vivo.

  16. Sodium channel Nav1.7 immunoreactivity in painful human dental pulp and burning mouth syndrome

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    Yiangou Yiangos

    2010-06-01

    Full Text Available Abstract Background Voltage gated sodium channels Nav1.7 are involved in nociceptor nerve action potentials and are known to affect pain sensitivity in clinical genetic disorders. Aims and Objectives To study Nav1.7 levels in dental pulpitis pain, an inflammatory condition, and burning mouth syndrome (BMS, considered a neuropathic orofacial pain disorder. Methods Two groups of patients were recruited for this study. One group consisted of patients with dental pulpitis pain (n = 5 and controls (n = 12, and the other patients with BMS (n = 7 and controls (n = 10. BMS patients were diagnosed according to the International Association for the Study of Pain criteria; a pain history was collected, including the visual analogue scale (VAS. Immunohistochemistry with visual intensity and computer image analysis were used to evaluate levels of Nav1.7 in dental pulp tissue samples from the dental pulpitis group, and tongue biopsies from the BMS group. Results There was a significantly increased visual intensity score for Nav1.7 in nerve fibres in the painful dental pulp specimens, compared to controls. Image analysis showed a trend for an increase of the Nav1.7 immunoreactive % area in the painful pulp group, but this was not statistically significant. When expressed as a ratio of the neurofilament % area, there was a strong trend for an increase of Nav1.7 in the painful pulp group. Nav1.7 immunoreactive fibres were seen in abundance in the sub-mucosal layer of tongue biopsies, with no significant difference between BMS and controls. Conclusion Nav1.7 sodium channel may play a significant role in inflammatory dental pain. Clinical trials with selective Nav1.7 channel blockers should prioritise dental pulp pain rather than BMS.

  17. Inhibitory effect of the recombinant Phoneutria nigriventer Tx1 toxin on voltage-gated sodium channels.

    Science.gov (United States)

    Silva, Anita O; Peigneur, Steve; Diniz, Marcelo R V; Tytgat, Jan; Beirão, Paulo S L

    2012-12-01

    Phoneutria nigriventer toxin Tx1 (PnTx1, also referred to in the literature as Tx1) exerts inhibitory effect on neuronal (Na(V)1.2) sodium channels in a way dependent on the holding potential, and competes with μ-conotoxins but not with tetrodotoxin for their binding sites. In the present study we investigated the electrophysiological properties of the recombinant toxin (rPnTx1), which has the complete amino acid sequence of the natural toxin with 3 additional residues: AM on the N-terminal and G on the C-terminal. At the concentration of 1.5 μM, the recombinant toxin inhibits Na(+) currents of dorsal root ganglia neurons (38.4 ± 6.1% inhibition at -80 mV holding potential) and tetrodotoxin-resistant Na(+) currents (26.2 ± 4.9% at the same holding potential). At -50 mV holding potential the inhibition of the total current reached 71.3 ± 2.3% with 1.5 μM rPnTx1. The selectivity of rPnTx1 was investigated on ten different isoforms of voltage-gated sodium channels expressed in Xenopus oocytes. The order of potency for rPnTx1 was: rNa(V)1.2 > rNa(V)1.7 ≈ rNa(V)1.4 ≥ rNa(V)1.3 > mNa(V)1.6 ≥ hNa(V)1.8. No effect was seen on hNa(V)1.5 and on the arthropods isoforms (DmNa(V)1, BGNa(V)1.1a and VdNa(V)1). The IC(50) for Na(V)1.2 was 33.7 ± 2.9 nM with a maximum inhibition of 83.3 ± 1.9%. The toxin did not alter the voltage-dependence of channel gating and was effective on Na(V)1.2 channels devoid of inactivation. It was ineffective on neuronal calcium channels. We conclude that rPnTx1 has a promising selectivity, and that it may be a valuable model to achieve pharmacological activities of interest for the treatment of channelopathies and neuropathic pain. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  18. Inverse Association between Sodium Channel-Blocking Antiepileptic Drug Use and Cancer: Data Mining of Spontaneous Reporting and Claims Databases.

    Science.gov (United States)

    Takada, Mitsutaka; Fujimoto, Mai; Motomura, Haruka; Hosomi, Kouichi

    2016-01-01

    Voltage-gated sodium channels (VGSCs) are drug targets for the treatment of epilepsy. Recently, a decreased risk of cancer associated with sodium channel-blocking antiepileptic drugs (AEDs) has become a research focus of interest. The purpose of this study was to test the hypothesis that the use of sodium channel-blocking AEDs are inversely associated with cancer, using different methodologies, algorithms, and databases. A total of 65,146,507 drug-reaction pairs from the first quarter of 2004 through the end of 2013 were downloaded from the US Food and Drug Administration Adverse Event Reporting System. The reporting odds ratio (ROR) and information component (IC) were used to detect an inverse association between AEDs and cancer. Upper limits of the 95% confidence interval (CI) of cancer over the period of January 2005 to May 2014. The upper limit of the 95% CI of adjusted sequence ratio (ASR) cancers. In the claims database analyses, sodium channel-blocking AED use was inversely associated with diagnoses of colorectal cancer, lung cancer, gastric cancer, and hematological malignancies, with ASRs of 0.72 (95% CI: 0.60 - 0.86), 0.65 (0.51 - 0.81), 0.80 (0.65 - 0.98), and 0.50 (0.37 - 0.66), respectively. Positive associations between sodium channel-blocking AEDs and cancer were not found in the study. Multi-methodological approaches using different methodologies, algorithms, and databases suggest that sodium channel-blocking AED use is inversely associated with colorectal cancer, lung cancer, gastric cancer, and hematological malignancies.

  19. Altered expression of sodium channel distribution in the dorsal root ganglion after gradual elongation of rat sciatic nerves.

    Science.gov (United States)

    Ohno, Katsunori; Yokota, Atsushi; Hirofuji, Shinji; Kanbara, Kiyoto; Ohtsuka, Hisashi; Kinoshita, Mitsuo

    2010-04-01

    To elucidate the pathophysiological mechanisms underlying chronic nerve-stretch injury, we gradually lengthened rat femurs by 15 mm at the rate of 0.5 mm/day (group L, n = 13). The control groups comprised sham-operated (group S, n = 10) and naive (group N, n = 8) rats. Immediately after the lengthening, we performed a conduction study on their sciatic nerves and harvested samples. Electrophysiological and histological analyses showed mild conduction slowing and axonal degeneration of unmyelinated fibers in group L rats. Altered mRNA expression of the voltage-gated sodium channels in the dorsal root ganglion was also observed. Tetrodotoxin-resistant (TTX-R) sodium-channel Nav1.8 mRNA expression was significantly decreased and TTX-R sodium-channel Nav1.9 mRNA expression showed a tendency to decrease when compared with the mRNA expressions in the control groups. However, tetrodotoxin-sensitive (TTX-S) sodium-channel Nav1.3 mRNA expression remained unaltered. The immunohistochemical alteration of Nav1.8 protein expression was parallel to the results of the mRNA expression. Previous studies involving neuropathic states have suggested that pain/paresthesia is modulated by a subset of sodium channels, including downregulation and/or upregulation of TTX-R and TTX-S sodium channels, respectively. Our findings indicate that Nav1.8 downregulation may be one of the pathophysiological mechanisms involved in limb lengthening-induced neuropathy.

  20. Trafficking regulates the subcellular distribution of voltage-gated sodium channels in primary sensory neurons.

    Science.gov (United States)

    Bao, Lan

    2015-09-30

    Voltage-gated sodium channels (Navs) comprise at least nine pore-forming α subunits. Of these, Nav1.6, Nav1.7, Nav1.8 and Nav1.9 are the most frequently studied in primary sensory neurons located in the dorsal root ganglion and are mainly localized to the cytoplasm. A large pool of intracellular Navs raises the possibility that changes in Nav trafficking could alter channel function. The molecular mediators of Nav trafficking mainly consist of signals within the Navs themselves, interacting proteins and extracellular factors. The surface expression of Navs is achieved by escape from the endoplasmic reticulum and proteasome degradation, forward trafficking and plasma membrane anchoring, and it is also regulated by channel phosphorylation and ubiquitination in primary sensory neurons. Axonal transport and localization of Navs in afferent fibers involves the motor protein KIF5B and scaffold proteins, including contactin and PDZ domain containing 2. Localization of Nav1.6 to the nodes of Ranvier in myelinated fibers of primary sensory neurons requires node formation and the submembrane cytoskeletal protein complex. These findings inform our understanding of the molecular and cellular mechanisms underlying Nav trafficking in primary sensory neurons.

  1. Venus Flytrap HKT1-Type Channel Provides for Prey Sodium Uptake into Carnivorous Plant Without Conflicting with Electrical Excitability.

    Science.gov (United States)

    Böhm, J; Scherzer, S; Shabala, S; Krol, E; Neher, E; Mueller, T D; Hedrich, R

    2016-03-07

    The animal diet of the carnivorous Venus flytrap, Dionaea muscipula, contains a sodium load that enters the capture organ via an HKT1-type sodium channel, expressed in special epithelia cells on the inner trap lobe surface. DmHKT1 expression and sodium uptake activity is induced upon prey contact. Here, we analyzed the HKT1 properties required for prey sodium osmolyte management of carnivorous Dionaea. Analyses were based on homology modeling, generation of model-derived point mutants, and their functional testing in Xenopus oocytes. We showed that the wild-type HKT1 and its Na(+)- and K(+)-permeable mutants function as ion channels rather than K(+) transporters driven by proton or sodium gradients. These structural and biophysical features of a high-capacity, Na(+)-selective ion channel enable Dionaea glands to manage prey-derived sodium loads without confounding the action potential-based information management of the flytrap. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Guanidinium Toxins and Their Interactions with Voltage-Gated Sodium Ion Channels

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    Lorena M. Durán-Riveroll

    2017-10-01

    Full Text Available Guanidinium toxins, such as saxitoxin (STX, tetrodotoxin (TTX and their analogs, are naturally occurring alkaloids with divergent evolutionary origins and biogeographical distribution, but which share the common chemical feature of guanidinium moieties. These guanidinium groups confer high biological activity with high affinity and ion flux blockage capacity for voltage-gated sodium channels (NaV. Members of the STX group, known collectively as paralytic shellfish toxins (PSTs, are produced among three genera of marine dinoflagellates and about a dozen genera of primarily freshwater or brackish water cyanobacteria. In contrast, toxins of the TTX group occur mainly in macrozoa, particularly among puffer fish, several species of marine invertebrates and a few terrestrial amphibians. In the case of TTX and analogs, most evidence suggests that symbiotic bacteria are the origin of the toxins, although endogenous biosynthesis independent from bacteria has not been excluded. The evolutionary origin of the biosynthetic genes for STX and analogs in dinoflagellates and cyanobacteria remains elusive. These highly potent molecules have been the subject of intensive research since the latter half of the past century; first to study the mode of action of their toxigenicity, and later as tools to characterize the role and structure of NaV channels, and finally as therapeutics. Their pharmacological activities have provided encouragement for their use as therapeutants for ion channel-related pathologies, such as pain control. The functional role in aquatic and terrestrial ecosystems for both groups of toxins is unproven, although plausible mechanisms of ion channel regulation and chemical defense are often invoked. Molecular approaches and the development of improved detection methods will yield deeper understanding of their physiological and ecological roles. This knowledge will facilitate their further biotechnological exploitation and point the way towards

  3. The pharmacology of voltage-gated sodium channels in sensory neurones.

    Science.gov (United States)

    Docherty, Reginald J; Farmer, Clare E

    2009-01-01

    Voltage-gated sodium channels (VGSCs) are vital for the normal functioning of most excitable cells. At least nine distinct functional subtypes of VGSCs are recognized, corresponding to nine genes for their pore-forming alpha-subunits. These have different developmental expression patterns, different tissue distributions in the adult and are differentially regulated at the cellular level by receptor-coupled cell signalling systems. Unsurprisingly, VGSC blockers are found to be useful as drugs in diverse clinical applications where excessive excitability of tissue leads to pathological dysfunction, e.g. epilepsy or cardiac tachyarrhythmias. The effects of most clinically useful VGSC blockers are use-dependent, i.e. their efficacy depends on channel activity. In addition, many natural toxins have been discovered that interact with VGSCs in complex ways and they have been used as experimental probes to study the structure and function of the channels and to better understand how drugs interact with the channels. Here we have attempted to summarize the properties of VGSCs in sensory neurones, discuss how they are regulated by cell signalling systems and we have considered briefly current concepts of their physiological function. We discuss in detail how drugs and toxins interact with archetypal VGSCs and where possible consider how they act on VGSCs in peripheral sensory neurones. Increasingly, drugs that block VGSCs are being used as systemic analgesic agents in chronic pain syndromes, but the full potential for VGSC blockers in this indication is yet to be realized and other applications in sensory dysfunction are also possible. Drugs targeting VGSC subtypes in sensory neurones are likely to provide novel systemic analgesics that are tissue-specific and perhaps even disease-specific, providing much-needed novel therapeutic approaches for the relief of chronic pain.

  4. Genomic organization of the human SCN5A gene encoding the cardiac sodium channel

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    Wang, Qing; Li, Zhizhong; Shen, Jiaxiang; Keating, M.T. [Univ. of Utah Health Sciences Center, Salt Lake City, UT (United States)

    1996-05-15

    The voltage-gated cardiac sodium channel, SCN5A, is responsible for the initial upstroke of the action potential. Mutations in the human SCN5A gene cause susceptibility to cardiac arrhythmias and sudden death in the long QT syndrome (LQT). In this report we characterize the genomic structure of SCN5A. SCN5A consists of 28 exons spanning approximately 80 kb on chromosome 3p21. We describe the sequences of all intron/exon boundaries and a dinucleotide repeat polymorphism in intron 16. Oligonucleotide primers based on exon-flanking sequences amplify all SCN5A exons by PCR. This work establishes the complete genomic organization of SCN5A and will enable high-resolution analyses of this locus for mutations associated with LQT and other phenotypes for which SCN5A may be a candidate gene. 40 refs., 4 figs., 2 tabs.

  5. Multiple mutations and mutation combinations in the sodium channel of permethrin resistant mosquitoes, Culex quinquefasciatus

    Science.gov (United States)

    Li, Ting; Zhang, Lee; Reid, William R.; Xu, Qiang; Dong, Ke; Liu, Nannan

    2012-10-01

    A previous study identified 3 nonsynonymous and 6 synonymous mutations in the entire mosquito sodium channel of Culex quinquefasciatus, the prevalence of which were strongly correlated with levels of resistance and increased dramatically following insecticide selection. However, it is unclear whether this is unique to this specific resistant population or is a common mechanism in field mosquito populations in response to insecticide pressure. The current study therefore further characterized these mutations and their combinations in other field and permethrin selected Culex mosquitoes, finding that the co-existence of all 9 mutations was indeed correlated with the high levels of permethrin resistance in mosquitoes. Comparison of mutation combinations revealed several common mutation combinations presented across different field and permethrin selected populations in response to high levels of insecticide resistance, demonstrating that the co-existence of multiple mutations is a common event in response to insecticide resistance across different Cx. quinquefasciatus mosquito populations.

  6. Malignant hyperthermia-like reaction in a family with a sodium channel mutation at residue 1306

    Energy Technology Data Exchange (ETDEWEB)

    Vita, G.M.; Jedlicka, A.E.; Levitt, R.C. [Johns Hopkins Medical Institute, Baltimore, MD (United States)] [and others

    1994-09-01

    Malignant hyperthermia susceptibility (MHS) is an autosomal dominant, hypermetabolic disorder, triggered by potent inhalational anesthetics. We have previously suggeste the skeletal muscle sodium channel {alpha}-subunit (SCN4A) as a gene candidate to explain some forms of MHS. To evaluate this gene for mutations that might lead to a MHS-like episode, we amplified genomic DNA by PCR and used SSCP to screen each exon. We studied multiple MHS families which may be linked to this gene. The proband and a sibling from one of these families suspected of having MHS experienced trismus and body rigidity after induction of anesthesia. The caffiene and halothane contracture test proved diagnostic in these individuals and EMG studies suggested a form of myotomia. A mutation co-segregating with the myotonia/MHS phenotype was found in the region of exon 22.

  7. Proteolytic activation of the epithelial sodium channel ENaC in preeclampsia examined with urinary exosomes

    DEFF Research Database (Denmark)

    Nielsen, Maria Ravn; Rytz, Mie; Frederiksen-Møller, Britta

    2015-01-01

    OBJECTIVES: Increased activity of the epithelial sodium channel (ENaC) in the kidneys may explain the coupling between proteinuria, edema, suppressed aldosterone and hypertension in preeclampsia. Preeclamptic women excrete plasminogen-plasmin in urine. In vitro, plasmin increases the activity...... as a positive control for the presence of collecting duct membrane. RESULTS: Urine plasmin-plasminogen/creatinine ratio was increased in the preeclampsia group (p... pregnancy and preeclampsia CONCLUSIONS: It is possible to examine collecting duct transport proteins in urine exosome from pregnant women including γ-ENaC, 2) Urine exosome fraction displays a variable pattern of γ-ENaC signal with a predominance of cleaved forms in both normal and preeclamptic women...

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

    Science.gov (United States)

    Patel, Reesha R; Barbosa, Cindy; Brustovetsky, Tatiana; Brustovetsky, Nickolay; Cummins, Theodore R

    2016-08-01

    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 Navβ4 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

  9. Biophysical and Pharmacological Characterization of Nav1.9 Voltage Dependent Sodium Channels Stably Expressed in HEK-293 Cells

    OpenAIRE

    Zhixin Lin; Sonia Santos; Karen Padilla; David Printzenhoff; Castle, Neil A.

    2016-01-01

    The voltage dependent sodium channel Nav1.9, is expressed preferentially in peripheral sensory neurons and has been linked to human genetic pain disorders, which makes it target of interest for the development of new pain therapeutics. However, characterization of Nav1.9 pharmacology has been limited due in part to the historical difficulty of functionally expressing recombinant channels. Here we report the successful generation and characterization of human, mouse and rat Nav1.9 stably expre...

  10. COMMD1 regulates the delta epithelial sodium channel ({delta}ENaC) through trafficking and ubiquitination

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Tina; Ke, Ying; Ly, Kevin [Department of Physiology, University of Otago, P.O. Box 913, Dunedin 9054 (New Zealand); McDonald, Fiona J., E-mail: fiona.mcdonald@otago.ac.nz [Department of Physiology, University of Otago, P.O. Box 913, Dunedin 9054 (New Zealand)

    2011-08-05

    Highlights: {yields} The COMM domain of COMMD1 mediates binding to {delta}ENaC. {yields} COMMD1 reduces the cell surface population of {delta}ENaC. {yields} COMMD1 increases the population of {delta}ENaC-ubiquitin. {yields} Both endogenous and transfected {delta}ENaC localize with COMMD1 and transferrin suggesting they are located in early/recycling endosomes. -- Abstract: The delta subunit of the epithelial sodium channel ({delta}ENaC) is a member of the ENaC/degenerin family of ion channels. {delta}ENaC is distinct from the related {alpha}-, {beta}- and {gamma}ENaC subunits, known for their role in sodium homeostasis and blood pressure control, as {delta}ENaC is expressed in brain neurons and activated by external protons. COMMD1 (copper metabolism Murr1 domain 1) was previously found to associate with and downregulate {delta}ENaC activity. Here, we show that COMMD1 interacts with {delta}ENaC through its COMM domain. Co-expression of {delta}ENaC with COMMD1 significantly reduced {delta}ENaC surface expression, and led to an increase in {delta}ENaC ubiquitination. Immunocytochemical and confocal microscopy studies show that COMMD1 promoted localization of {delta}ENaC to the early/recycling endosomal pool where the two proteins were localized together. These results suggest that COMMD1 downregulates {delta}ENaC activity by reducing {delta}ENaC surface expression through promoting internalization of surface {delta}ENaC to an intracellular recycling pool, possibly via enhanced ubiquitination.

  11. Cardiac sodium channel antagonism - Translation of preclinical in vitro assays to clinical QRS prolongation.

    Science.gov (United States)

    Jenkinson, Stephen; Bassyouni, Asser; Cordes, Jason; Fermini, Bernard; Guo, Donglin; Potter, David M; Ramirez, David S; Steidl-Nichols, Jill; Sun, Sunny; Wisialowski, Todd

    Cardiac sodium channel antagonists have historically been used to treat cardiac arrhythmias by preventing the reentry of the electrical impulse that could occur following myocardial damage. However, clinical studies have highlighted a significant increase in mortality associated with such treatment. Cardiac sodium channel antagonist activity is now seen as an off-target pharmacology that should be mitigated during the drug development process. The aim of this study was to examine the correlation between in vitro/ex vivo assays that are routinely used to measure Nav1.5 activity and determine the translatability of the individual assays to QRS prolongation in the clinic. A set of clinical compounds with known Nav1.5 activity was profiled in several in vitro/ex vivo assays (binding, membrane potential, patch clamp and the Langendorff isolated heart). Clinical data comprising compound exposure levels and changes in QRS interval were obtained from the literature. Sensitivity/specificity analysis was performed with respect to the clinical outcome. The in vitro assays showed utility in predicting QRS prolongation in the clinic. Optimal thresholds were defined for each assay (binding: IC 20 ; membrane potential: IC 10 ; patch clamp: IC 20 ) and sensitivity (69-88%) and specificity (53-84%) values were shown to be similar between assay formats. The data provide clear statistical insight into the translatability of Nav1.5 antagonism data generated in vitro to potential clinical outcomes. These results improve our ability to understand the liability posed by such activity in novel development compounds at an early stage. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Modulation of the Epithelial Sodium Channel (ENaC) by Bacterial Metalloproteases and Protease Inhibitors

    Science.gov (United States)

    Butterworth, Michael B.; Zhang, Liang; Liu, Xiaoning; Shanks, Robert M.; Thibodeau, Patrick H.

    2014-01-01

    The serralysin family of metalloproteases is associated with the virulence of multiple gram-negative human pathogens, including Pseudomonas aeruginosa and Serratia marcescens. The serralysin proteases share highly conserved catalytic domains and show evolutionary similarity to the mammalian matrix metalloproteases. Our previous studies demonstrated that alkaline protease (AP) from Pseudomonas aeruginosa is capable of activating the epithelial sodium channel (ENaC), leading to an increase in sodium absorption in airway epithelia. The serralysin proteases are often co-expressed with endogenous, intracellular or periplasmic inhibitors, which putatively protect the bacterium from unwanted or unregulated protease activities. To evaluate the potential use of these small protein inhibitors in regulating the serralysin induced activation of ENaC, proteases from Pseudomonas aeruginosa and Serratia marcescens were purified for characterization along with a high affinity inhibitor from Pseudomonas. Both proteases showed activity against in vitro substrates and could be blocked by near stoichiometric concentrations of the inhibitor. In addition, both proteases were capable of activating ENaC when added to the apical surfaces of multiple epithelial cells with similar slow activation kinetics. The high-affinity periplasmic inhibitor from Pseudomonas effectively blocked this activation. These data suggest that multiple metalloproteases are capable of activating ENaC. Further, the endogenous, periplasmic bacterial inhibitors may be useful for modulating the downstream effects of the serralysin virulence factors under physiological conditions. PMID:24963801

  13. Modulation of the epithelial sodium channel (ENaC by bacterial metalloproteases and protease inhibitors.

    Directory of Open Access Journals (Sweden)

    Michael B Butterworth

    Full Text Available The serralysin family of metalloproteases is associated with the virulence of multiple gram-negative human pathogens, including Pseudomonas aeruginosa and Serratia marcescens. The serralysin proteases share highly conserved catalytic domains and show evolutionary similarity to the mammalian matrix metalloproteases. Our previous studies demonstrated that alkaline protease (AP from Pseudomonas aeruginosa is capable of activating the epithelial sodium channel (ENaC, leading to an increase in sodium absorption in airway epithelia. The serralysin proteases are often co-expressed with endogenous, intracellular or periplasmic inhibitors, which putatively protect the bacterium from unwanted or unregulated protease activities. To evaluate the potential use of these small protein inhibitors in regulating the serralysin induced activation of ENaC, proteases from Pseudomonas aeruginosa and Serratia marcescens were purified for characterization along with a high affinity inhibitor from Pseudomonas. Both proteases showed activity against in vitro substrates and could be blocked by near stoichiometric concentrations of the inhibitor. In addition, both proteases were capable of activating ENaC when added to the apical surfaces of multiple epithelial cells with similar slow activation kinetics. The high-affinity periplasmic inhibitor from Pseudomonas effectively blocked this activation. These data suggest that multiple metalloproteases are capable of activating ENaC. Further, the endogenous, periplasmic bacterial inhibitors may be useful for modulating the downstream effects of the serralysin virulence factors under physiological conditions.

  14. Sodium channel current loss of function in induced pluripotent stem cell-derived cardiomyocytes from a Brugada syndrome patient.

    Science.gov (United States)

    Selga, Elisabet; Sendfeld, Franziska; Martinez-Moreno, Rebecca; Medine, Claire N; Tura-Ceide, Olga; Wilmut, Sir Ian; Pérez, Guillermo J; Scornik, Fabiana S; Brugada, Ramon; Mills, Nicholas L

    2017-10-09

    Brugada syndrome predisposes to sudden death due to disruption of normal cardiac ion channel function, yet our understanding of the underlying cellular mechanisms is incomplete. Commonly used heterologous expression models lack many characteristics of native cardiomyocytes and, in particular, the individual genetic background of a patient. Patient-specific induced pluripotent stem (iPS) cell-derived cardiomyocytes (iPS-CM) may uncover cellular phenotypical characteristics not observed in heterologous models. Our objective was to determine the properties of the sodium current in iPS-CM with a mutation in SCN5A associated with Brugada syndrome. Dermal fibroblasts from a Brugada syndrome patient with a mutation in SCN5A (c.1100G>A, leading to Nav1.5_p.R367H) were reprogrammed to iPS cells. Clones were characterized and differentiated to form beating clusters and sheets. Patient and control iPS-CM were structurally indistinguishable. Sodium current properties of patient and control iPS-CM were compared. These results were contrasted with those obtained in tsA201 cells heterologously expressing sodium channels with the same mutation. Patient-derived iPS-CM showed a 33.1-45.5% reduction in INa density, a shift in both activation and inactivation voltage-dependence curves, and faster recovery from inactivation. Co-expression of wild-type and mutant channels in tsA201 cells did not compromise channel trafficking to the membrane, but resulted in a reduction of 49.8% in sodium current density without affecting any other parameters. Cardiomyocytes derived from iPS cells from a Brugada syndrome patient with a mutation in SCN5A recapitulate the loss of function of sodium channel current associated with this syndrome; including pro-arrhythmic changes in channel function not detected using conventional heterologous expression systems. Copyright © 2017. Published by Elsevier Ltd.

  15. The cardiac sodium channel gene SCN5A and its gene product NaV1.5: Role in physiology and pathophysiology

    NARCIS (Netherlands)

    Veerman, Christiaan C.; Wilde, Arthur A. M.; Lodder, Elisabeth M.

    2015-01-01

    The gene SCN5A encodes the main cardiac sodium channel NaV1.5. This channel predominates the cardiac sodium current, INa, which underlies the fast upstroke of the cardiac action potential. As such, it plays a crucial role in cardiac electrophysiology. Over the last 60years a tremendous amount of

  16. Blocking voltage-gated sodium channels as a strategy to suppress pathological cough.

    Science.gov (United States)

    Sun, Hui; Kollarik, Marian; Undem, Bradley J

    2017-12-01

    Pathological cough is thought to be secondary to inappropriate activation of vagal sensory nerves. Sensory nerves can be activated by a large number of disparate stimuli. The most relevant stimuli to block for effective anti-tussive therapy likely depend on the specific underlying pathology that is leading to the coughing. Blocking voltage-gated sodium channels (NaV) will prevent action potential initiation and conduction and therefore prevent sensory communication between the airways and brainstem. In so doing, they would be expected to inhibit evoked cough independently of the nature of the stimulus and underlying pathology. There are nine subtypes of NaVs each with distinct pore-forming alpha subunits referred to NaV1.1-1.9. Among these channels, based on functional and genetic analysis of cough causing vagal afferent nerve subtypes, we hypothesize that targeting NaV1.7 and NaV1.8 is a rational strategy forward for the effective treatment of pathological cough. Copyright © 2017. Published by Elsevier Ltd.

  17. Activity-dependent depression of neuronal sodium channels by the general anaesthetic isoflurane

    Science.gov (United States)

    Purtell, K.; Gingrich, K. J.; Ouyang, W.; Herold, K. F.; Hemmings, H. C.

    2015-01-01

    Background The mechanisms by which volatile anaesthetics such as isoflurane alter neuronal function are poorly understood, in particular their presynaptic mechanisms. Presynaptic voltage-gated sodium channels (Nav) have been implicated as a target for anaesthetic inhibition of neurotransmitter release. We hypothesize that state-dependent interactions of isoflurane with Nav lead to increased inhibition of Na+ current (INa) during periods of high-frequency neuronal activity. Methods The electrophysiological effects of isoflurane, at concentrations equivalent to those used clinically, were measured on recombinant brain-type Nav1.2 expressed in ND7/23 neuroblastoma cells and on endogenous Nav in isolated rat neurohypophysial nerve terminals. Rate constants determined from experiments on the recombinant channel were used in a simple model of Nav gating. Results At resting membrane potentials, isoflurane depressed peak INa and shifted steady-state inactivation in a hyperpolarizing direction. After membrane depolarization, isoflurane accelerated entry (τcontrol=0.36 [0.03] ms compared with τisoflurane=0.33 [0.05] ms, P1.9] ms, PNav. A simple model of Nav gating involving stabilisation of fast inactivation, accounts for this novel form of activity-dependent block. Conclusions Isoflurane stabilises the fast-inactivated state of neuronal Nav leading to greater depression of INa during high-frequency stimulation, consistent with enhanced inhibition of fast firing neurones. PMID:26089447

  18. Conotoxins Targeting Neuronal Voltage-Gated Sodium Channel Subtypes: Potential Analgesics?

    Directory of Open Access Journals (Sweden)

    Jeffrey R. McArthur

    2012-11-01

    Full Text Available Voltage-gated sodium channels (VGSC are the primary mediators of electrical signal amplification and propagation in excitable cells. VGSC subtypes are diverse, with different biophysical and pharmacological properties, and varied tissue distribution. Altered VGSC expression and/or increased VGSC activity in sensory neurons is characteristic of inflammatory and neuropathic pain states. Therefore, VGSC modulators could be used in prospective analgesic compounds. VGSCs have specific binding sites for four conotoxin families: μ-, μO-, δ- and ί-conotoxins. Various studies have identified that the binding site of these peptide toxins is restricted to well-defined areas or domains. To date, only the μ- and μO-family exhibit analgesic properties in animal pain models. This review will focus on conotoxins from the μ- and μO-families that act on neuronal VGSCs. Examples of how these conotoxins target various pharmacologically important neuronal ion channels, as well as potential problems with the development of drugs from conotoxins, will be discussed.

  19. Biophysical characterization of the Varroa destructor NaV1 sodium channel and its affinity for τ-fluvalinate insecticide.

    Science.gov (United States)

    Gosselin-Badaroudine, Pascal; Chahine, Mohamed

    2017-07-01

    The decline of the western honeybee (Apis mellifera) has been reported to be due to parasitism by Varroa destructor mites and to colony collapse disorder in which these mites may be involved. In-hive chemicals such as τ-fluvalinate are being used to control Vdestructor populations. This approach may lead to the chronic exposure of bees to this liposoluble chemical, which tends to accumulate in hives. We cloned a variant of the V. destructor voltage-dependent sodium (VdNaV1) channel and studied its biophysical characteristics and sensitivity to τ-fluvalinate using the Xenopus oocyte expression system and the 2-microelectrode voltage-clamp technique. We compared the affinity of VdNaV1 for τ-fluvalinate with the honeybee voltage-dependent sodium ortholog. Our results showed that the honeybee sodium channel is more sensitive to τ-fluvalinate than the V. destructor channel, suggesting that care must be taken when treating hives with this chemical.-Gosselin-Badaroudine, P., Chahine, M. Biophysical characterization of the Varroa destructor NaV1 sodium channel and its affinity for τ-fluvalinate insecticide. © FASEB.

  20. Mutations in sodium channel {beta}-subunit SCN3B are associated with early-onset lone atrial fibrillation

    DEFF Research Database (Denmark)

    Olesen, Morten Salling; Jespersen, Thomas; Nielsen, Jonas Bille

    2011-01-01

    across species. Electrophysiological studies on the SCN3B mutation were carried out and all three SCN3B mutations caused a functionally reduced sodium channel current. One synonymous variant was found in SCN4B. CONCLUSION: In 192 young lone AF patients, we found three patients with suspected disease...

  1. A new Nav1.7 sodium channel mutation I234T in a child with severe pain.

    NARCIS (Netherlands)

    Ahn, H.S.; Dib-Hajj, S.D.; Cox, J.J.; Tyrrell, L.; Elmslie, F.V.; Clarke, A.A.; Drenth, J.P.H.; Woods, C.G.; Waxman, S.G.

    2010-01-01

    Dominant gain-of-function mutations that hyperpolarize activation of the Na(v)1.7 sodium channel have been linked to inherited erythromelalgia (IEM), a disorder characterized by severe pain and redness in the feet and hands in response to mild warmth. Pharmacotherapy remains largely ineffective for

  2. Characterization of an insect heterodimeric voltage-gated sodium channel with unique alternative splicing mode.

    Science.gov (United States)

    Jiang, Xuan-Zhao; Pei, Yu-Xia; Lei, Wei; Wang, Ke-Yi; Shang, Feng; Jiang, Hong-Bo; Wang, Jin-Jun

    2017-01-01

    Recent discovery of the heterodimeric voltage-gated sodium channels (Na v ) in two aphid species, Acyrthosiphon pisum and Myzus persicae, aroused interest in exploring whether this kind of channel is conserved for aphids. Herewith, we aim to provide evidence for the conservation of heterodimeric Na v s in aphids and investigate whether they have unique splicing patterns. We found that the only identifiable Na v from Toxoptera citricida consisted of two subunits, forming a heterodimeric Na v , which carried an atypical "DENS" ion selectivity filter and a conventional "MFM" inactivation gate, confirming the heterodimeric Na v s' conservation within aphids. These unique heterodimeric channels may form a new Na v subfamily, specific to aphids. A more ancient member of four-domain Na v homolog was well preserved in T. citricida, carrying a typical "DEEA" and "MFL" motif. The presence of "DENS" in mammalian Na x s and "DEKT" in a fungus Na v suggested that the heterodimeric Na v s may still preserve Na + permeability. Sequencing 46 clones from nymphs and adults exposed unique splicing patterns for this heterodimeric Na v from T. citricida, revealing 7 alternatively spliced exons, evidencing that exon 5 was no longer unique to Bombyx mori, and exon k/l was semi-mutually exclusive. Two previously undescribed optional exons and a SNP site seemingly unique to aphids were identified. In conclusion, the dimeric Na v s might form a new aphids-specific heterodimeric N a v subfamily. This dimeric Na v from T. citricida was characterized with distinguishable alternative splicing modes, exemplified by the discovery of two novel alternative exons and unique usage patterns of alternative exons. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Lidocaine reduces the transition to slow inactivation in Nav1.7 voltage-gated sodium channels

    Science.gov (United States)

    Sheets, Patrick L; Jarecki, Brian W; Cummins, Theodore R

    2011-01-01

    BACKGROUND AND PURPOSE The primary use of local anaesthetics is to prevent or relieve pain by reversibly preventing action potential propagation through the inhibition of voltage-gated sodium channels. The tetrodotoxin-sensitive voltage-gated sodium channel subtype Nav1.7, abundantly expressed in pain-sensing neurons, plays a crucial role in perception and transmission of painful stimuli and in inherited chronic pain syndromes. Understanding the interaction of lidocaine with Nav1.7 channels could provide valuable insight into the drug's action in alleviating pain in distinct patient populations. The aim of this study was to determine how lidocaine interacts with multiple inactivated conformations of Nav1.7 channels. EXPERIMENTAL APPROACH We investigated the interactions of lidocaine with wild-type Nav1.7 channels and a paroxysmal extreme pain disorder mutation (I1461T) that destabilizes fast inactivation. Whole cell patch clamp recordings were used to examine the activity of channels expressed in human embryonic kidney 293 cells. KEY RESULTS Depolarizing pulses that increased slow inactivation of Nav1.7 channels also reduced lidocaine inhibition. Lidocaine enhanced recovery of Nav1.7 channels from prolonged depolarizing pulses by decreasing slow inactivation. A paroxysmal extreme pain disorder mutation that destabilizes fast inactivation of Nav1.7 channels decreased lidocaine inhibition. CONCLUSIONS AND IMPLICATIONS Lidocaine decreased the transition of Nav1.7 channels to the slow inactivated state. The fast inactivation gate (domain III–IV linker) is important for potentiating the interaction of lidocaine with the Nav1.7 channel. PMID:21232038

  4. Molecular Insights into the Local Anesthetic Receptor within Voltage-Gated Sodium Channels Using Hydroxylated Analogs of Mexiletine.

    Science.gov (United States)

    Desaphy, Jean-François; Dipalma, Antonella; Costanza, Teresa; Carbonara, Roberta; Dinardo, Maria Maddalena; Catalano, Alessia; Carocci, Alessia; Lentini, Giovanni; Franchini, Carlo; Camerino, Diana Conte

    2012-01-01

    We previously showed that the β-adrenoceptor modulators, clenbuterol and propranolol, directly blocked voltage-gated sodium channels, whereas salbutamol and nadolol did not (Desaphy et al., 2003), suggesting the presence of two hydroxyl groups on the aromatic moiety of the drugs as a molecular requisite for impeding sodium channel block. To verify such an hypothesis, we synthesized five new mexiletine analogs by adding one or two hydroxyl groups to the aryloxy moiety of the sodium channel blocker and tested these compounds on hNav1.4 channels expressed in HEK293 cells. Concentration-response relationships were constructed using 25-ms-long depolarizing pulses at -30 mV applied from an holding potential of -120 mV at 0.1 Hz (tonic block) and 10 Hz (use-dependent block) stimulation frequencies. The half-maximum inhibitory concentrations (IC(50)) were linearly correlated to drug lipophilicity: the less lipophilic the drug, minor was the block. The same compounds were also tested on F1586C and Y1593C hNav1.4 channel mutants, to gain further information on the molecular interactions of mexiletine with its receptor within the sodium channel pore. In particular, replacement of Phe1586 and Tyr1593 by non-aromatic cysteine residues may help in the understanding of the role of π-π or π-cation interactions in mexiletine binding. Alteration of tonic block suggests that the aryloxy moiety of mexiletine may interact either directly or indirectly with Phe1586 in the closed sodium channel to produce low-affinity binding block, and that this interaction depends on the electrostatic potential of the drug aromatic tail. Alteration of use-dependent block suggests that addition of hydroxyl groups to the aryloxy moiety may modify high-affinity binding of the drug amine terminal to Phe1586 through cooperativity between the two pharmacophores, this effect being mainly related to drug lipophilicity. Mutation of Tyr1593 further impaired such cooperativity. In conclusion, these

  5. Classification of drugs based on properties of sodium channel inhibition: a comparative automated patch-clamp study.

    Directory of Open Access Journals (Sweden)

    Nora Lenkey

    Full Text Available BACKGROUND: There is only one established drug binding site on sodium channels. However, drug binding of sodium channels shows extreme promiscuity: ∼25% of investigated drugs have been found to potently inhibit sodium channels. The structural diversity of these molecules suggests that they may not share the binding site, and/or the mode of action. Our goal was to attempt classification of sodium channel inhibitors by measuring multiple properties of inhibition in electrophysiology experiments. We also aimed to investigate if different properties of inhibition correlate with specific chemical properties of the compounds. METHODOLOGY/PRINCIPAL FINDINGS: A comparative electrophysiological study of 35 compounds, including classic sodium channel inhibitors (anticonvulsants, antiarrhythmics and local anesthetics, as well as antidepressants, antipsychotics and neuroprotective agents, was carried out using rNav1.2 expressing HEK-293 cells and the QPatch automatic patch-clamp instrument. In the multi-dimensional space defined by the eight properties of inhibition (resting and inactivated affinity, potency, reversibility, time constants of onset and offset, use-dependence and state-dependence, at least three distinct types of inhibition could be identified; these probably reflect distinct modes of action. The compounds were clustered similarly in the multi-dimensional space defined by relevant chemical properties, including measures of lipophilicity, aromaticity, molecular size, polarity and electric charge. Drugs of the same therapeutic indication typically belonged to the same type. We identified chemical properties, which were important in determining specific properties of inhibition. State-dependence correlated with lipophilicity, the ratio of the neutral form of molecules, and aromaticity: We noticed that the highly state dependent inhibitors had at least two aromatic rings, logP>4.0, and pKa<8.0. CONCLUSIONS/SIGNIFICANCE: The correlations of

  6. Spontaneous and CRH-Induced Excitability and Calcium Signaling in Mice Corticotrophs Involves Sodium, Calcium, and Cation-Conducting Channels.

    Science.gov (United States)

    Zemkova, Hana; Tomić, Melanija; Kucka, Marek; Aguilera, Greti; Stojilkovic, Stanko S

    2016-04-01

    Transgenic mice expressing the tdimer2(12) form of Discosoma red fluorescent protein under control of the proopiomelanocortin gene's regulatory elements are a useful model for studying corticotrophs. Using these mice, we studied the ion channels and mechanisms controlling corticotroph excitability. Corticotrophs were either quiescent or electrically active, with a 22-mV difference in the resting membrane potential (RMP) between the 2 groups. In quiescent cells, CRH depolarized the membrane, leading to initial single spiking and sustained bursting; in active cells, CRH further facilitated or inhibited electrical activity and calcium spiking, depending on the initial activity pattern and CRH concentration. The stimulatory but not inhibitory action of CRH on electrical activity was mimicked by cAMP independently of the presence or absence of arachidonic acid. Removal of bath sodium silenced spiking and hyperpolarized the majority of cells; in contrast, the removal of bath calcium did not affect RMP but reduced CRH-induced depolarization, which abolished bursting electrical activity and decreased the spiking frequency but not the amplitude of single spikes. Corticotrophs with inhibited voltage-gated sodium channels fired calcium-dependent action potentials, whereas cells with inhibited L-type calcium channels fired sodium-dependent spikes; blockade of both channels abolished spiking without affecting the RMP. These results indicate that the background voltage-insensitive sodium conductance influences RMP, the CRH-depolarization current is driven by a cationic conductance, and the interplay between voltage-gated sodium and calcium channels plays a critical role in determining the status and pattern of electrical activity and calcium signaling.

  7. Transient Receptor Potential Vanilloid 4-Induced Modulation of Voltage-Gated Sodium Channels in Hippocampal Neurons.

    Science.gov (United States)

    Hong, Zhiwen; Jie, Pinghui; Tian, Yujing; Chen, Tingting; Chen, Lei; Chen, Ling

    2016-01-01

    Transient receptor potential vanilloid 4 (TRPV4) is reported to control the resting membrane potential and increase excitability in many types of cells. Voltage-gated sodium channels (VGSCs) play an important role in initiating action potentials in neurons. However, whether VGSCs can be modulated by the activation of TRPV4 in hippocampal pyramidal neurons remains unknown. In this study, we tested the effect of TRPV4 agonists (GSK1016790A and 4α-PDD) on voltage-gated sodium current (I Na) in hippocampal CA1 pyramidal neurons and the protein levels of α/β-subunit of VGSCs in the hippocampus of mice subjected to intracerebroventricular (icv.) injection of GSK1016790A (GSK-injected mice). Herein, we report that I Na was inhibited by acute application of GSK1016790A or 4α-PDD. In the presence of TRPV4 agonists, the voltage-dependent inactivation curve shifted to the hyperpolarization, whereas the voltage-dependent activation curve remained unchanged. The TRPV4 agonist-induced inhibition of I Na was blocked by the TRPV4 antagonist or tetrodotoxin. Moreover, blocking protein kinase A (PKA) markedly attenuated the GSK1016790A-induced inhibition of I Na, whereas antagonism of protein kinase C or p38 mitogen-activated protein kinase did not change GSK1016790A action. Finally, the protein levels of Nav1.1, Nav1.2, and Nav1.6 in the hippocampus increased in GSK-injected mice, whereas those of Nav1.3 and Navβ1 remained nearly unchanged. We conclude that I Na is inhibited by the acute activation of TRPV4 through PKA signaling pathway in hippocampal pyramidal neurons, but protein expression of α-subunit of VGSCs is increased by sustained TRPV4 activation, which may compensate for the acute inhibition of I Na and provide a possibility for hyper-excitability upon sustained TRPV4 activation.

  8. Modulation of epithelial sodium channel (ENaC expression in mouse lung infected with Pseudomonas aeruginosa

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    Radzioch Danuta

    2005-01-01

    Full Text Available Abstract Background The intratracheal instillation of Pseudomonas aeruginosa entrapped in agar beads in the mouse lung leads to chronic lung infection in susceptible mouse strains. As the infection generates a strong inflammatory response with some lung edema, we tested if it could modulate the expression of genes involved in lung liquid clearance, such as the α, β and γ subunits of the epithelial sodium channel (ENaC and the catalytic subunit of Na+-K+-ATPase. Methods Pseudomonas aeruginosa entrapped in agar beads were instilled in the lung of resistant (BalB/c and susceptible (DBA/2, C57BL/6 and A/J mouse strains. The mRNA expression of ENaC and Na+-K+-ATPase subunits was tested in the lung by Northern blot following a 3 hours to 14 days infection. Results The infection of the different mouse strains evoked regulation of α and β ENaC mRNA. Following Pseudomonas instillation, the expression of αENaC mRNA decreased to a median of 43% on days 3 and 7 after infection and was still decreased to a median of 45% 14 days after infection (p 1Na+-K+-ATPase mRNA, the catalytic subunit of the sodium pump, was recorded. The distinctive expression profiles of the three subunits were not different, between the susceptible and resistant mouse strains. Conclusions These results show that Pseudomonas infection, by modulating ENaC subunit expression, could influence edema formation and clearance in infected lungs.

  9. Common variants in epithelial sodium channel genes contribute to salt sensitivity of blood pressure: The GenSalt study.

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    Zhao, Qi; Gu, Dongfeng; Hixson, James E; Liu, De-Pei; Rao, Dabeeru C; Jaquish, Cashell E; Kelly, Tanika N; Lu, Fanghong; Ma, Jixiang; Mu, Jianjun; Shimmin, Lawrence C; Chen, Jichun; Mei, Hao; Hamm, L Lee; He, Jiang

    2011-08-01

    Rare mutations of the epithelial sodium channel (ENaC) lead to mendelian forms of salt-sensitive hypertension or salt-wasting hypotension. We aimed to examine the association between common variants in the ENaC genes and salt sensitivity of blood pressure (BP). A total of 1906 Han Chinese participated in the Genetic Epidemiology Network of Salt Sensitivity (GenSalt) study, which includes a 7-day low-sodium intake (51.3 mmol sodium/d) followed by a 7-day high-sodium intake (307.8 mmol sodium/d). Nine BP measurements were obtained at baseline and each intervention period using a random-zero sphygmomanometer. Single-nucleotide polymorphisms, both tagging and functional, from the 3 ENaC subunits, α, β, and γ (SCNN1A, SCNN1B, and SCNN1G), were genotyped. Multiple common single-nucleotide polymorphisms in SCNN1G were significantly associated with BP response to low-sodium intervention (rs4073930, P=1.7×10(-5); rs4073291, P=1.1×10(-5); rs7404408, P=1.9×10(-5); rs5735, P=3.0×10(-4); rs4299163, P=0.004; and rs4499238, P=0.002) even after correcting for multiple testing. For example, under an additive model, the minor allele G of SNP rs4073291 was associated with 1.33 mm Hg lower systolic BP reduction during low-sodium intervention. This large dietary sodium intervention study indicates that common variants of ENaC subunits may contribute to the variation of BP response to dietary sodium intake. Future studies are warranted to confirm these findings in an independent population and to identify functional variants for salt sensitivity. URL: http://www.clinicaltrials.gov. Unique identifier: NCT00721721.

  10. Neurotrophin-3 significantly reduces sodium channel expression linked to neuropathic pain states.

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    Wilson-Gerwing, Tracy D; Stucky, Cheryl L; McComb, Geoffrey W; Verge, Valerie M K

    2008-10-01

    Neuropathic pain resulting from chronic constriction injury (CCI) is critically linked to sensitization of peripheral nociceptors. Voltage gated sodium channels are major contributors to this state and their expression can be upregulated by nerve growth factor (NGF). We have previously demonstrated that neurotrophin-3 (NT-3) acts antagonistically to NGF in modulation of aspects of CCI-induced changes in trkA-associated nociceptor phenotype and thermal hyperalgesia. Thus, we hypothesized that exposure of neurons to increased levels of NT-3 would reduce expression of Na(v)1.8 and Na(v)1.9 in DRG neurons subject to CCI. In adult male rats, Na(v)1.8 and Na(v)1.9 mRNAs are expressed at high levels in predominantly small to medium size neurons. One week following CCI, there is reduced incidence of neurons expressing detectable Na(v)1.8 and Na(v)1.9 mRNA, but without a significant decline in mean level of neuronal expression, and similar findings observed immunohistochemically. There is also increased accumulation/redistribution of channel protein in the nerve most apparent proximal to the first constriction site. Intrathecal infusion of NT-3 significantly attenuates neuronal expression of Na(v)1.8 and Na(v)1.9 mRNA contralateral and most notably, ipsilateral to CCI, with a similar impact on relative protein expression at the level of the neuron and constricted nerve. We also observe reduced expression of the common neurotrophin receptor p75 in response to CCI that is not reversed by NT-3 in small to medium sized neurons and may confer an enhanced ability of NT-3 to signal via trkA, as has been previously shown in other cell types. These findings are consistent with an analgesic role for NT-3.

  11. Activity-dependent depression of neuronal sodium channels by the general anaesthetic isoflurane.

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    Purtell, K; Gingrich, K J; Ouyang, W; Herold, K F; Hemmings, H C

    2015-07-01

    The mechanisms by which volatile anaesthetics such as isoflurane alter neuronal function are poorly understood, in particular their presynaptic mechanisms. Presynaptic voltage-gated sodium channels (Na(v)) have been implicated as a target for anaesthetic inhibition of neurotransmitter release. We hypothesize that state-dependent interactions of isoflurane with Na(v) lead to increased inhibition of Na(+) current (I(Na)) during periods of high-frequency neuronal activity. The electrophysiological effects of isoflurane, at concentrations equivalent to those used clinically, were measured on recombinant brain-type Na(v)1.2 expressed in ND7/23 neuroblastoma cells and on endogenous Na(v) in isolated rat neurohypophysial nerve terminals. Rate constants determined from experiments on the recombinant channel were used in a simple model of Na(v) gating. At resting membrane potentials, isoflurane depressed peak I(Na) and shifted steady-state inactivation in a hyperpolarizing direction. After membrane depolarization, isoflurane accelerated entry (τ(control)=0.36 [0.03] ms compared with τ(isoflurane)=0.33 [0.05] ms, P1.9] ms, P<0.005) from apparent fast inactivation, resulting in enhanced depression of I(Na), during high-frequency stimulation of both recombinant and endogenous nerve terminal Na(v). A simple model of Na(v) gating involving stabilisation of fast inactivation, accounts for this novel form of activity-dependent block. Isoflurane stabilises the fast-inactivated state of neuronal Na(v) leading to greater depression of I(Na) during high-frequency stimulation, consistent with enhanced inhibition of fast firing neurones. © The Author 2015. Published by Oxford University Press on behalf of the British Journal of Anaesthesia. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  12. Neurotrophin-3 significantly reduces sodium channel expression linked to neuropathic pain states

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    Wilson-Gerwing, Tracy D.; Stucky, Cheryl L.; McComb, Geoffrey W.; Verge, Valerie M. K.

    2009-01-01

    Neuropathic pain resulting from chronic constriction injury (CCI) is critically linked to sensitization of peripheral nociceptors. Voltage gated sodium channels are major contributors to this state and their expression can be upregulated by nerve growth factor (NGF). We have previously demonstrated that neurotrophin-3 (NT-3) acts antagonistically to NGF in modulation of aspects of CCI-induced changes in trkA-associated nociceptor phenotype and thermal hyperalgesia. Thus, we hypothesized that exposure of neurons to increased levels of NT-3 would reduce expression of Nav1.8 and Nav1.9 in DRG neurons subject to CCI. In adult male rats, Nav1.8 and Nav1.9 mRNAs are expressed at high levels in predominantly small to medium size neurons. One week following CCI, there is reduced incidence of neurons expressing detectable Nav1.8 and Nav1.9 mRNA, but without a significant decline in mean level of neuronal expression, and similar findings observed immunohistochemically. There is also increased accumulation/redistribution of channel protein in the nerve most apparent proximal to the first constriction site. Intrathecal infusion of NT-3 significantly attenuates neuronal expression of Nav1.8 and Nav1.9 mRNA contralateral and most notably, ipsilateral to CCI, with a similar impact on relative protein expression at the level of the neuron and constricted nerve. We also observe reduced expression of the common neurotrophin receptor p75 in response to CCI that is not reversed by NT-3 in small to medium sized neurons and may confer an enhanced ability of NT-3 to signal via trkA, as has been previously shown in other cell types. These findings are consistent with an analgesic role for NT-3. PMID:18601922

  13. Carisbamate, a novel neuromodulator, inhibits voltage-gated sodium channels and action potential firing of rat hippocampal neurons.

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    Liu, Yi; Yohrling, George J; Wang, Yan; Hutchinson, Tasha L; Brenneman, Douglas E; Flores, Christopher M; Zhao, Boyu

    2009-01-01

    Carisbamate (RWJ-333369; (S)-2-O-carbamoyl-1-o-chlorophenyl-ethanol) is a novel investigational antiepileptic drug that exhibits a broad-spectrum of activity in a number of animal models of seizure and drug refractory epilepsy. In an effort to understand the molecular mechanism by which carisbamate produces its antiepileptic actions, we studied its effects on the function of voltage-gated, rat brain sodium and potassium channels and on the repetitive firing of action potentials in cultured rat hippocampal neurons. In whole-cell patch clamp recording, carisbamate resulted in a concentration-, voltage- and use-dependent inhibition of rat Nav1.2, with an IC(50) value of 68 microM at -67 mV. In rat hippocampal neurons, carisbamate similarly blocked voltage-gated sodium channels, with an IC(50) value of 89 microM at -67 mV, and inhibited repetitive firing of action potentials in a concentration-dependent manner (by 46% at 30 microM and 87% at 100 microM, respectively). Carisbamate had no effect on the steady-state membrane potential or voltage-gated potassium channels (K(v)) in these neurons. These inhibitory effects of carisbamate occurred at therapeutically relevant concentrations in vivo, raising the possibility that block of voltage-gated sodium channels by carisbamate contributes to its antiepileptic activity.

  14. Differential expression of neuronal voltage-gated sodium channel mRNAs during the development of the rat trigeminal ganglion.

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    Thun, Jonas; Persson, Anna-Karin; Fried, Kaj

    2009-05-07

    The developmental pattern of sodium channel expression in neurons of primary sensory ganglia is likely reflected in the electrical behavior of these cells. Little information is available on how voltage-gated sodium channels in sensory neurons are expressed during development in the trigeminal-innervated craniofacial region, where sensitivity is fundamental during early stages of life. Using in situ hybridization, we here demonstrate a differential both regional and transcript-dependent distribution of sodium channel alpha- and beta-subunits between Embryonic day (E)15 and Postnatal day (P)5/6 in the rat trigeminal ganglion. Na(v)1.3 mRNA was strongly expressed at E15, but declined to low levels at P5/P6. Na(v)1.8 was expressed at E15, increased to reach maximum levels at P1 and then decreased. Na(v)1.9 mRNA was detected at E19, reached a maximum at P1, and was then reduced. beta1 mRNA showed a steady rise in expression from E17, while beta2 mRNA was widely expressed from P1. beta 3 mRNA was detected at E15, reached a maximum at E19 followed by a decrease in expression. In the ophthalmic TG portion, there was a higher expression level of Na(v)1.8 and Na(v)1.9 between E19 and P5/P6 as compared to the maxillary/mandibular part, indicating an unexpected positional difference in channel distribution. mRNA levels of p11, which facilitates the expression of Na(v)1.8, were high at all stages. These findings show that trigeminal ganglion sodium channel transcripts mature in steps that are specific for each transcript. They also raise the possibility that different facial regions could differ in the ability to transmit sensory signals during early life.

  15. The voltage-gated sodium channel NaV 1.9 in visceral pain.

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    Hockley, J R F; Winchester, W J; Bulmer, D C

    2016-03-01

    Visceral pain is a common symptom for patients with gastrointestinal (GI) disease. It is unpleasant, debilitating, and represents a large unmet medical need for effective clinical treatments. Recent studies have identified NaV 1.9 as an important regulator of afferent sensitivity in visceral pain pathways to mechanical and inflammatory stimuli, suggesting that NaV 1.9 could represent an important therapeutic target for the treatment of visceral pain. This potential has been highlighted by the identification of patients who have an insensitivity to pain or painful neuropathies associated with mutations in SCN11A, the gene encoding voltage-gated sodium channel subtype 1.9 (NaV 1.9). Here, we address the role of NaV 1.9 in visceral pain and what known human NaV 1.9 mutants can tell us about NaV 1.9 function in gut physiology and pathophysiology. © 2015 John Wiley & Sons Ltd.

  16. Multiple sodium channel isoforms mediate the pathological effects of Pacific ciguatoxin-1.

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    Inserra, Marco C; Israel, Mathilde R; Caldwell, Ashlee; Castro, Joel; Deuis, Jennifer R; Harrington, Andrea M; Keramidas, Angelo; Garcia-Caraballo, Sonia; Maddern, Jessica; Erickson, Andelain; Grundy, Luke; Rychkov, Grigori Y; Zimmermann, Katharina; Lewis, Richard J; Brierley, Stuart M; Vetter, Irina

    2017-02-22

    Human intoxication with the seafood poison ciguatoxin, a dinoflagellate polyether that activates voltage-gated sodium channels (NaV), causes ciguatera, a disease characterised by gastrointestinal and neurological disturbances. We assessed the activity of the most potent congener, Pacific ciguatoxin-1 (P-CTX-1), on NaV1.1-1.9 using imaging and electrophysiological approaches. Although P-CTX-1 is essentially a non-selective NaV toxin and shifted the voltage-dependence of activation to more hyperpolarising potentials at all NaV subtypes, an increase in the inactivation time constant was observed only at NaV1.8, while the slope factor of the conductance-voltage curves was significantly increased for NaV1.7 and peak current was significantly increased for NaV1.6. Accordingly, P-CTX-1-induced visceral and cutaneous pain behaviours were significantly decreased after pharmacological inhibition of NaV1.8 and the tetrodotoxin-sensitive isoforms NaV1.7 and NaV1.6, respectively. The contribution of these isoforms to excitability of peripheral C- and A-fibre sensory neurons, confirmed using murine skin and visceral single-fibre recordings, reflects the expression pattern of NaV isoforms in peripheral sensory neurons and their contribution to membrane depolarisation, action potential initiation and propagation.

  17. Expression and distribution of epithelial sodium channel in nasal polyp and nasal mucosa.

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    Jiang, Yiming; Xu, Ji; Chen, Yanqing; Shi, Jiali; Zhang, Chun; Li, Jiping

    2015-11-01

    To evaluate the expression and location of epithelial sodium channels (ENaCs) in human nasal polyp and normal nasal mucosa, and to characterize the relevance of ENaCs to the development of NPs. Nasal polyp tissue from 17 patients and nasal mucosa from ten patients were obtained through endoscopic sinus surgery. The mRNA concentrations of ENaC-α, β, and γ were detected by real-time polymerase chain reaction. The expression of ENaC-α was detected using western blot and immunofluorescence techniques. The distribution of ENaC-α in mucosal tissue was observed using a laser scanning confocal microscope. The transcriptional expression of three subunits of ENaC was in the following order: α > β > γ, in both groups. The transcriptional expression of α, β, and γ subunits of ENaC was elevated in nasal polyp compared to nasal mucosa (p distributed in the epithelial cell apical membrane. The expression of ENaC is upregulated in human nasal polyp, which might in turn facilitate the formation and development of nasal polyp.

  18. The Segregated Expression of Voltage-Gated Potassium and Sodium Channels in Neuronal Membranes: Functional Implications and Regulatory Mechanisms.

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    Duménieu, Maël; Oulé, Marie; Kreutz, Michael R; Lopez-Rojas, Jeffrey

    2017-01-01

    Neurons are highly polarized cells with apparent functional and morphological differences between dendrites and axon. A critical determinant for the molecular and functional identity of axonal and dendritic segments is the restricted expression of voltage-gated ion channels (VGCs). Several studies show an uneven distribution of ion channels and their differential regulation within dendrites and axons, which is a prerequisite for an appropriate integration of synaptic inputs and the generation of adequate action potential (AP) firing patterns. This review article will focus on the signaling pathways leading to segmented expression of voltage-gated potassium and sodium ion channels at the neuronal plasma membrane and the regulatory mechanisms ensuring segregated functions. We will also discuss the relevance of proper ion channel targeting for neuronal physiology and how alterations in polarized distribution contribute to neuronal pathology.

  19. Biophysical and Pharmacological Characterization of Nav1.9 Voltage Dependent Sodium Channels Stably Expressed in HEK-293 Cells.

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    Lin, Zhixin; Santos, Sonia; Padilla, Karen; Printzenhoff, David; Castle, Neil A

    2016-01-01

    The voltage dependent sodium channel Nav1.9, is expressed preferentially in peripheral sensory neurons and has been linked to human genetic pain disorders, which makes it target of interest for the development of new pain therapeutics. However, characterization of Nav1.9 pharmacology has been limited due in part to the historical difficulty of functionally expressing recombinant channels. Here we report the successful generation and characterization of human, mouse and rat Nav1.9 stably expressed in human HEK-293 cells. These cells exhibit slowly activating and inactivating inward sodium channel currents that have characteristics of native Nav1.9. Optimal functional expression was achieved by coexpression of Nav1.9 with β1/β2 subunits. While recombinantly expressed Nav1.9 was found to be sensitive to sodium channel inhibitors TC-N 1752 and tetracaine, potency was up to 100-fold less than reported for other Nav channel subtypes despite evidence to support an interaction with the canonical local anesthetic (LA) binding region on Domain 4 S6. Nav1.9 Domain 2 S6 pore domain contains a unique lysine residue (K799) which is predicted to be spatially near the local anesthetic interaction site. Mutation of this residue to the consensus asparagine (K799N) resulted in an increase in potency for tetracaine, but a decrease for TC-N 1752, suggesting that this residue can influence interaction of inhibitors with the Nav1.9 pore. In summary, we have shown that stable functional expression of Nav1.9 in the widely used HEK-293 cells is possible, which opens up opportunities to better understand channel properties and may potentially aid identification of novel Nav1.9 based pharmacotherapies.

  20. Biophysical and Pharmacological Characterization of Nav1.9 Voltage Dependent Sodium Channels Stably Expressed in HEK-293 Cells.

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    Zhixin Lin

    Full Text Available The voltage dependent sodium channel Nav1.9, is expressed preferentially in peripheral sensory neurons and has been linked to human genetic pain disorders, which makes it target of interest for the development of new pain therapeutics. However, characterization of Nav1.9 pharmacology has been limited due in part to the historical difficulty of functionally expressing recombinant channels. Here we report the successful generation and characterization of human, mouse and rat Nav1.9 stably expressed in human HEK-293 cells. These cells exhibit slowly activating and inactivating inward sodium channel currents that have characteristics of native Nav1.9. Optimal functional expression was achieved by coexpression of Nav1.9 with β1/β2 subunits. While recombinantly expressed Nav1.9 was found to be sensitive to sodium channel inhibitors TC-N 1752 and tetracaine, potency was up to 100-fold less than reported for other Nav channel subtypes despite evidence to support an interaction with the canonical local anesthetic (LA binding region on Domain 4 S6. Nav1.9 Domain 2 S6 pore domain contains a unique lysine residue (K799 which is predicted to be spatially near the local anesthetic interaction site. Mutation of this residue to the consensus asparagine (K799N resulted in an increase in potency for tetracaine, but a decrease for TC-N 1752, suggesting that this residue can influence interaction of inhibitors with the Nav1.9 pore. In summary, we have shown that stable functional expression of Nav1.9 in the widely used HEK-293 cells is possible, which opens up opportunities to better understand channel properties and may potentially aid identification of novel Nav1.9 based pharmacotherapies.

  1. PKA phosphorylation reshapes the pharmacological kinetics of BmK AS, a unique site-4 sodium channel-specific modulator.

    Science.gov (United States)

    Liu, Z R; Zhang, H; Wu, J Q; Zhou, J J; Ji, Y H

    2014-01-16

    Although modulation of the activity of voltage-gated sodium channels (VGSCs) by protein kinase A (PKA) phosphorylation has been investigated in multiple preparations, the pharmacological sensitivity of VGSCs to scorpion toxins after PKA phosphorylation has rarely been approached. In this study, the effects of BmK AS, a sodium channel-specific modulator from Chinese scorpion Buthus martensi Karsch, on the voltage-dependent activation and inactivation of Nav1.2 were examined before and after PKA activation. After PKA phosphorylation, the pattern of dose-dependent modulation of BmK AS, on both Nav1.2α and Nav1.2 (α + β1) was reshaped. Meanwhile, the shifts in voltage-dependency of activation and inactivation induced by BmK AS were attenuated. The results suggested that PKA might play a role in different patterns how β-like toxins such as BmK AS modulate gating properties and peak currents of VGSCs.

  2. Clinical and electrocardiographic predictors of positive response to the intravenous sodium channel blockers in patients suspected of the Brugada syndrome.

    Science.gov (United States)

    Shahrzad, Shahab; Khoramshahi, Maryam; Aslani, Amir; Fazelifar, Amir Farjam; Haghjoo, Majid

    2013-05-10

    Sodium channel blockers are used to unmask the diagnostic ECG pattern of the Brugada syndrome (BrS) in case of a non-diagnostic baseline ECG. The aim of the study was to determine clinical and ECG predictors of a positive challenge test in patients suspected to the BrS. A total of 106 consecutive patients (91 men; mean age, 35 ± 12 years) suspected of the BrS underwent the intravenous sodium channel blocker challenge test with procainamide or flecainide. Of the 106 patients, positive tests were detected in 20 (19%) patients. During test, a transient episode of a second-degree atrioventricular block and isolated ventricular ectopies were observed in 1 (0.9%) and 2 (1.9%) patients, respectively. A QRS prolongation ≥ 30% was observed in 4 (3.8%) patients. Baseline QRS duration in V1 ≥ 110 ms had a sensitivity of 70% and a specificity of 80% for a positive response. An ST-segment elevation ≥ 0.17 mV in V2 had a sensitivity of 60% and a specificity of 82% for a positive response. Of the multiple clinical and ECG variables entered into a binary logistic regression analysis, a history of syncope (P=0.001), previous cardiac arrest (P=0.001), baseline QRS duration in V1 ≥ 110 ms (P=0.001), and baseline ST-segment elevation in V2 ≥ 0.17 mV (P=0.012) emerged as the independent predictors of a positive response to the intravenous challenge with sodium channel blockers. The results of the sodium channel blocker challenge test can be predicted by clinical presentation and baseline ECG features. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  3. Lack of variation in voltage-gated sodium channels of common bottlenose dolphins (Tursiops truncatus) exposed to neurotoxic algal blooms.

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    Cammen, Kristina M; Rosel, Patricia E; Wells, Randall S; Read, Andrew J

    2014-12-01

    In coastal marine ecosystems, neurotoxins produced by harmful algal blooms (HABs) often result in large-scale mortality events of many marine species. Historical and frequent exposure to HABs therefore may provide a strong selective pressure for adaptations that result in toxin resistance. Neurotoxin resistance has independently evolved in a variety of terrestrial and marine species via mutations in genes encoding the toxin binding sites within the voltage-gated sodium channel gene complex. Accordingly, we tested the hypothesis that genetic variation in the putative binding site of brevetoxins in common bottlenose dolphins (Tursiops truncatus) explains differences among individuals or populations in resistance to harmful Karenia brevis blooms in the Gulf of Mexico. We found very little variation in the sodium channel exons encoding the putative brevetoxin binding site among bottlenose dolphins from central-west Florida and the Florida Panhandle. Our study included samples from several bottlenose dolphin mortality events associated with HABs, but we found no association between genetic variation and survival. We observed a significant effect of geographic region on genetic variation for some sodium channel isoforms, but this can be primarily explained by rare private alleles and is more likely a reflection of regional genetic differentiation than the cause of different levels of HAB resistance between regions. In contrast to many other previously studied neurotoxin-resistant species, we conclude that bottlenose dolphins have not evolved resistance to HABs via mutations in genes encoding the brevetoxin binding site on the voltage-gated sodium channels. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Localization of receptor site on insect sodium channel for depressant β-toxin BmK IT2.

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    Huiqiong He

    Full Text Available BACKGROUND: BmK IT2 is regarded as a receptor site-4 modulator of sodium channels with depressant insect toxicity. It also displays anti-nociceptive and anti-convulsant activities in rat models. In this study, the potency and efficacy of BmK IT2 were for the first time assessed and compared among four sodium channel isoforms expressed in Xenopus oocytes. Combined with molecular approach, the receptor site of BmK IT2 was further localized. PRINCIPAL FINDINGS: 2 µM BmK IT2 strongly shifted the activation of DmNa(v1, the sodium channel from Drosophila, to more hyperpolarized potentials; whereas it hardly affected the gating properties of rNa(v1.2, rNa(v1.3 and mNa(v1.6, three mammalian central neuronal sodium channel subtypes. (1 Mutations of Glu(896, Leu(899, Gly(904 in extracellular loop Domain II S3-S4 of DmNa(v1 abolished the functional action of BmK IT2. (2 BmK IT2-preference for DmNa(v1 could be conferred by Domain III. Analysis of subsequent DmNa(v1 mutants highlighted the residues in Domain III pore loop, esp. Ile(1529 was critical for recognition and binding of BmK IT2. CONCLUSIONS/SIGNIFICANCE: In this study, BmK IT2 displayed total insect-selectivity. Two binding regions, comprising domains II and III of DmNa(v1, play separated but indispensable roles in the interaction with BmK IT2. The insensitivity of Na(v1.2, Na(v1.3 and Na(v1.6 to BmK IT2 suggests other isoforms or mechanism might be involved in the suppressive activity of BmK IT2 in rat pathological models.

  5. Seeing the forest through the trees: towards a unified view on physiological calcium regulation of voltage-gated sodium channels.

    Science.gov (United States)

    Van Petegem, Filip; Lobo, Paolo A; Ahern, Christopher A

    2012-12-05

    Voltage-gated sodium channels (Na(V)s) underlie the upstroke of the action potential in the excitable tissues of nerve and muscle. After opening, Na(V)s rapidly undergo inactivation, a crucial process through which sodium conductance is negatively regulated. Disruption of inactivation by inherited mutations is an established cause of lethal cardiac arrhythmia, epilepsy, or painful syndromes. Intracellular calcium ions (Ca(2+)) modulate sodium channel inactivation, and multiple players have been suggested in this process, including the cytoplasmic Na(V) C-terminal region including two EF-hands and an IQ motif, the Na(V) domain III-IV linker, and calmodulin. Calmodulin can bind to the IQ domain in both Ca(2+)-bound and Ca(2+)-free conditions, but only to the DIII-IV linker in a Ca(2+)-loaded state. The mechanism of Ca(2+) regulation, and its composite effect(s) on channel gating, has been shrouded in much controversy owing to numerous apparent experimental inconsistencies. Herein, we attempt to summarize these disparate data and propose a novel, to our knowledge, physiological mechanism whereby calcium ions promote sodium current facilitation due to Ca(2+) memory at high-action-potential frequencies where Ca(2+) levels may accumulate. The available data suggest that this phenomenon may be disrupted in diseases where cytoplasmic calcium ion levels are chronically high and where targeted phosphorylation may decouple the Ca(2+) regulatory machinery. Many Na(V) disease mutations associated with electrical dysfunction are located in the Ca(2+)-sensing machinery and misregulation of Ca(2+)-dependent channel modulation is likely to contribute to disease phenotypes. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  6. Effects of 15 Hz square wave magnetic fields on the voltage-gated sodium and potassium channels in prefrontal cortex pyramidal neurons.

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    Zheng, Yu; Dou, Jun-Rong; Gao, Yang; Dong, Lei; Li, Gang

    2017-04-01

    Although magnetic fields have significant effects on neurons, little is known about the mechanisms behind their effects. The present study aimed to measure the effects of magnetic fields on ion channels in cortical pyramidal neurons. Cortical pyramidal neurons of Kunming mice were isolated and then subjected to 15 Hz, 1 mT square wave (duty ratio 50%) magnetic fields stimulation. Sodium currents (INa), transient potassium currents (IA) and delayed rectifier potassium currents (IK) were recorded by whole-cell patch clamp method. We found that magnetic field exposure depressed channel current densities, and altered the activation kinetics of sodium and potassium channels. The inactivation properties of INa and IA were also altered. Magnetic field exposure alters ion channel function in neurons. It is likely that the structures of sodium and potassium channels were influenced by the applied field. Sialic acid, which is an important component of the channels, could be the molecule responsible for the reported results.

  7. Predictive 3D modelling of the interactions of pyrethroids with the voltage-gated sodium channels of ticks and mites.

    Science.gov (United States)

    O'Reilly, Andrias O; Williamson, Martin S; González-Cabrera, Joel; Turberg, Andreas; Field, Linda M; Wallace, B A; Davies, T G Emyr

    2014-03-01

    The pyrethroid insecticides are a very successful group of compounds that target invertebrate voltage-gated sodium channels and are widely used in the control of insects, ticks and mites. It is well established that some pyrethroids are good insecticides whereas others are more effective as acaricides. This species specificity is advantageous for controlling particular pest(s) in the presence of another non-target invertebrate, for example controlling the Varroa mite in honeybee colonies. We applied in silico techniques to compare the voltage-gated sodium channels of insects versus ticks and mites and their interactions with a range of pyrethroids and DDT analogues. We identified a single amino acid difference within the pyrethroid binding pocket of ticks/mites that may have significant impact on the effectiveness of pyrethroids as acaricides. Other individual amino acid differences within the binding pocket in distinct tick and mite species may provide a basis for future acaricidal selectivity. Three-dimensional modelling of the pyrethroid/DDT receptor site has led to a new hypothesis to explain the preferential binding of acaricidal pyrethroids to the sodium channels of ticks/mites. This is important for understanding pyrethroid selectivity and the potential effects of mutations that can give rise to resistance to pyrethroids in commercially-important pest species. © 2013 Society of Chemical Industry.

  8. Influence of the sodium channel band on retinal ganglion cell excitation during electric stimulation--a modeling study.

    Science.gov (United States)

    Werginz, P; Fried, S I; Rattay, F

    2014-04-25

    Electric stimulation using retinal implants allows blind people to re-experience a rudimentary kind of vision. The elicited percepts or so called 'phosphenes' are highly inconstant and therefore do not restore vision properly. The better knowledge of how retinal neurons, especially retinal ganglion cells, respond to electric stimulation will help to develop more sophisticated stimulation strategies. Special anatomic and physiologic properties like a band of highly dense sodium channels in retinal ganglion cells may help to achieve a focal activation of target cells and as a result better restoration of vision. A portion of retinal ganglion cell axons, about 40μm from the soma and between 25 and 40μm in length, shows a specific biophysical property. Electrode locations close to a band of highly dense sodium channels which were identified immunochemically show lowest thresholds during electric stimulation. The (modeled) thresholds for this kind of structure result in lowest thresholds as well. The influence on the location where action potentials are generated within the axon is far reaching. When a stimulating electrode is positioned far outside the actual band region the site of spike initiation still remains within the sodium channel band. These findings suggest to further examine the key mechanisms of activation for retinal ganglion cells because focal activation without influencing passing axons of neurons located far away can improve the outcome of electric stimulation and therefore the development of retinal implants. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

  9. The voltage-gated sodium channel Nav1.9 is required for inflammation-based urinary bladder dysfunction.

    Science.gov (United States)

    Ritter, Amy M; Martin, William J; Thorneloe, Kevin S

    2009-03-06

    Tetrodotoxin (TTX)-resistant sodium channels are found in small diameter primary sensory neurons and are thought to be important in the maintenance of inflammatory pain. Here we examined bladder urodynamics of Nav1.9 voltage-gated sodium channel knock out (KO) mice, and the contribution of Nav1.9 to the development of inflammation-based bladder dysfunction. Basal urodynamics were not different between wildtype (WT) mice and those lacking Nav1.9. Peripheral nerve recordings from pelvic afferents in Nav1.9 KO mice revealed a lack of sensitization to intravesicularly applied prostaglandin E2 (PGE2). Consistent with this, cyclophosphamide treatment in vivo, which is associated with an enhancement of PGE2 production, evoked a reduction in bladder capacity of WT, but not Nav1.9 KO mice. We conclude that the Nav1.9 sodium channel provides an important link between inflammatory processes and changes in urodynamic properties that occur during urinary bladder inflammation.

  10. Functional properties and toxin pharmacology of a dorsal root ganglion sodium channel viewed through its voltage sensors

    Science.gov (United States)

    Puopolo, Michelino; Martin-Eauclaire, Marie-France; Bean, Bruce P.

    2011-01-01

    The voltage-activated sodium (Nav) channel Nav1.9 is expressed in dorsal root ganglion (DRG) neurons where it is believed to play an important role in nociception. Progress in revealing the functional properties and pharmacological sensitivities of this non-canonical Nav channel has been slow because attempts to express this channel in a heterologous expression system have been unsuccessful. Here, we use a protein engineering approach to dissect the contributions of the four Nav1.9 voltage sensors to channel function and pharmacology. We define individual S3b–S4 paddle motifs within each voltage sensor, and show that they can sense changes in membrane voltage and drive voltage sensor activation when transplanted into voltage-activated potassium channels. We also find that the paddle motifs in Nav1.9 are targeted by animal toxins, and that these toxins alter Nav1.9-mediated currents in DRG neurons. Our results demonstrate that slowly activating and inactivating Nav1.9 channels have functional and pharmacological properties in common with canonical Nav channels, but also show distinctive pharmacological sensitivities that can potentially be exploited for developing novel treatments for pain. PMID:21670206

  11. Functional properties and toxin pharmacology of a dorsal root ganglion sodium channel viewed through its voltage sensors.

    Science.gov (United States)

    Bosmans, Frank; Puopolo, Michelino; Martin-Eauclaire, Marie-France; Bean, Bruce P; Swartz, Kenton J

    2011-07-01

    The voltage-activated sodium (Nav) channel Nav1.9 is expressed in dorsal root ganglion (DRG) neurons where it is believed to play an important role in nociception. Progress in revealing the functional properties and pharmacological sensitivities of this non-canonical Nav channel has been slow because attempts to express this channel in a heterologous expression system have been unsuccessful. Here, we use a protein engineering approach to dissect the contributions of the four Nav1.9 voltage sensors to channel function and pharmacology. We define individual S3b-S4 paddle motifs within each voltage sensor, and show that they can sense changes in membrane voltage and drive voltage sensor activation when transplanted into voltage-activated potassium channels. We also find that the paddle motifs in Nav1.9 are targeted by animal toxins, and that these toxins alter Nav1.9-mediated currents in DRG neurons. Our results demonstrate that slowly activating and inactivating Nav1.9 channels have functional and pharmacological properties in common with canonical Nav channels, but also show distinctive pharmacological sensitivities that can potentially be exploited for developing novel treatments for pain.

  12. Gain-of-function mutations in sodium channel Na(v)1.9 in painful neuropathy.

    Science.gov (United States)

    Huang, Jianying; Han, Chongyang; Estacion, Mark; Vasylyev, Dymtro; Hoeijmakers, Janneke G J; Gerrits, Monique M; Tyrrell, Lynda; Lauria, Giuseppe; Faber, Catharina G; Dib-Hajj, Sulayman D; Merkies, Ingemar S J; Waxman, Stephen G

    2014-06-01

    Sodium channel Nav1.9 is expressed in peripheral nociceptive neurons, as well as visceral afferents, and has been shown to act as a threshold channel. Painful peripheral neuropathy represents a significant public health challenge and may involve gain-of-function variants in sodium channels that are preferentially expressed in peripheral sensory neurons. Although gain-of-function variants of peripheral sodium channels Nav1.7 and Nav1.8 have recently been found in painful small fibre neuropathy, the aetiology of peripheral neuropathy in many cases remains unknown. We evaluated 459 patients who were referred for possible painful peripheral neuropathy, and confirmed the diagnosis of small fibre neuropathy in a cohort of 393 patients (369 patients with pure small fibre neuropathy, and small fibre neuropathy together with large fibre involvement in an additional 24 patients). From this cohort of 393 patients with peripheral neuropathy, we sequenced SCN11A in 345 patients without mutations in SCN9A and SCN10A, and found eight variants in 12 patients. Functional profiling by electrophysiological recordings showed that these Nav1.9 mutations confer gain-of-function attributes to the channel, depolarize resting membrane potential of dorsal root ganglion neurons, enhance spontaneous firing, and increase evoked firing of these neurons. Our data show, for the first time, missense mutations of Nav1.9 in individuals with painful peripheral neuropathy. These genetic and functional observations identify missense mutations of Nav1.9 as a cause of painful peripheral neuropathy. © The Author (2014). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  13. Ischemia-related subcellular redistribution of sodium channels enhances the proarrhythmic effect of class I antiarrhythmic drugs: a simulation study.

    Directory of Open Access Journals (Sweden)

    Kunichika Tsumoto

    Full Text Available Cardiomyocytes located at the ischemic border zone of infarcted ventricle are accompanied by redistribution of gap junctions, which mediate electrical transmission between cardiomyocytes. This ischemic border zone provides an arrhythmogenic substrate. It was also shown that sodium (Na+ channels are redistributed within myocytes located in the ischemic border zone. However, the roles of the subcellular redistribution of Na+ channels in the arrhythmogenicity under ischemia remain unclear.Computer simulations of excitation conduction were performed in a myofiber model incorporating both subcellular Na+ channel redistribution and the electric field mechanism, taking into account the intercellular cleft potentials.We found in the myofiber model that the subcellular redistribution of the Na+ channels under myocardial ischemia, decreasing in Na+ channel expression of the lateral cell membrane of each myocyte, decreased the tissue excitability, resulting in conduction slowing even without any ischemia-related electrophysiological change. The conventional model (i.e., without the electric field mechanism did not reproduce the conduction slowing caused by the subcellular Na+ channel redistribution. Furthermore, Na+ channel blockade with the coexistence of a non-ischemic zone with an ischemic border zone expanded the vulnerable period for reentrant tachyarrhythmias compared to the model without the ischemic border zone. Na+ channel blockade tended to cause unidirectional conduction block at sites near the ischemic border zone. Thus, such a unidirectional conduction block induced by a premature stimulus at sites near the ischemic border zone is associated with the initiation of reentrant tachyarrhythmias.Proarrhythmia of Na+ channel blockade in patients with old myocardial infarction might be partly attributable to the ischemia-related subcellular Na+ channel redistribution.

  14. Point mutations in the sodium channel gene conferring tau-fluvalinate resistance in Varroa destructor.

    Science.gov (United States)

    Hubert, Jan; Nesvorna, Marta; Kamler, Martin; Kopecky, Jan; Tyl, Jan; Titera, Dalibor; Stara, Jitka

    2014-06-01

    Sodium channels (SCs) in mites and insects are target sites for pesticides, including pyrethroids. Point mutations in the SC gene have been reported to change the structural conformation of the protein and its sensitivity to pesticides. To find mutations in the SC gene of the mite Varroa destructor (VmNa), the authors analysed the VmNa gene sequences available in GenBank and prepared specific primers for the amplification of two fragments containing the regions coding for (i) the domain II S4-S6 region (bp 2805-3337) and (ii) the domain III S4-3' terminus region (bp 4737-6500), as determined according to the VmNa cDNA sequence AY259834. Sensitive and resistant mite populations did not differ in the amino acid sequences of the III S4-3' terminus VmNa region. However, differences were found in the IIS4-IIS6 fragment. In the resistant population, the mutation C(3004) → G resulted in the substitution L(1002) → V (codon ctg → gtg) at the position equivalent to that of the housefly L925 in the domain II S5 helix. Additionally, the mutation F(1052) → L (codon ttc → ctc) at the position equivalent to that of the housefly F975 in the domain II P-loop connecting segments S5 and S6 was detected in both the resistant and sensitive populations. All individuals that survived the tau-fluvalinate treatment in the bioassay harboured the L(1002) → V mutation combined with the F(1052), while dead individuals from both the sensitive and resistant populations harboured mostly the L(1002) residue and either of the two residues at position 1052. © 2013 Society of Chemical Industry.

  15. Selective genotyping reveals association between the epithelial sodium channel gamma-subunit and systolic blood pressure.

    Science.gov (United States)

    Büsst, Cara J; Scurrah, Katrina J; Ellis, Justine A; Harrap, Stephen B

    2007-10-01

    Systolic blood pressure is determined in large part by genes. Six independent studies have reported evidence of linkage between systolic pressure and chromosome 16p12 that incorporates SCNN1G, the gene encoding the gamma-subunit of the epithelial sodium channel. We undertook the first comprehensive association analysis of SCNN1G and systolic pressure. To achieve genetic contrast, we sampled unrelated subjects within the upper (mean: 166 mm Hg; n=96) and lower (mean: 98 mm Hg; n=94) 10% of the systolic pressure distribution of 2911 subjects from the Victorian Family Heart Study. We examined genotypes and haplotypes related to 26 single nucleotide polymorphisms across SCNN1G and its promoter. Each of 3 single nucleotide polymorphisms (rs13331086, rs11074553, and rs4299163) in introns 5 and 6 showed evidence of association with systolic pressure in logistic regression analyses adjusted for age, sex, and body mass index. Considered as a haplotype block, these single nucleotide polymorphisms were significantly associated with systolic pressure (haplo.score global: P=0.0001). In permutation analyses to account for multiple testing, a result such as this was observed only once in 10,000 permutations. The estimated frequency of 1 haplotype (TGC) was substantially greater in high (13.3%) than low (0.6%) systolic pressure subjects (P=0.0001). Three other haplotypes (TGG, TAC, and GGC) showed associations with high or low systolic pressure consistent with the observed associations of their composite alleles. These findings identify relatively common polymorphisms in the SCNN1G gene that are associated with high systolic blood pressure in the general Australian white population.

  16. Juvenile growth reduces the influence of epithelial sodium channels on myogenic tone in skeletal muscle arterioles.

    Science.gov (United States)

    Kang, Lori S; Masilamani, Shyama; Boegehold, Matthew A

    2016-12-01

    Previous studies have documented that rapid juvenile growth is accompanied by functional changes in the arteriolar endothelium, but much less is known about functional changes in arteriolar smooth muscle over this period. In this study, we investigate the possible contribution of epithelial sodium channels (ENaC) to the myogenic behaviour of arterioles at two stages of juvenile growth. The effects of the ENaC inhibitor benzamil on different levels of myogenic tone were studied in isolated gracilis muscle arterioles from rats aged 21-28 days ("weanlings") and 42-49 days ("juveniles"). ENaC subunit expression in the arteriolar wall was also determined, and the interaction between ENaC and nitric oxide (NO) in regulating vascular tone was explored by combined use of benzamil and NG -monomethyl-l-arginine (l-NMMA). At physiological pressures, both steady-state myogenic tone and the dynamic adjustments in this tone triggered by acute pressure changes were less in juvenile arterioles than in weanling arterioles. α, β and γ ENaC protein was present in arterioles at both ages, but benzamil only had an effect on myogenic tone in weanling arterioles. In these vessels, benzamil increased, rather than decreased, myogenic tone, and this effect was prevented by l-NMMA or endothelial removal. These findings suggest that although ENaC is present in gracilis muscle arterioles of both weanling and juvenile rats, it is not obligatory for the genesis of myogenic activity in these vessels at either age. However, ENaC activity can significantly modulate the level of myogenic tone through stimulation of endothelial NO release at an early stage of growth. © 2016 John Wiley & Sons Australia, Ltd.

  17. Activation of the Epithelial Sodium Channel (ENaC) by the Alkaline Protease from Pseudomonas aeruginosa*

    Science.gov (United States)

    Butterworth, Michael B.; Zhang, Liang; Heidrich, Elisa M.; Myerburg, Michael M.; Thibodeau, Patrick H.

    2012-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen that significantly contributes to the mortality of patients with cystic fibrosis. Chronic infection by Pseudomonas induces sustained immune and inflammatory responses and damage to the airway. The ability of Pseudomonas to resist host defenses is aided, in part, by secreted proteases, which act as virulence factors in multiple modes of infection. Recent studies suggest that misregulation of protease activity in the cystic fibrosis lung may alter fluid secretion and pathogen clearance by proteolytic activation of the epithelial sodium channel (ENaC). To evaluate the possibility that proteolytic activation of ENaC may contribute to the virulence of Pseudomonas, primary human bronchial epithelial cells were exposed to P. aeruginosa and ENaC function was assessed by short circuit current measurements. Apical treatment with a strain known to express high levels of alkaline protease (AP) resulted in an increase in basal ENaC current and a loss of trypsin-inducible ENaC current, consistent with sustained activation of ENaC. To further characterize this AP-induced ENaC activation, AP was purified, and its folding, activity, and ability to activate ENaC were assessed. AP folding was efficient under pH and calcium conditions thought to exist in the airway surface liquid of normal and cystic fibrosis (CF) lungs. Short circuit measurements of ENaC in polarized monolayers indicated that AP activated ENaC in immortalized cell lines as well as post-transplant, primary human bronchial epithelial cells from both CF and non-CF patients. This activation was mapped to the γ-subunit of ENaC. Based on these data, patho-mechanisms associated with AP in the CF lung are proposed wherein secretion of AP leads to decreased airway surface liquid volume and a corresponding decrease in mucocilliary clearance of pulmonary pathogens. PMID:22859302

  18. Changes of voltage-gated sodium channels in sensory nerve regeneration and neuropathic pain models.

    Science.gov (United States)

    Casals-Díaz, Laura; Casas, Caty; Navarro, Xavier

    2015-01-01

    The present study was conducted to determine changes in the expression of voltage-gated sodium channels (VGSCs) α-subunits after nerve injury and their relation with development of neuropathic pain. We used the crush injury model of regeneration of the sciatic nerve (Crush) and the spared nerve injury (SNI) model of neuropathic pain in the rat. Measurements of thermal and mechanical pain thresholds were performed until 3 months after injury. Real-time PCR and immunohistochemistry of VGSC α-subunits were used to evaluate the mRNA and protein expression in the DRG. Both nerve injuries induced similar alterations in the VGSCs expression at 7 dpi, with upregulation of Nav1.3, and downregulation of Nav1.7, Nav1.8 and Nav1.9. These changes persisted until 28 days, when hyperalgesia was still present in SNI but not in Crush rats. At 90 days, mRNA expression of all analyzed α-subunits returned to basal levels in the Crush group. However, SNI rats still showed altered expression of VGSCs, and neuropathic pain responses. Immunohistochemical staining revealed that Nav1.8 and Nav1.9 were widely expressed in IB4-positive neurons of the DRG, relevant in pain processing. The population of neurons coexpressing each α-subunit and IB4 was also affected by the injury, more markedly after the Crush. Shifts in VGSCs expression occur in parallel to neuropathic pain behavior in rats early after injury, while at later times they appear to be more related to sensory nerve degeneration and regeneration processes.

  19. Rate of action of Anemonia sulcata toxin II on sodium channels in myelinated nerve fibres.

    Science.gov (United States)

    Schmidtmayer, J; Stoye-Herzog, M; Ulbricht, W

    1982-10-01

    1. The effect of Anemonia sulcata toxin II (ATX II) on single myelinated nerve fibres of the frog, Rana esculenta, was investigated. 2. ATX II promptly and reversibly increased the duration of action potentials; on applying 9.5 micro M the time, t0.5, to reach half of the final effect was 2.6 s. In the presence of 10 mM tetraethylammonium the duration was very sensitive to ATX II and as little as 10 nM could be detected. 3. The underlying mechanism was a diphasic incomplete inactivation of sodium channels which, at 15 degrees C, caused a sizeable INa to persist after 15 ms depolarization (I15ms). 4. On applying 5 micro M (1.25 micro M) ATX II at ca. 15 degrees C, I15ms developed with a sigmoid time course whose t0.5 was 1.5 s (2.6 s) and on washing declined in a near-exponential fashion with tau off = 6.1 s (6.4 s). Washing after a short (1-2s) application led to a transient considerable increase in I15ms followed by a faster decline with tau'off less than tau off. 5. Cooling decreased the rate of action with a Q10 of 1/tau0.5 = 1.9 and of 1/tau off = 2.0 (between 7 and 14 degrees C). ATX I (up to 15 micro M) was ineffective and did not antagonize ATX II. 6. Both rates of diffusional access and reaction seem to contribute to the rate of action. The results suggest a superficial binding site.

  20. Value of the sodium-channel blocker challenge in Brugada syndrome.

    Science.gov (United States)

    Therasse, Dylan; Sacher, Frederic; Babuty, Dominique; Mabo, Philippe; Mansourati, Jacques; Kyndt, Florence; Redon, Richard; Schott, Jean Jacques; Barc, Julien; Probst, Vincent; Gourraud, Jean-Baptiste

    2017-10-15

    Intravenous drug challenge is frequently performed to unmask Brugada syndrome (BrS). However, its true sensitivity has never been assessed. We used the obligate BrS transmitters in families affected by BrS to evaluate the true accuracy of drug challenge. All consecutive patients from 2000 to 2014 who underwent drug challenge during familial screening for BrS were included in the study. Obligate BrS transmitters were defined as the presence of a descendant and non-descendant first-degree relative affected by BrS. Two physicians blinded to the clinical and genetic status reviewed the data. Among 705 drug challenges performed in 149 families, 50 were performed in obligate transmitters from 42 different families. SCN5A mutations were identified in 20 families. Two obligate transmitters were not carrier of the familial mutation. Based on obligate transmitters, sensitivity was 100% for Ajmaline vs 77% for Flecainide (P=0.002). Based on the presence of the familial SCN5A mutation in all family relatives, sensitivity and specificity of sodium channel blocker challenge were respectively 78% (95/122) and 46% (68/148). During a median follow-up of 91 (26-136) months, 2 ventricular fibrillations occurred in obligate transmitters. We demonstrated that Ajmaline challenge presents an excellent sensitivity that may rule out the diagnosis of BrS when negative. Conversely, a negative Flecainide challenge may not prevent from Brs inheritance and risk of SCD. This may lead to suggest systematic use of Ajmaline during drug challenge. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Selective Voltage-Gated Sodium Channel Peptide Toxins from Animal Venom: Pharmacological Probes and Analgesic Drug Development.

    Science.gov (United States)

    Wu, Ying; Ma, Hui; Zhang, Fan; Zhang, Chunlei; Zou, Xiaohan; Cao, Zhengyu

    2017-12-08

    Voltage-gated sodium channels (Navs) play critical roles in action potential generation and propagation. Nav channelopathy as well as pathological sensitization contribute to allodynia and hyperalgesia. Recent evidence has demonstrated the significant roles of Nav subtypes (Nav1.3, 1.7, 1.8, and 1.9) in nociceptive transduction, and therefore these Navs may represent attractive targets for analgesic drug discovery. Animal toxins are structurally diverse peptides that are highly potent yet selective on ion channel subtypes and therefore represent valuable probes to elucidate the structures, gating properties, and cellular functions of ion channels. In this review, we summarize recent advances on peptide toxins from animal venom that selectively target Nav1.3, 1.7, 1.8, and 1.9, along with their potential in analgesic drug discovery.

  2. Structural Basis for the Inhibition of Voltage-gated Sodium Channels by Conotoxin μO§-GVIIJ.

    Science.gov (United States)

    Green, Brad R; Gajewiak, Joanna; Chhabra, Sandeep; Skalicky, Jack J; Zhang, Min-Min; Rivier, Jean E; Bulaj, Grzegorz; Olivera, Baldomero M; Yoshikami, Doju; Norton, Raymond S

    2016-03-25

    Cone snail toxins are well known blockers of voltage-gated sodium channels, a property that is of broad interest in biology and therapeutically in treating neuropathic pain and neurological disorders. Although most conotoxin channel blockers function by direct binding to a channel and disrupting its normal ion movement, conotoxin μO§-GVIIJ channel blocking is unique, using both favorable binding interactions with the channel and a direct tether via an intermolecular disulfide bond. Disulfide exchange is possible because conotoxin μO§-GVIIJ contains anS-cysteinylated Cys-24 residue that is capable of exchanging with a free cysteine thiol on the channel surface. Here, we present the solution structure of an analog of μO§-GVIIJ (GVIIJ[C24S]) and the results of structure-activity studies with synthetic μO§-GVIIJ variants. GVIIJ[C24S] adopts an inhibitor cystine knot structure, with two antiparallel β-strands stabilized by three disulfide bridges. The loop region linking the β-strands (loop 4) presents residue 24 in a configuration where it could bind to the proposed free cysteine of the channel (Cys-910, rat NaV1.2 numbering; at site 8). The structure-activity study shows that three residues (Lys-12, Arg-14, and Tyr-16) located in loop 2 and spatially close to residue 24 were also important for functional activity. We propose that the interaction of μO§-GVIIJ with the channel depends on not only disulfide tethering via Cys-24 to a free cysteine at site 8 on the channel but also the participation of key residues of μO§-GVIIJ on a distinct surface of the peptide. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. The voltage-gated sodium channel Na(v)1.9 is an effector of peripheral inflammatory pain hypersensitivity.

    Science.gov (United States)

    Amaya, Fumimasa; Wang, Haibin; Costigan, Michael; Allchorne, Andrew J; Hatcher, Jon P; Egerton, Julie; Stean, Tania; Morisset, Valerie; Grose, David; Gunthorpe, Martin J; Chessell, Iain P; Tate, Simon; Green, Paula J; Woolf, Clifford J

    2006-12-13

    We used a mouse with deletion of exons 4, 5, and 6 of the SCN11A (sodium channel, voltage-gated, type XI, alpha) gene that encodes the voltage-gated sodium channel Na(v)1.9 to assess its contribution to pain. Na(v)1.9 is present in nociceptor sensory neurons that express TRPV1, bradykinin B2, and purinergic P2X3 receptors. In Na(v)1.9-/- mice, the non-inactivating persistent tetrodotoxin-resistant sodium TTXr-Per current is absent, whereas TTXr-Slow is unchanged. TTXs currents are unaffected by the mutation of Na(v)1.9. Pain hypersensitivity elicited by intraplantar administration of prostaglandin E2, bradykinin, interleukin-1beta, capsaicin, and P2X3 and P2Y receptor agonists, but not NGF, is either reduced or absent in Na(v)1.9-/- mice, whereas basal thermal and mechanical pain sensitivity is unchanged. Thermal, but not mechanical, hypersensitivity produced by peripheral inflammation (intraplanatar complete Freund's adjuvant) is substantially diminished in the null allele mutant mice, whereas hypersensitivity in two neuropathic pain models is unchanged in the Na(v)1.9-/- mice. Na(v)1.9 is, we conclude, an effector of the hypersensitivity produced by multiple inflammatory mediators on nociceptor peripheral terminals and therefore plays a key role in mediating peripheral sensitization.

  4. Steady-state properties of sodium channels from healthy and tumorous human brain

    NARCIS (Netherlands)

    Frenkel, C.; Wartenberg, H. C.; Duch, D. S.; Urban, B. W.

    1998-01-01

    This extensive bilayer study of unpurified human brain channels from non-diseased and tumorous human brain involves more than 300 lipid bilayer experiments. Single channel conductances and subconductances, single channel fractional open times, the voltage-dependence of tetrodotoxin (TTX) block and

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

    Directory of Open Access Journals (Sweden)

    Cédric James Laedermann

    2015-11-01

    Full Text Available In the peripheral sensory nervous system the neuronal expression of voltage-gated sodium channels (Navs is a very important for the transmission of nociceptive information since they give rise to the upstroke of the action potential. Navs are composed of 9 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 action potential and consequently modify pain 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 the subunit of 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

  6. Lacosamide and sodium channel-blocking antiepileptic drug cross-titration against levetiracetam background therapy.

    Science.gov (United States)

    Baulac, M; Byrnes, W; Williams, P; Borghs, S; Webster, E; De Backer, M; Dedeken, P

    2017-04-01

    To assess prospectively the effectiveness of lacosamide (LCM) added to levetiracetam (LEV) after down-titration of a concomitant sodium channel blocker (SCB) among patients with focal epilepsy not adequately controlled on LEV and SCB. In this open-label trial, LCM was initiated at 100 mg/day and up-titrated to 200-600 mg/day over 9 weeks; SCB down-titration started when LCM dose reached 200 mg/day. Patients remained on stable LCM/LEV doses for 12 weeks' maintenance (21-week treatment period). The primary outcome was retention rate on LCM. Due to recruitment challenges, fewer than the planned 300 patients participated in the trial, resulting in the trial being underpowered. Overall, 120 patients (mean age 39.7 years) started and 93 completed the trial. The most frequently used SCBs were lamotrigine (39.2%), carbamazepine (30.8%) and oxcarbazepine (27.5%). Eighty-four patients adhered to protocol and discontinued their SCB after cross-titration, but there was insufficient evidence for 36 patients. Retention rate was 73.3% (88/120) for all patients and 83.3% (70/84) for those with evidence of SCB discontinuation. Seizure freedom for patients completing maintenance was 14.0% (13/93). Discontinuation due to adverse events (6.7%) and lack of efficacy (3.3%) occurred primarily during cross-titration. Most frequently reported adverse events during treatment were dizziness (23.3%), headache (15.0%) and fatigue (8.3%). In patients with uncontrolled seizures on LEV/SCB, the LCM/LEV combination appeared to be effective and well tolerated. A cross-titration schedule-flexible LCM up-titration, concomitant SCB down-titration and stable background LEV-could present a feasible and practical approach to initiating LCM while minimizing pharmacodynamic interactions with a SCB. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Inhibition of voltage-gated sodium channels by emulsified isoflurane may contribute to its subarachnoid anesthetic effect in beagle dogs.

    Science.gov (United States)

    Zhou, Cheng; Wu, Wei; Liu, Jin; Liao, Da-qing; Kang, Yi; Chen, Xiang-dong

    2011-01-01

    Volatile anesthetics, in addition to their general anesthesia action, have been proven to produce regional anesthetic effect in various animal models. The major aim of this study was to examine whether emulsified isoflurane (EI) could also produce subarachnoid anesthesia and to investigate its possible mechanism. Beagle dogs were randomly assigned into 5 groups (n = 6/group): intrathecally receiving 1% lidocaine 0.1 mL/kg, 30% intralipid 0.1 mL/kg (control), or 8% EI at doses of 0.05, 0.075, or 0.1 mL/kg, respectively. Consciousness state, motor function of limbs, and response to nociceptive stimulus were observed after drug administration. The effect of EI on voltage-gated Na channel was recorded from isolated spinal neurons of rats, using the whole-cell patch-clamp technique. Inhibition of peak sodium currents and effect of EI on Na channel gating were analyzed. Emulsified isoflurane produced subarachnoid anesthesia in a dose-dependent manner, and at the dose of 0.1 mL/kg, the effect of 8% EI was similar to 1% lidocaine. Sodium channel currents were inhibited by EI at clinically relevant concentrations, with the IC50 (median inhibitory concentration) at 0.69 ± 0.08 mM. Voltage activation of Na channels was positive, shifted by isoflurane at the concentration of 0.77 mM, and V½ of activation (voltage for half-maximal activation) shifted from -12.4 ± 2.7 mV to -7.3 ± 2.3 mV (P channels in the spinal cord.

  8. Inhibitors of arachidonate-regulated calcium channel signaling suppress triggered activity induced by the late sodium current.

    Science.gov (United States)

    Wolkowicz, Paul; Umeda, Patrick K; Sharifov, Oleg F; White, C Roger; Huang, Jian; Mahtani, Harry; Urthaler, Ferdinand

    2014-02-05

    Disturbances in myocyte calcium homeostasis are hypothesized to be one cause for cardiac arrhythmia. The full development of this hypothesis requires (i) the identification of all sources of arrhythmogenic calcium and (ii) an understanding of the mechanism(s) through which calcium initiates arrhythmia. To these ends we superfused rat left atria with the late sodium current activator type II Anemonia sulcata toxin (ATXII). This toxin prolonged atrial action potentials, induced early afterdepolarization, and provoked triggered activity. The calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93 (N-[2-[[[3-(4-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulphon-amide) suppressed ATXII triggered activity but its inactive congener KN-92 (2-[N-(4-methoxy benzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) did not. Neither drug affected normal atrial contractility. Calcium entry via L-type channels or calcium leakage from sarcoplasmic reticulum stores are not critical for this type of ectopy as neither verapamil ((RS)-2-(3,4-dimethoxyphenyl)-5-{[2-(3,4-dimethoxyphenyl)ethyl]-(methyl)amino}-2-prop-2-ylpentanenitrile) nor ryanodine affected ATXII triggered activity. By contrast, inhibitors of the voltage independent arachidonate-regulated calcium (ARC) channel and the store-operated calcium channel specifically suppressed ATXII triggered activity without normalizing action potentials or affecting atrial contractility. Inhibitors of cytosolic calcium-dependent phospholipase A2 also suppressed triggered activity suggesting that this lipase, which generates free arachidonate, plays a key role in ATXII ectopy. Thus, increased left atrial late sodium current appears to activate atrial Orai-linked ARC and store operated calcium channels, and these voltage-independent channels may be unexpected sources for the arrhythmogenic calcium that underlies triggered activity. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Neonatal maternal deprivation sensitizes voltage-gated sodium channel currents in colon-specific dorsal root ganglion neurons in rats.

    Science.gov (United States)

    Hu, Shufen; Xiao, Ying; Zhu, Liyan; Li, Lin; Hu, Chuang-Ying; Jiang, Xinghong; Xu, Guang-Yin

    2013-02-15

    Irritable bowel syndrome (IBS) is a common gastrointestinal disorder characterized by abdominal pain in association with altered bowel movements. The underlying mechanisms of visceral hypersensitivity remain elusive. This study was designed to examine the role for sodium channels in a rat model of chronic visceral hyperalgesia induced by neonatal maternal deprivation (NMD). Abdominal withdrawal reflex (AWR) scores were performed on adult male rats. Colon-specific dorsal root ganglion (DRG) neurons were labeled with DiI and acutely dissociated for measuring excitability and sodium channel current under whole-cell patch-clamp configurations. The expression of Na(V)1.8 was analyzed by Western blot and quantitative real-time PCR. NMD significantly increased AWR scores, which lasted for ~6 wk in an association with hyperexcitability of colon DRG neurons. TTX-resistant but not TTX-sensitive sodium current density was greatly enhanced in colon DRG neurons in NMD rats. Compared with controls, activation curves showed a leftward shift in NMD rats whereas inactivation curves did not differ significantly. NMD markedly accelerated the activation time of peak current amplitude without any changes in inactivation time. Furthermore, NMD remarkably enhanced expression of Na(V)1.8 at protein levels but not at mRNA levels in colon-related DRGs. The expression of Na(V)1.9 was not altered after NMD. These data suggest that NMD enhances TTX-resistant sodium activity of colon DRG neurons, which is most likely mediated by a leftward shift of activation curve and by enhanced expression of Na(V)1.8 at protein levels, thus identifying a specific molecular mechanism underlying chronic visceral pain and sensitization in patients with IBS.

  10. Regulation of Podosome Formation in Macrophages by a Splice Variant of the Sodium Channel SCN8A*S⃞

    Science.gov (United States)

    Carrithers, Michael D.; Chatterjee, Gouri; Carrithers, Lisette M.; Offoha, Roosevelt; Iheagwara, Uzoma; Rahner, Christoph; Graham, Morven; Waxman, Stephen G.

    2009-01-01

    Voltage-gated sodium channels initiate electrical signaling in excitable cells such as muscle and neurons. They also are expressed in non-excitable cells such as macrophages and neoplastic cells. Previously, in macrophages, we demonstrated expression of SCN8A, the gene that encodes the channel NaV1.6, and intracellular localization of NaV1.6 to regions near F-actin bundles, particularly at areas of cell attachment. Here we show that a splice variant of NaV1.6 regulates cellular invasion through its effects on podosome and invadopodia formation in macrophages and melanoma cells. cDNA sequence analysis of SCN8A from THP-1 cells, a human monocyte-macrophage cell line, confirmed the expression of a full-length splice variant that lacks exon 18. Immunoelectron microscopy demonstrated NaV1.6-positive staining within the electron dense podosome rosette structure. Pharmacologic antagonism with tetrodotoxin (TTX) in differentiated THP-1 cells or absence of functional NaV1.6 through a naturally occurring mutation (med) in mouse peritoneal macrophages inhibited podosome formation. Agonist-mediated activation of the channel with veratridine caused release of sodium from cationic vesicular compartments, uptake by mitochondria, and mitochondrial calcium release through the Na/Ca exchanger. Invasion by differentiated THP-1 and HTB-66 cells, an invasive melanoma cell line, through extracellular matrix was inhibited by TTX. THP-1 invasion also was inhibited by small hairpin RNA knockdown of SCN8A. These results demonstrate that a variant of NaV1.6 participates in the control of podosome and invadopodia formation and suggest that intracellular sodium release mediated by NaV1.6 may regulate cellular invasion of macrophages and melanoma cells. PMID:19136557

  11. Regulation of podosome formation in macrophages by a splice variant of the sodium channel SCN8A.

    Science.gov (United States)

    Carrithers, Michael D; Chatterjee, Gouri; Carrithers, Lisette M; Offoha, Roosevelt; Iheagwara, Uzoma; Rahner, Christoph; Graham, Morven; Waxman, Stephen G

    2009-03-20

    Voltage-gated sodium channels initiate electrical signaling in excitable cells such as muscle and neurons. They also are expressed in non-excitable cells such as macrophages and neoplastic cells. Previously, in macrophages, we demonstrated expression of SCN8A, the gene that encodes the channel NaV1.6, and intracellular localization of NaV1.6 to regions near F-actin bundles, particularly at areas of cell attachment. Here we show that a splice variant of NaV1.6 regulates cellular invasion through its effects on podosome and invadopodia formation in macrophages and melanoma cells. cDNA sequence analysis of SCN8A from THP-1 cells, a human monocyte-macrophage cell line, confirmed the expression of a full-length splice variant that lacks exon 18. Immunoelectron microscopy demonstrated NaV1.6-positive staining within the electron dense podosome rosette structure. Pharmacologic antagonism with tetrodotoxin (TTX) in differentiated THP-1 cells or absence of functional NaV1.6 through a naturally occurring mutation (med) in mouse peritoneal macrophages inhibited podosome formation. Agonist-mediated activation of the channel with veratridine caused release of sodium from cationic vesicular compartments, uptake by mitochondria, and mitochondrial calcium release through the Na/Ca exchanger. Invasion by differentiated THP-1 and HTB-66 cells, an invasive melanoma cell line, through extracellular matrix was inhibited by TTX. THP-1 invasion also was inhibited by small hairpin RNA knockdown of SCN8A. These results demonstrate that a variant of NaV1.6 participates in the control of podosome and invadopodia formation and suggest that intracellular sodium release mediated by NaV1.6 may regulate cellular invasion of macrophages and melanoma cells.

  12. A SCN9A gene-encoded dorsal root ganglia sodium channel polymorphism associated with severe fibromyalgia

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    Vargas-Alarcon Gilberto

    2012-02-01

    Full Text Available Abstract Background A consistent line of investigation suggests that autonomic nervous system dysfunction may explain the multi-system features of fibromyalgia (FM; and that FM is a sympathetically maintained neuropathic pain syndrome. Dorsal root ganglia (DRG are key sympathetic-nociceptive short-circuit sites. Sodium channels located in DRG (particularly Nav1.7 act as molecular gatekeepers for pain detection. Nav1.7 is encoded in gene SCN9A of chromosome 2q24.3 and is predominantly expressed in the DRG pain-sensing neurons and sympathetic ganglia neurons. Several SCN9A sodium channelopathies have been recognized as the cause of rare painful dysautonomic syndromes such as paroxysmal extreme pain disorder and primary erythromelalgia. The aim of this study was to search for an association between fibromyalgia and several SCN9A sodium channels gene polymorphisms. Methods We studied 73 Mexican women suffering from FM and 48 age-matched women who considered themselves healthy. All participants filled out the Fibromyalgia Impact Questionnaire (FIQ. Genomic DNA from whole blood containing EDTA was extracted by standard techniques. The following SCN9A single-nucleotide polymorphisms (SNP were determined by 5' exonuclease TaqMan assays: rs4371369; rs4387806; rs4453709; rs4597545; rs6746030; rs6754031; rs7607967; rs12620053; rs12994338; and rs13017637. Results The frequency of the rs6754031 polymorphism was significantly different in both groups (P = 0.036 mostly due to an absence of the GG genotype in controls. Interestingly; patients with this rs6754031 GG genotype had higher FIQ scores (median = 80; percentile 25/75 = 69/88 than patients with the GT genotype (median = 63; percentile 25/75 = 58/73; P = 0.002 and the TT genotype (median = 71; percentile 25/75 = 64/77; P = 0.001. Conclusion In this ethnic group; a disabling form of FM is associated to a particular SCN9A sodium channel gene variant. These preliminary results raise the possibility that

  13. Mechanism of sodium hydrosulfide modulation of L-type calcium channels in rat colonic smooth muscle cells.

    Science.gov (United States)

    Tang, Qincai; Quan, Xiaojing; Yan, Lin; Ren, Haixia; Chen, Wei; Xia, Hong; Luo, Hesheng

    2018-01-05

    Hydrogen sulfide (H 2 S) can exert different effects on the gastrointestinal tract by modulating ion channels. Previously, we found that H 2 S donor sodium hydrosulfide (NaHS) regulates colonic motility through L-type calcium channels, but the molecular mechanism remains unknown. The present study was designed to investigate possible mechanisms underlying the modulation of L-type calcium channels by NaHS in rat colonic smooth muscle cells. L-type calcium currents in colonic smooth muscle cells were recorded using the whole-cell patch-clamp technique. Spontaneous contractions of mid-colonic smooth muscle strips were measured in an organ bath system and a biological signal acquisition system. NaHS evoked a significant rightward shift in the steady-state activation curve of L-type calcium channels, changed the shape of the current-voltage (I-V) curve, and decreased the peak current density at 0mV, although it significantly increased with higher stimulatory voltage. The sulfhydryl-modifying reagent DL-dithiothreitol (DTT) enhanced the effects of NaHS on L-type calcium channels, while diamide (DM) and reduced L-glutathione (GSH) alleviated the effects of NaHS. Additionally, NaHS inhibited the spontaneous high-amplitude contractions of both longitudinal and circular smooth muscle strips in a dose-dependent manner. The inhibitory effects were reversible. DTT and GSH enhanced the effects of NaHS, while DM attenuated the effects of NaHS. In conclusion, NaHS modulates L-type calcium channels in rat colonic smooth muscle cells and regulates the contractile activity of colonic smooth muscle, potentially by modifying the free sulfhydryl groups of L-type calcium channels. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Electrophysiological and Pharmacological Analyses of Nav1.9 Voltage-Gated Sodium Channel by Establishing a Heterologous Expression System

    OpenAIRE

    Xi Zhou; Zhen Xiao; Yan Xu; Yunxiao Zhang; Dongfang Tang; Xinzhou Wu; Cheng Tang; Minzhi Chen,; Xiaoliu Shi; Ping Chen; Songping Liang; Zhonghua Liu

    2017-01-01

    Nav1. 9 voltage-gated sodium channel is preferentially expressed in peripheral nociceptive neurons. Recent progresses have proved its role in pain sensation, but our understanding of Nav1.9, in general, has lagged behind because of limitations in heterologous expression in mammal cells. In this work, functional expression of human Nav1.9 (hNav1.9) was achieved by fusing GFP to the C-terminal of hNav1.9 in ND7/23 cells, which has been proved to be a reliable method to the electrophysiological ...

  15. Enhancement of carbon nanotube FET performance via direct synthesis of poly (sodium 4-styrenesulfonate) in the transistor channel

    Science.gov (United States)

    Toader, M.; Schubel, R.; Hartmann, M.; Scharfenberg, L.; Jordan, R.; Mertig, M.; Schulz, S. E.; Gessner, T.; Hermann, S.

    2016-09-01

    Direct synthesis of poly (sodium 4-styrenesulfonate) (P(NaSS)) inside the channel of single-walled carbon nanotube (SWCNT) field-effect transistors (FETs), is shown to be highly beneficial in improving the device parameters. Starting with monomeric compounds, the FET-channel was in-situ polymerized, using the self-initiated photografting and photopolymerization process. Upon formation of the P(NaSS) polymer matrix, we report improved device-to-device consistency, lower variability in the threshold voltage, higher drain currents and higher on/off ratios. Annealing in vacuum was shown to further improve the device performance and induce an ambipolar behavior. Moreover, those FET devices showed a long-term stability even under ambient environment.

  16. Cardiac sodium channel NaV1.5 distribution in myocytes via interacting proteins: the multiple pool model.

    Science.gov (United States)

    Shy, Diana; Gillet, Ludovic; Abriel, Hugues

    2013-04-01

    The cardiac sodium current (INa) is responsible for the rapid depolarization of cardiac cells, thus allowing for their contraction. It is also involved in regulating the duration of the cardiac action potential (AP) and propagation of the impulse throughout the myocardium. Cardiac INa is generated by the voltage-gated Na(+) channel, NaV1.5, a 2016-residue protein which forms the pore of the channel. Over the past years, hundreds of mutations in SCN5A, the human gene coding for NaV1.5, have been linked to many cardiac electrical disorders, including the congenital and acquired long QT syndrome, Brugada syndrome, conduction slowing, sick sinus syndrome, atrial fibrillation, and dilated cardiomyopathy. Similar to many membrane proteins, NaV1.5 has been found to be regulated by several interacting proteins. In some cases, these different proteins, which reside in distinct membrane compartments (i.e. lateral membrane vs. intercalated disks), have been shown to interact with the same regulatory domain of NaV1.5, thus suggesting that several pools of NaV1.5 channels may co-exist in cardiac cells. The aim of this review article is to summarize the recent works that demonstrate its interaction with regulatory proteins and illustrate the model that the sodium channel NaV1.5 resides in distinct and different pools in cardiac cells. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Sodium channel SCN8A (Nav1.6: properties and de novo mutations in epileptic encephalopathy and intellectual disability

    Directory of Open Access Journals (Sweden)

    Janelle Elizabeth O'Brien

    2013-10-01

    Full Text Available The sodium channel Nav1.6, encoded by the gene SCN8A, is one of the major voltage-gated channels in human brain. The sequences of sodium channels have been highly conserved during evolution, and minor changes in biophysical properties can have a major impact in vivo. Insight into the role of Nav1.6 has come from analysis of spontaneous and induced mutations of mouse Scn8a during the past 18 years. Only within the past year has the role of SCN8A in human disease become apparent from whole exome and genome sequences of patients with sporadic disease. Unique features of Nav1.6 include its contribution to persistent current, resurgent current, repetitive neuronal firing, and subcellular localization at the axon initial segment and nodes of Ranvier. Loss of Nav1.6 activity results in reduced neuronal excitability, while gain-of-function mutations can increase neuronal excitability. Mouse Scn8a (med mutants exhibit movement disorders including ataxia, tremor and dystonia. Thus far, more than ten human de novo mutations have been identified in patients with two types of disorders, epileptic encephalopathy and intellectual disability. We review these human mutations as well as the unique features of Nav1.6 that contribute to its role in determining neuronal excitability in vivo. A supplemental figure illustrating the positions of amino acid residues within the 4 domains and 24 transmembrane segments of Nav1.6 is provided to facilitate the location of novel mutations within the channel protein.

  18. Role of voltage-gated sodium, potassium and calcium channels in the development of cocaine-associated cardiac arrhythmias

    Science.gov (United States)

    O'Leary, Michael E; Hancox, Jules C

    2010-01-01

    Cocaine is a highly active stimulant that alters dopamine metabolism in the central nervous system resulting in a feeling of euphoria that with time can lead to addictive behaviours. Cocaine has numerous deleterious effects in humans including seizures, vasoconstriction, ischaemia, increased heart rate and blood pressure, cardiac arrhythmias and sudden death. The cardiotoxic effects of cocaine are indirectly mediated by an increase in sympathomimetic stimulation to the heart and coronary vasculature and by a direct effect on the ion channels responsible for maintaining the electrical excitability of the heart. The direct and indirect effects of cocaine work in tandem to disrupt the co-ordinated electrical activity of the heart and have been associated with life-threatening cardiac arrhythmias. This review focuses on the direct effects of cocaine on cardiac ion channels, with particular focus on sodium, potassium and calcium channels, and on the contributions of these channels to cocaine-induced arrhythmias. Companion articles in this edition of the journal examine the epidemiology of cocaine use (Wood & Dargan [1]) and the treatment of cocaine-associated arrhythmias (Hoffmann [2]). PMID:20573078

  19. Voltage-gated sodium channels: (NaV )igating the field to determine their contribution to visceral nociception.

    Science.gov (United States)

    Erickson, Andelain; Deiteren, Annemie; Harrington, Andrea M; Garcia-Caraballo, Sonia; Castro, Joel; Caldwell, Ashlee; Grundy, Luke; Brierley, Stuart M

    2018-01-09

    Chronic visceral pain, altered motility and bladder dysfunction are common, yet poorly managed symptoms of functional and inflammatory disorders of the gastrointestinal and urinary tracts. Recently, numerous human channelopathies of the voltage-gated sodium (NaV ) channel family have been identified, which induce either painful neuropathies, an insensitivity to pain, or alterations in smooth muscle function. The identification of these disorders, in addition to the recent utilisation of genetically modified NaV mice and specific NaV channel modulators, has shed new light on how NaV channels contribute to the function of neuronal and non-neuronal tissues within the gastrointestinal tract and bladder. Here we review the current pre-clinical and clinical evidence to reveal how the nine NaV channel family members (NaV 1.1 - NaV 1.9) contribute to abdominal visceral function in normal and disease states. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  20. Reduced expression and activation of voltage-gated sodium channels contributes to blunted baroreflex sensitivity in heart failure rats.

    Science.gov (United States)

    Tu, Huiyin; Zhang, Libin; Tran, Thai P; Muelleman, Robert L; Li, Yu-Long

    2010-11-15

    Voltage-gated sodium (Na(v)) channels are responsible for initiation and propagation of action potential in the neurons. To explore the mechanisms of chronic heart failure (CHF)-induced baroreflex dysfunction, we measured the expression and current density of Na(v) channel subunits (Na(v)1.7, Na(v)1.8, and Na(v)1.9) in the aortic baroreceptor neurons and investigated the role of Na(v) channels in aortic baroreceptor neuron excitability and baroreflex sensitivity in sham and CHF rats. CHF was induced by left coronary artery ligation. The development of CHF (6-8 weeks after the coronary ligation) was confirmed by hemodynamic and morphological characteristics. Immunofluorescent data indicated that Na(v)1.7 was expressed in A-type (myelinated) and C-type (unmyelinated) nodose neurons, but Na(v)1.8 and Na(v)1.9 were expressed only in C-type nodose neurons. Real-time RT-PCR and Western blot data showed that CHF reduced mRNA and protein expression levels of Na(v) channels in nodose neurons. In addition, using the whole-cell patch-clamp technique, we found that Na(v) current density and cell excitability of the aortic baroreceptor neurons were lower in CHF rats than that in sham rats. Aortic baroreflex sensitivity was blunted in anesthetized CHF rats, compared with that in sham rats. Furthermore, Na(v) channel activator (rATX II, 100 nM) significantly enhanced Na(v) current density and cell excitability of aortic baroreceptor neurons and improved aortic baroreflex sensitivity in CHF rats. These results suggest that reduced expression and activation of the Na(v) channels are involved in the attenuation of baroreceptor neuron excitability, which subsequently contributes to the impairment of baroreflex in CHF state.

  1. Systematic Study of Binding of μ-Conotoxins to the Sodium Channel NaV1.4

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    Somayeh Mahdavi

    2014-12-01

    Full Text Available Voltage-gated sodium channels (NaV are fundamental components of the nervous system. Their dysfunction is implicated in a number of neurological disorders, such as chronic pain, making them potential targets for the treatment of such disorders. The prominence of the NaV channels in the nervous system has been exploited by venomous animals for preying purposes, which have developed toxins that can block the NaV channels, thereby disabling their function. Because of their potency, such toxins could provide drug leads for the treatment of neurological disorders associated with NaV channels. However, most toxins lack selectivity for a given target NaV channel, and improving their selectivity profile among the NaV1 isoforms is essential for their development as drug leads. Computational methods will be very useful in the solution of such design problems, provided accurate models of the protein-ligand complex can be constructed. Using docking and molecular dynamics simulations, we have recently constructed a model for the NaV1.4-μ-conotoxin-GIIIA complex and validated it with the ample mutational data available for this complex. Here, we use the validated NaV1.4 model in a systematic study of binding other μ-conotoxins (PIIIA, KIIIA and BuIIIB to NaV1.4. The binding mode obtained for each complex is shown to be consistent with the available mutation data and binding constants. We compare the binding modes of PIIIA, KIIIA and BuIIIB to that of GIIIA and point out the similarities and differences among them. The detailed information about NaV1.4-μ-conotoxin interactions provided here will be useful in the design of new NaV channel blocking peptides.

  2. Menthol pain relief through cumulative inactivation of voltage-gated sodium channels.

    Science.gov (United States)

    Gaudioso, Christelle; Hao, Jizhe; Martin-Eauclaire, Marie-France; Gabriac, Mélanie; Delmas, Patrick

    2012-02-01

    Menthol is a natural compound of plant origin known to produce cool sensation via the activation of the TRPM8 channel. It is also frequently part of topical analgesic drugs available in a pharmacy, although its mechanism of action is still unknown. Compelling evidence indicates that voltage-gated Na(+) channels are critical for experiencing pain sensation. We tested the hypothesis that menthol may block voltage-gated Na(+) channels in dorsal root ganglion (DRG) neurons. By use of a patch clamp, we evaluated the effects of menthol application on tetrodotoxin (TTX)-resistant Nav1.8 and Nav1.9 channel subtypes in DRG neurons, and on TTX-sensitive Na(+) channels in immortalized DRG neuron-derived F11 cells. The results indicate that menthol inhibits Na(+) channels in a concentration-, voltage-, and frequency-dependent manner. Menthol promoted fast and slow inactivation states, causing use-dependent depression of Na(+) channel activity. In current clamp recordings, menthol inhibited firing at high-frequency stimulation with minimal effects on normal neuronal activity. We found that low concentrations of menthol cause analgesia in mice, relieving pain produced by a Na(+) channel-targeting toxin. We conclude that menthol is a state-selective blocker of Nav1.8, Nav1.9, and TTX-sensitive Na(+) channels, indicating a role for Na(+) channel blockade in the efficacy of menthol as topical analgesic compound. Copyright © 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

  3. Expression of alternatively spliced sodium channel alpha-subunit genes. Unique splicing patterns are observed in dorsal root ganglia.

    Science.gov (United States)

    Raymond, Christopher K; Castle, John; Garrett-Engele, Philip; Armour, Christopher D; Kan, Zhengyan; Tsinoremas, Nicholas; Johnson, Jason M

    2004-10-29

    Molecular medicine requires the precise definition of drug targets, and tools are now in place to provide genome-wide information on the expression and alternative splicing patterns of any known gene. DNA microarrays were used to monitor transcript levels of the nine well-characterized alpha-subunit sodium channel genes across a broad range of tissues from cynomolgus monkey, a non-human primate model. Alternative splicing of human transcripts for a subset of the genes that are expressed in dorsal root ganglia, SCN8A (Na(v)1.6), SCN9A (Na(v)1.7), and SCN11A (Na(v)1.9) was characterized in detail. Genomic sequence analysis among gene family paralogs and between cross-species orthologs suggested specific alternative splicing events within transcripts of these genes, all of which were experimentally confirmed in human tissues. Quantitative PCR revealed that certain alternative splice events are uniquely expressed in dorsal root ganglia. In addition to characterization of human transcripts, alternatively spliced sodium channel transcripts were monitored in a rat model for neuropathic pain. Consistent down-regulation of all transcripts was observed, as well as significant changes in the splicing patterns of SCN8A and SCN9A.

  4. BACE1-/- mice exhibit seizure activity that does not correlate with sodium channel level or axonal localization

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    Hitt Brian D

    2010-08-01

    Full Text Available Abstract Background BACE1 is a key enzyme in the generation of the Aβ peptide that plays a central role in the pathogenesis of Alzheimer's disease. While BACE1 is an attractive therapeutic target, its normal physiological function remains largely unknown. Examination of BACE1-/- mice can provide insight into this function and also help anticipate consequences of BACE1 inhibition. Here we report a seizure-susceptibility phenotype that we have identified and characterized in BACE1-/- mice. Results We find that electroencephalographic recordings reveal epileptiform abnormalities in some BACE1-/- mice, occasionally including generalized tonic-clonic and absence seizures. In addition, we find that kainic acid injection induces seizures of greater severity in BACE1-/- mice relative to BACE1+/+ littermates, and causes excitotoxic cell death in a subset of BACE1-/- mice. This hyperexcitability phenotype is variable and appears to be manifest in approximately 30% of BACE1-/- mice. Finally, examination of the expression and localization of the voltage-gated sodium channel α-subunit Nav1.2 reveals no correlation with BACE1 genotype or any measure of seizure susceptibility. Conclusions Our data indicate that BACE1 deficiency predisposes mice to spontaneous and pharmacologically-induced seizure activity. This finding has implications for the development of safe therapeutic strategies for reducing Aβ levels in Alzheimer's disease. Further, we demonstrate that altered sodium channel expression and axonal localization are insufficient to account for the observed effect, warranting investigation of alternative mechanisms.

  5. BACE1-/- mice exhibit seizure activity that does not correlate with sodium channel level or axonal localization.

    Science.gov (United States)

    Hitt, Brian D; Jaramillo, Thomas C; Chetkovich, Dane M; Vassar, Robert

    2010-08-23

    BACE1 is a key enzyme in the generation of the Abeta peptide that plays a central role in the pathogenesis of Alzheimer's disease. While BACE1 is an attractive therapeutic target, its normal physiological function remains largely unknown. Examination of BACE1-/- mice can provide insight into this function and also help anticipate consequences of BACE1 inhibition. Here we report a seizure-susceptibility phenotype that we have identified and characterized in BACE1-/- mice. We find that electroencephalographic recordings reveal epileptiform abnormalities in some BACE1-/- mice, occasionally including generalized tonic-clonic and absence seizures. In addition, we find that kainic acid injection induces seizures of greater severity in BACE1-/- mice relative to BACE1+/+ littermates, and causes excitotoxic cell death in a subset of BACE1-/- mice. This hyperexcitability phenotype is variable and appears to be manifest in approximately 30% of BACE1-/- mice. Finally, examination of the expression and localization of the voltage-gated sodium channel alpha-subunit Nav1.2 reveals no correlation with BACE1 genotype or any measure of seizure susceptibility. Our data indicate that BACE1 deficiency predisposes mice to spontaneous and pharmacologically-induced seizure activity. This finding has implications for the development of safe therapeutic strategies for reducing Abeta levels in Alzheimer's disease. Further, we demonstrate that altered sodium channel expression and axonal localization are insufficient to account for the observed effect, warranting investigation of alternative mechanisms.

  6. Voltage-gated sodium channels in nociceptive versus non-nociceptive nodose vagal sensory neurons innervating guinea pig lungs

    Science.gov (United States)

    Kwong, Kevin; Carr, Michael J; Gibbard, Anna; Savage, Tony J; Singh, Kuljit; Jing, Junping; Meeker, Sonya; Undem, Bradley J

    2008-01-01

    Lung vagal sensory fibres are broadly categorized as C fibres (nociceptors) and A fibres (non-nociceptive; rapidly and slowly adapting low-threshold stretch receptors). These afferent fibre types differ in degree of myelination, conduction velocity, neuropeptide content, sensitivity to chemical and mechanical stimuli, as well as evoked reflex responses. Recent studies in nociceptive fibres of the somatosensory system indicated that the tetrodotoxin-resistant (TTX-R) voltage-gated sodium channels (VGSC) are preferentially expressed in the nociceptive fibres of the somatosensory system (dorsal root ganglia). Whereas TTX-R sodium currents have been documented in lung vagal sensory nerves fibres, a rigorous comparison of their expression in nociceptive versus non-nociceptive vagal sensory neurons has not been carried out. Using multiple approaches including patch clamp electrophysiology, immunohistochemistry, and single-cell gene expression analysis in the guinea pig, we obtained data supporting the hypothesis that the TTX-R sodium currents are similarly distributed between nodose ganglion A-fibres and C-fibres innervating the lung. Moreover, mRNA and immunoreactivity for the TTX-R VGSC molecules NaV1.8 and NaV1.9 were present in nearly all neurons. We conclude that contrary to findings in the somatosensory neurons, TTX-R VGSCs are not preferentially expressed in the nociceptive C-fibre population innervating the lungs. PMID:18187475

  7. PITX2 Modulates Atrial Membrane Potential and the Antiarrhythmic Effects of Sodium-Channel Blockers.

    Science.gov (United States)

    Syeda, Fahima; Holmes, Andrew P; Yu, Ting Y; Tull, Samantha; Kuhlmann, Stefan Michael; Pavlovic, Davor; Betney, Daniel; Riley, Genna; Kucera, Jan P; Jousset, Florian; de Groot, Joris R; Rohr, Stephan; Brown, Nigel A; Fabritz, Larissa; Kirchhof, Paulus

    2016-10-25

    Antiarrhythmic drugs are widely used to treat patients with atrial fibrillation (AF), but the mechanisms conveying their variable effectiveness are not known. Recent data suggested that paired like homeodomain-2 transcription factor (PITX2) might play an important role in regulating gene expression and electrical function of the adult left atrium (LA). After determining LA PITX2 expression in AF patients requiring rhythm control therapy, the authors assessed the effects of Pitx2c on LA electrophysiology and the effect of antiarrhythmic drugs. LA PITX2 messenger ribonucleic acid (mRNA) levels were measured in 95 patients undergoing thoracoscopic AF ablation. The effects of flecainide, a sodium (Na + )-channel blocker, and d,l-sotalol, a potassium channel blocker, were studied in littermate mice with normal and reduced Pitx2c mRNA by electrophysiological study, optical mapping, and patch clamp studies. PITX2-dependent mechanisms of antiarrhythmic drug action were studied in human embryonic kidney (HEK) cells expressing human Na channels and by modeling human action potentials. Flecainide 1 μmol/l was more effective in suppressing atrial arrhythmias in atria with reduced Pitx2c mRNA levels (Pitx2c +/- ). Resting membrane potential was more depolarized in Pitx2c +/- atria, and TWIK-related acid-sensitive K + channel 2 (TASK-2) gene and protein expression were decreased. This resulted in enhanced post-repolarization refractoriness and more effective Na-channel inhibition. Defined holding potentials eliminated differences in flecainide's effects between wild-type and Pitx2c +/- atrial cardiomyocytes. More positive holding potentials replicated the increased effectiveness of flecainide in blocking human Na v 1.5 channels in HEK293 cells. Computer modeling reproduced an enhanced effectiveness of Na-channel block when resting membrane potential was slightly depolarized. PITX2 mRNA modulates atrial resting membrane potential and thereby alters the effectiveness of Na-channel

  8. A mutation in segment I-S6 alters slow inactivation of sodium channels.

    Science.gov (United States)

    Wang, S Y; Wang, G K

    1997-01-01

    Slow inactivation occurs in voltage-gated Na+ channels when the membrane is depolarized for several seconds, whereas fast inactivation takes place rapidly within a few milliseconds. Unlike fast inactivation, the molecular entity that governs the slow inactivation of Na+ channels has not been as well defined. Some regions of Na+ channels, such as mu1-W402C and mu1-T698M, have been reported to affect slow inactivation. A mutation in segment I-S6 of mu1 Na+ channels, N434A, shifts the voltage dependence of activation and fast inactivation toward the depolarizing direction. The mutant Na+ current at +50 mV is diminished by 60-80% during repetitive stimulation at 5 Hz, resulting in a profound use-dependent phenomenon. This mutant phenotype is due to the enhancement of slow inactivation, which develops faster than that of wild-type channels (tau = 0.46 +/- 0.01 s versus 2.11 +/- 0.10 s at +30 mV, n = 9). An oxidant, chloramine-T, abolishes fast inactivation and yet greatly accelerates slow inactivation in both mutant and wild-type channels (tau = 0.21 +/- 0.02 s and 0.67 +/- 0.05 s, respectively, n = 6). These findings together demonstrate that N434 of mu1 Na+ channels is also critical for slow inactivation. We propose that this slow form of Na+ channel inactivation is analogous to the "C-type" inactivation in Shaker K+ channels. PMID:9083667

  9. Identification of binding domains in the sodium channel Na(V)1.8 intracellular N-terminal region and annexin II light chain p11.

    Science.gov (United States)

    Poon, W-Y Louisa; Malik-Hall, Misbah; Wood, John N; Okuse, Kenji

    2004-01-30

    The interaction of p11 (annexin II light chain) with the N-terminal domain of Na(V)1.8, a tetrodotoxin-resistant sodium channel, is essential for the functional expression of the channel. Here we show that p11 binds to Na(V)1.8 but not to sodium channel isoforms Na(V)1.2, 1.5, 1.7 or Na(V)1.9. The binding of amino acids 74-103 of Na(V)1.8 to p11 residues 33-78 occurs in a random coiled region flanked by two EF hand motifs whose crystal structure has been established. As Na(V)1.8 channel expression is associated with pain pathways, drugs that disrupt the Na(V)1.8-p11 interaction and down-regulate channel expression may have analgesic activity.

  10. Modification of sodium channel inactivation in single myelinated nerve fibers by methionine-reactive chemicals.

    Science.gov (United States)

    Wang, G K

    1984-01-01

    Several methionine-reactive reagents, including N-bromoacetamide, N-bromosuccinimide, chloramine-T, and N-chlorosuccinimide, irreversibly slowed and prevented Na channel inactivation in single myelinated nerve fibers, whereas sulfhydryl- or tyrosine-modifying reagents had little effect. The activation process was not modified by the reagents that altered inactivation and could be modulated normally by Ca++ ions and Centruroides scorpion toxin II alpha. These results suggest that externally applied N-bromoacetamide and its related compounds specifically affect Na channel inactivation by modifying methionine residues of the channel. PMID:6331542

  11. Three-dimensional solution structure of mu-conotoxin GIIIB, a specific blocker of skeletal muscle sodium channels.

    Science.gov (United States)

    Hill, J M; Alewood, P F; Craik, D J

    1996-07-09

    The three-dimensional solution structure of mu-conotoxin GIIIB, a 22-residue polypeptide from the venom of the piscivorous cone snail Conus geographus, has been determined using 2D 1H NMR spectroscopy. GIIIB binds with high affinity and selectivity to skeletal muscle sodium channels and is a valuable tool for characterizing both the structure and function of these channels. Structural restraints consisting of 289 interproton distances inferred from NOEs and 9 backbone and 5 side chain dihedral angle restraints from spin-spin coupling constants were used as input for simulated annealing calculations and energy minimization in the program X-PLOR. In addition to the 1H NMR derived information, the 13C resonances of GIIIB were assigned at natural abundance, and hydroxyproline C beta and C gamma chemical shifts were used to distinguish between the cis and trans peptide bond conformations. The final set of 20 structures had mean pairwise rms differences over the whole molecule of 1.22 A for the backbone atoms and 2.48 A for all heavy atoms. For the well-defined region encompassing residues 3-21, the corresponding values were 0.74 and 2.54 A, respectively. GIIIB adopts a compact structure consisting of a distorted 310-helix, a small beta-hairpin, a cis-hydroxyproline, and several turns. The molecule is stabilized by three disulfide bonds, two of which connect the helix and the beta-sheet, forming a structural core with similarities to the CS alpha beta motif [Cornet, B., Bonmatin, J.-M., Hetru, C., Hoffmann, J. A., Ptak, M., & Vovelle, F. (1995) Structure 3, 435-448]. This motif is common to several families of small proteins including scorpion toxins and insect defensins. Other structural features of GIIIB include the presence of eight arginine and lysine side chains that project into the solvent in a radial orientation relative to the core of the molecule. These cationic side chains form potential sites of interaction with anionic sites on sodium channels. The global

  12. Salt-Induced Hypertension in a Mouse Model of Liddle's Syndrome is Mediated by Epithelial Sodium Channels in the Brain

    Science.gov (United States)

    Van Huysse, James W.; Amin, Md. Shahrier; Yang, Baoli; Leenen, Frans H. H.

    2012-01-01

    Neural precursor cell expressed and developmentally downregulated 4-2 protein (Nedd4-2) facilitates the endocytosis of epithelial Na channels (ENaC). Both mice and humans with a loss of regulation of ENaC by Nedd4-2 have salt-induced hypertension. ENaC is also expressed in the brain, where it is critical for hypertension on high salt diet in salt-sensitive rats. In the present studies we assessed whether Nedd4-2 knockout (−/−) mice have: 1) increased brain ENaC; 2) elevated CSF sodium on high salt diet; and 3) enhanced pressor responses to CSF sodium and hypertension on high salt diet, both mediated by brain ENaC. Prominent choroid plexus and neuronal ENaC staining was present in −/− but not in wild-type (W/T) mice. In chronically instrumented mice, intracerebroventricular (icv) infusion of Na-rich aCSF increased MAP 3-fold higher in −/− than W/T. Icv infusion of the ENaC blocker benzamil abolished this enhancement. In telemetered −/− mice on high salt diet (8% NaCl), CSF [Na+], MAP and HR increased significantly, MAP by 30-35 mmHg. These MAP and HR responses were largely prevented by icv benzamil, but only to a minor extent by sc benzamil at the icv rate. We conclude that increased ENaC expression in the brain of Nedd 4-2 −/− mice mediates their hypertensive response to high salt diet, by causing increased sodium levels in the CSF as well as hyper-responsiveness to CSF sodium. These findings highlight the possible causative contribution of CNS ENaC in the etiology of salt-induced hypertension. PMID:22802227

  13. Differential contribution of sodium channel subtypes to action potential generation in unmyelinated human C-type nerve fibers.

    Science.gov (United States)

    Lang, Philip M; Hilmer, Verena B; Grafe, Peter

    2007-09-01

    Multiple voltage-dependent sodium channels (Na(v)) contribute to action potentials and excitability of primary nociceptive neurons. The aim of the current study was to characterize subtypes of Na(v) that contribute to action potential generation in peripheral unmyelinated human C-type nerve fibers. Registration of C-fiber compound action potentials and determination of membrane threshold was performed by a computerized threshold tracking program. Nerve fibers were stimulated with a 1-ms current pulse either alone or after a small ramp current lasting 300 ms. Compound C-fiber action potentials elicited by supramaximal 1-ms current pulses were rather resistant to application of tetrodotoxin (30-90 nM). However, the same concentrations of tetrodotoxin strongly reduced the peak height and elevated membrane threshold of action potentials evoked at the end of a 300-ms current ramp. A similar effect was observed during application of lidocaine and mexiletine (50 microM each). These data indicate that more than one type of Na(v) contributes to the generation of action potentials in unmyelinated human C-type nerve fibers. The peak height of an action potential produced by a short electrical impulse is dependent on the activation of tetrodotoxin-resistant ion channels. In contrast, membrane threshold and action potential peak height at the end of a slow membrane depolarization are regulated by a subtype of Na(v) with high sensitivity to low concentrations of tetrodotoxin, lidocaine, and mexiletine. The electrophysiologic and pharmacologic characteristics may indicate the functional activity of the Na(v) 1.7 subtype of voltage-dependent sodium channels.

  14. Voltage-gated sodium channels contribute to action potentials and spontaneous contractility in isolated human lymphatic vessels.

    Science.gov (United States)

    Telinius, Niklas; Majgaard, Jens; Kim, Sukhan; Katballe, Niels; Pahle, Einar; Nielsen, Jørn; Hjortdal, Vibeke; Aalkjaer, Christian; Boedtkjer, Donna Briggs

    2015-07-15

    Voltage-gated sodium channels (VGSC) play a key role for initiating action potentials (AP) in excitable cells. VGSC in human lymphatic vessels have not been investigated. In the present study, we report the electrical activity and APs of small human lymphatic collecting vessels, as well as mRNA expression and function of VGSC in small and large human lymphatic vessels. The VGSC blocker TTX inhibited spontaneous contractions in six of 10 spontaneously active vessels, whereas ranolazine, which has a narrower VGSC blocking profile, had no influence on spontaneous activity. TTX did not affect noradrenaline-induced contractions. The VGSC opener veratridine induced contractions in a concentration-dependent manner (0.1-30 μm) eliciting a stable tonic contraction and membrane depolarization to -18 ± 0.6 mV. Veratridine-induced depolarizations and contractions were reversed ∼80% by TTX, and were dependent on Ca(2+) influx via L-type calcium channels and the sodium-calcium exchanger in reverse mode. Molecular analysis determined NaV 1.3 to be the predominantly expressed VGSC isoform. Electrophysiology of mesenteric lymphatics determined the resting membrane potential to be -45 ± 1.7 mV. Spontaneous APs were preceded by a slow depolarization of 5.3 ± 0.6 mV after which a spike was elicited that almost completely repolarized before immediately depolarizing again to plateau. Vessels transiently hyperpolarized prior to returning to the resting membrane potential. TTX application blocked APs. We have shown that VGSC are necessary for initiating and maintaining APs and spontaneous contractions in human lymphatic vessels and our data suggest the main contribution from comes NaV 1.3. We have also shown that activation of these channels augments the contractile activity of the vessels. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  15. A sodium channel knockin mutant (NaV1.4-R669H) mouse model of hypokalemic periodic paralysis.

    Science.gov (United States)

    Wu, Fenfen; Mi, Wentao; Burns, Dennis K; Fu, Yu; Gray, Hillery F; Struyk, Arie F; Cannon, Stephen C

    2011-10-01

    Hypokalemic periodic paralysis (HypoPP) is an ion channelopathy of skeletal muscle characterized by attacks of muscle weakness associated with low serum K+. HypoPP results from a transient failure of muscle fiber excitability. Mutations in the genes encoding a calcium channel (CaV1.1) and a sodium channel (NaV1.4) have been identified in HypoPP families. Mutations of NaV1.4 give rise to a heterogeneous group of muscle disorders, with gain-of-function defects causing myotonia or hyperkalemic periodic paralysis. To address the question of specificity for the allele encoding the NaV1.4-R669H variant as a cause of HypoPP and to produce a model system in which to characterize functional defects of the mutant channel and susceptibility to paralysis, we generated knockin mice carrying the ortholog of the gene encoding the NaV1.4-R669H variant (referred to herein as R669H mice). Homozygous R669H mice had a robust HypoPP phenotype, with transient loss of muscle excitability and weakness in low-K+ challenge, insensitivity to high-K+ challenge, dominant inheritance, and absence of myotonia. Recovery was sensitive to the Na+/K+-ATPase pump inhibitor ouabain. Affected fibers had an anomalous inward current at hyperpolarized potentials, consistent with the proposal that a leaky gating pore in R669H channels triggers attacks, whereas a reduction in the amplitude of action potentials implies additional loss-of-function changes for the mutant NaV1.4 channels.

  16. Neuroplastic alteration of TTX-resistant sodium channel with visceral pain and morphine-induced hyperalgesia

    Directory of Open Access Journals (Sweden)

    Chen J

    2012-11-01

    Full Text Available Jinghong Chen,1,2,4 Ze-hui Gong,4 Hao Yan,2 Zhijun Qiao,3 Bo-yi Qin41Department of Internal Medicine, Neuroscience Program, The University of Texas Medical Branch, Galveston, TX, USA; 2The Divisions of Pharmacy, Pharmacology core lab, MD Anderson Cancer Center, Houston, TX, USA; 3University of Texas-Pan American, Edinburg, TX, USA; 4Beijing Institute of Pharmacology and Toxicology, Beijing, China Abstract: The discovery of the tetrodotoxin-resistant (TTX-R Na+ channel in nociceptive neurons has provided a special target for analgesic intervention. In a previous study we found that both morphine tolerance and persistent visceral inflammation resulted in visceral hyperalgesia. It has also been suggested that hyperexcitability of sensory neurons due to altered TTX-R Na+ channel properties and expression contributes to hyperalgesia; however, we do not know if some TTX-R Na+ channel property changes can be triggered by visceral hyperalgesia and morphine tolerance, or whether there are similar molecular or channel mechanisms in both situations. To evaluate the effects of morphine tolerance and visceral inflammation on the channel, we investigated the dorsal root ganglia (DRG neuronal change following these chronic treatments. Using whole-cell patch clamp recording, we recorded TTX-R Na+ currents in isolated adult rat lumbar and sacral (L6-S2 DRG neurons from normal and pathologic rats with colon inflammatory pain or chronic morphine treatment. We found that the amplitudes of TTX-R Na+ currents were signiflcantly increased in small-diameter DRG neurons with either morphine tolerance or visceral inflammatory pain. Meanwhile, the result also showed that those treatments altered the kinetics properties of the electrical current (ie, the activating and inactivating speed of the channel was accelerated. Our current results suggested that in both models, visceral chronic inflammatory pain and morphine tolerance causes electrophysiological changes in voltage

  17. Novel synthetic sulfoglycolipid IG20 facilitates exocytosis in chromaffin cells through the regulation of sodium channels.

    Science.gov (United States)

    Crespo-Castrillo, Andrea; Punzón, Eva; de Pascual, Ricardo; Maroto, Marcos; Padín, Juan Fernando; García-Álvarez, Isabel; Nanclares, Carmen; Ruiz-Pascual, Lucía; Gandía, Luis; Fernández-Mayoralas, Alfonso; García, Antonio G

    2015-12-01

    In search of druggable synthetic lipids that function as potential modulators of synaptic transmission and plasticity, we synthesized sulfoglycolipid IG20, which stimulates neuritic outgrowth. Here, we have explored its effects on ion channels and exocytosis in bovine chromaffin cells. IG20 augmented the rate of basal catecholamine release. Such effect did not depend on Ca(2+) mobilization from intracellular stores; rather, IG20-elicited secretion entirely dependent on Ca(2+) entry through L-subtype voltage-activated Ca(2+) channels. Those channels were recruited by cell depolarization mediated by IG20 likely through its ability to enhance the recruitment of Na(+) channels at more hyperpolarizing potentials. Confocal imaging with fluorescent derivative IG20-NBD revealed its rapid incorporation and confinement into the plasmalemma, supporting the idea that IG20 effects are exerted through a plasmalemmal-delimited mechanism. Thus, synthetic IG20 seems to mimic several physiological effects of endogenous lipids such as regulation of ion channels, Ca(2+) signaling, and exocytosis. Therefore, sulfoglycolipid IG20 may become a pharmacological tool for investigating the role of the lipid environment on neuronal excitability, ion channels, neurotransmitter release, synaptic efficacy, and neuronal plasticity. It may also inspire the synthesis of druggable sulfoglycolipids aimed at increasing synaptic plasticity and efficacy in neurodegenerative diseases and traumatic brain-spinal cord injury. The novel synthetic sulfoglycolipid IG20 mimics several physiological effects of endogenous lipids such as regulation of ion channels, Ca(2+) signaling, and exocytosis. This profile may eventually drive enhanced synaptic plasticity and efficacy. © 2015 International Society for Neurochemistry.

  18. Mechanism of sodium channel NaV1.9 potentiation by G-protein signaling.

    Science.gov (United States)

    Vanoye, Carlos G; Kunic, Jennifer D; Ehring, George R; George, Alfred L

    2013-02-01

    Tetrodotoxin (TTX)-resistant voltage-gated Na (Na(V)) channels have been implicated in nociception. In particular, Na(V)1.9 contributes to expression of persistent Na current in small diameter, nociceptive sensory neurons in dorsal root ganglia and is required for inflammatory pain sensation. Using ND7/23 cells stably expressing human Na(V)1.9, we elucidated the biophysical mechanisms responsible for potentiation of channel activity by G-protein signaling to better understand the response to inflammatory mediators. Heterologous Na(V)1.9 expression evoked TTX-resistant Na current with peak activation at -40 mV with extensive overlap in voltage dependence of activation and inactivation. Inactivation kinetics were slow and incomplete, giving rise to large persistent Na currents. Single-channel recording demonstrated long openings and correspondingly high open probability (P(o)) accounting for the large persistent current amplitude. Channels exposed to intracellular GTPγS, a proxy for G-protein signaling, exhibited twofold greater current density, slowing of inactivation, and a depolarizing shift in voltage dependence of inactivation but no change in activation voltage dependence. At the single-channel level, intracellular GTPγS had no effect on single-channel amplitude but caused an increased mean open time and greater P(o) compared with recordings made in the absence of GTPγS. We conclude that G-protein activation potentiates human Na(V)1.9 activity by increasing channel open probability and mean open time, causing the larger peak and persistent current, respectively. Our results advance our understanding about the mechanism of Na(V)1.9 potentiation by G-protein signaling during inflammation and provide a cellular platform useful for the discovery of Na(V)1.9 modulators with potential utility in treating inflammatory pain.

  19. Anaesthetic tricaine acts preferentially on neural voltage-gated sodium channels and fails to block directly evoked muscle contraction.

    Directory of Open Access Journals (Sweden)

    Seetharamaiah Attili

    Full Text Available Movements in animals arise through concerted action of neurons and skeletal muscle. General anaesthetics prevent movement and cause loss of consciousness by blocking neural function. Anaesthetics of the amino amide-class are thought to act by blockade of voltage-gated sodium channels. In fish, the commonly used anaesthetic tricaine methanesulphonate, also known as 3-aminobenzoic acid ethyl ester, metacaine or MS-222, causes loss of consciousness. However, its role in blocking action potentials in distinct excitable cells is unclear, raising the possibility that tricaine could act as a neuromuscular blocking agent directly causing paralysis. Here we use evoked electrical stimulation to show that tricaine efficiently blocks neural action potentials, but does not prevent directly evoked muscle contraction. Nifedipine-sensitive L-type Cav channels affecting movement are also primarily neural, suggesting that muscle Nav channels are relatively insensitive to tricaine. These findings show that tricaine used at standard concentrations in zebrafish larvae does not paralyse muscle, thereby diminishing concern that a direct action on muscle could mask a lack of general anaesthesia.

  20. Cardiac sodium channels expressed in a peripheral neurotumor-derived cell line, RT4-B8.

    Science.gov (United States)

    Zeng, D; Kyle, J W; Martin, R L; Ambler, K S; Hanck, D A

    1996-05-01

    RT4-B is one of several cell lines derived from a multipotent stem cell line, RT4-AC, which originated from a rat peripheral neurotumor. Based on Northern blot and ribonuclease protection experiments, RT4-B8 cells have been proposed to express rat cardiac Na channel mRNA as the major isoform. We report here direct electrophysiological evidence that the expressed voltage-gated Na channels in the RT4-B8 cell line are of the cardiac phenotype with no evidence for subpopulations expressing other Na channel isoforms. Current activation half point (conductance) was -41 +/- 5 mV (n = 7) and the steady-state voltage-dependent availability half point was -89 +/- 1 mV. As expected for cardiac Na channels, the half concentration of block for tetrodotoxin block was 0.74 microM, for saxitoxin (STX) was 0.15 microM, and for the class 2B divalent cation Cd2+ was 67 microM. Block was well described by single-site dose-response relationships with no indication of a subpopulation with "neuronal" affinity. Single-channel conductance (140 mM Na+) was 10 pS and predicted the average number of channels open at peak Na current to be 3 channels/microns2. [3H]STX binding data were also consistent with a single population of low-affinity STX binding sites and predicted channel density to be 11 sites/microns2. No inwardly or outwardly rectifying K or Ca currents were detected electrophysiologically, although in some cells a small time-independent Cl current was detected. Reverse transcription-polymerase chain reaction of mRNA isolated from RT4-B8 cells demonstrated the presence of rat cardiac (rH1) and brain IIa alpha-subunit mRNA, as well as mRNA for the Na channel beta 1-subunit. Northern blot analysis confirmed the predominance of the rat cardiac Na mRNA compared with brain IIa. The beta 1-subunit mRNA levels were significantly lower than those detected in rat brain and heart mRNA but were comparable to the low level of beta 1-subunit mRNA detected in isolated rat ventricular myocytes.

  1. Regulatory role of voltage-gated sodium channel β subunits in sensory neurons

    Directory of Open Access Journals (Sweden)

    Mohamed eChahine

    2011-11-01

    Full Text Available Voltage-gated Na+ channels are transmembrane-bound proteins incorporating aqueous conduction pores that are highly selective for Na+. The opening of these channels results in the rapid influx of Na+ ions that depolarize the cell and drive the rapid upstroke of nerve and muscle action potentials. While the concept of a Na+-selective ion channel had been formulated in the 1940s, it was not until the 1980s that the biochemical properties of the 260-kDa and 36-kDa auxiliary β subunits (β1, β2 were first described. Subsequent cloning and heterologous expression studies revealed that the  subunit forms the core of the channel and is responsible for both voltage-dependent gating and ionic selectivity. To date, ten isoforms of the Na+ channel α subunit have been identified that vary in their primary structures, tissue distribution, biophysical properties, and sensitivity to neurotoxins. Four β subunits (β1-β4 and two splice variants (β1A, β1B have been identified that modulate the subcellular distribution, cell surface expression, and functional properties of the α subunits. The purpose of this review is to provide a broad overview of β subunit expression and function in peripheral sensory neurons and examine their contributions to neuropathic pain.

  2. Inhibition of collagen synthesis by select calcium and sodium channel blockers can be mitigated by ascorbic acid and ascorbyl palmitate.

    Science.gov (United States)

    Ivanov, Vadim; Ivanova, Svetlana; Kalinovsky, Tatiana; Niedzwiecki, Aleksandra; Rath, Matthias

    2016-01-01

    Calcium, sodium and potassium channel blockers are widely prescribed medications for a variety of health problems, most frequently for cardiac arrhythmias, hypertension, angina pectoris and other disorders. However, chronic application of channel blockers is associated with numerous side effects, including worsening cardiac pathology. For example, nifedipine, a calcium-channel blocker was found to be associated with increased mortality and increased risk for myocardial infarction. In addition to the side effects mentioned above by different channel blockers, these drugs can cause arterial wall damage, thereby contributing to vascular wall structure destabilization and promoting events facilitating rupture of plaques. Collagen synthesis is regulated by ascorbic acid, which is also essential for its optimum structure as a cofactor in lysine and proline hydroxylation, a precondition for optimum crosslinking of collagen and elastin. Therefore, the main objective in this study was to evaluate effects of various types of channel blockers on intracellular accumulation and cellular functions of ascorbate, specifically in relation to formation and extracellular deposition of major collagen types relevant for vascular function. Effects of select Na- and Ca- channel blockers on collagen synthesis and deposition were evaluated in cultured human dermal fibroblasts and aortic smooth muscle cells by immunoassay. All channel blockers tested demonstrated inhibitory effects on collagen type I deposition to the ECM by fibroblasts, each to a different degree. Ascorbic acid significantly increased collagen I ECM deposition. Nifedipine (50 µM), a representative of channel blockers tested, significantly reduced ascorbic acid and ascorbyl palmitate-dependent ECM deposition of collagen type l and collagen type lV by cultured aortic smooth muscle cells. In addition, nifedipine (50 µM) significantly reduced ascorbate-dependent collagen type l and type lV synthesis by cultured aortic smooth

  3. Distribution of TTX-sensitive voltage-gated sodium channels in primary sensory endings of mammalian muscle spindles.

    Science.gov (United States)

    Carrasco, Dario I; Vincent, Jacob A; Cope, Timothy C

    2017-04-01

    Knowledge of the molecular mechanisms underlying signaling of mechanical stimuli by muscle spindles remains incomplete. In particular, the ionic conductances that sustain tonic firing during static muscle stretch are unknown. We hypothesized that tonic firing by spindle afferents depends on sodium persistent inward current (INaP) and tested for the necessary presence of the appropriate voltage-gated sodium (NaV) channels in primary sensory endings. The NaV 1.6 isoform was selected for both its capacity to produce INaP and for its presence in other mechanosensors that fire tonically. The present study shows that NaV 1.6 immunoreactivity (IR) is concentrated in heminodes, presumably where tonic firing is generated, and we were surprised to find NaV 1.6 IR strongly expressed also in the sensory terminals, where mechanotransduction occurs. This spatial pattern of NaV 1.6 IR distribution was consistent for three mammalian species (rat, cat, and mouse), as was tonic firing by primary spindle afferents. These findings meet some of the conditions needed to establish participation of INaP in tonic firing by primary sensory endings. The study was extended to two additional NaV isoforms, selected for their sensitivity to TTX, excluding TTX-resistant NaV channels, which alone are insufficient to support firing by primary spindle endings. Positive immunoreactivity was found for NaV 1.1 , predominantly in sensory terminals together with NaV 1.6 and for NaV 1.7 , mainly in preterminal axons. Differential distribution in primary sensory endings suggests specialized roles for these three NaV isoforms in the process of mechanosensory signaling by muscle spindles. NEW & NOTEWORTHY The molecular mechanisms underlying mechanosensory signaling responsible for proprioceptive functions are not completely elucidated. This study provides the first evidence that voltage-gated sodium channels (NaVs) are expressed in the spindle primary sensory ending, where NaVs are found at every site

  4. Statistical Metamodeling and Sequential Design of Computer Experiments to Model Glyco-Altered Gating of Sodium Channels in Cardiac Myocytes.

    Science.gov (United States)

    Du, Dongping; Yang, Hui; Ednie, Andrew R; Bennett, Eric S

    2016-09-01

    Glycan structures account for up to 35% of the mass of cardiac sodium ( Nav ) channels. To question whether and how reduced sialylation affects Nav activity and cardiac electrical signaling, we conducted a series of in vitro experiments on ventricular apex myocytes under two different glycosylation conditions, reduced protein sialylation (ST3Gal4(-/-)) and full glycosylation (control). Although aberrant electrical signaling is observed in reduced sialylation, realizing a better understanding of mechanistic details of pathological variations in INa and AP is difficult without performing in silico studies. However, computer model of Nav channels and cardiac myocytes involves greater levels of complexity, e.g., high-dimensional parameter space, nonlinear and nonconvex equations. Traditional linear and nonlinear optimization methods have encountered many difficulties for model calibration. This paper presents a new statistical metamodeling approach for efficient computer experiments and optimization of Nav models. First, we utilize a fractional factorial design to identify control variables from the large set of model parameters, thereby reducing the dimensionality of parametric space. Further, we develop the Gaussian process model as a surrogate of expensive and time-consuming computer models and then identify the next best design point that yields the maximal probability of improvement. This process iterates until convergence, and the performance is evaluated and validated with real-world experimental data. Experimental results show the proposed algorithm achieves superior performance in modeling the kinetics of Nav channels under a variety of glycosylation conditions. As a result, in silico models provide a better understanding of glyco-altered mechanistic details in state transitions and distributions of Nav channels. Notably, ST3Gal4(-/-) myocytes are shown to have higher probabilities accumulated in intermediate inactivation during the repolarization and yield a

  5. Particulate matter induces cardiac arrhythmias via dysregulation of carotid body sensitivity and cardiac sodium channels.

    Science.gov (United States)

    Wang, Ting; Lang, Gabriel D; Moreno-Vinasco, Liliana; Huang, Yong; Goonewardena, Sascha N; Peng, Ying-Jie; Svensson, Eric C; Natarajan, Viswanathan; Lang, Roberto M; Linares, Jered D; Breysse, Patrick N; Geyh, Alison S; Samet, Jonathan M; Lussier, Yves A; Dudley, Samuel; Prabhakar, Nanduri R; Garcia, Joe G N

    2012-04-01

    The mechanistic links between exposure to airborne particulate matter (PM) pollution and the associated increases in cardiovascular morbidity and mortality, particularly in people with congestive heart failure (CHF), have not been identified. To advance understanding of this issue, genetically engineered mice (CREB(A133)) exhibiting severe dilated cardiomyopathic changes were exposed to ambient PM collected in Baltimore. CREB(A133) mice, which display aberrant cardiac physiology and anatomy reminiscent of human CHF, displayed evidence of basal autonomic aberrancies (compared with wild-type mice) with PM exposure via aspiration, producing significantly reduced heart rate variability, respiratory dysynchrony, and increased ventricular arrhythmias. Carotid body afferent nerve responses to hypoxia and hyperoxia-induced respiratory depression were pronounced in PM-challenged CREB(A133) mice, and denervation of the carotid bodies significantly reduced PM-mediated cardiac arrhythmias. Genome-wide expression analyses of CREB(A133) left ventricular tissues demonstrated prominent Na(+) and K(+) channel pathway gene dysregulation. Subsequent PM challenge increased tyrosine phosphorylation and nitration of the voltage-gated type V cardiac muscle α-subunit of the Na(+) channel encoded by SCN5A. Ranolazine, a Na(+) channel modulator that reduces late cardiac Na(+) channel currents, attenuated PM-mediated cardiac arrhythmias and shortened PM-elongated QT intervals in vivo. These observations provide mechanistic insights into the epidemiologic findings in susceptibility of human CHF populations to PM exposure. Our results suggest a multiorgan pathobiology inherent to the CHF phenotype that is exaggerated by PM exposure via heightened carotid body sensitivity and cardiac Na(+) channel dysfunction.

  6. Particulate Matter Induces Cardiac Arrhythmias via Dysregulation of Carotid Body Sensitivity and Cardiac Sodium Channels

    Science.gov (United States)

    Wang, Ting; Lang, Gabriel D.; Moreno-Vinasco, Liliana; Huang, Yong; Goonewardena, Sascha N.; Peng, Ying–Jie; Svensson, Eric C.; Natarajan, Viswanathan; Lang, Roberto M.; Linares, Jered D.; Breysse, Patrick N.; Geyh, Alison S.; Samet, Jonathan M.; Lussier, Yves A.; Dudley, Samuel; Prabhakar, Nanduri R.

    2012-01-01

    The mechanistic links between exposure to airborne particulate matter (PM) pollution and the associated increases in cardiovascular morbidity and mortality, particularly in people with congestive heart failure (CHF), have not been identified. To advance understanding of this issue, genetically engineered mice (CREBA133) exhibiting severe dilated cardiomyopathic changes were exposed to ambient PM collected in Baltimore. CREBA133 mice, which display aberrant cardiac physiology and anatomy reminiscent of human CHF, displayed evidence of basal autonomic aberrancies (compared with wild-type mice) with PM exposure via aspiration, producing significantly reduced heart rate variability, respiratory dysynchrony, and increased ventricular arrhythmias. Carotid body afferent nerve responses to hypoxia and hyperoxia-induced respiratory depression were pronounced in PM-challenged CREBA133 mice, and denervation of the carotid bodies significantly reduced PM-mediated cardiac arrhythmias. Genome-wide expression analyses of CREBA133 left ventricular tissues demonstrated prominent Na+ and K+ channel pathway gene dysregulation. Subsequent PM challenge increased tyrosine phosphorylation and nitration of the voltage-gated type V cardiac muscle α-subunit of the Na+ channel encoded by SCN5A. Ranolazine, a Na+ channel modulator that reduces late cardiac Na+ channel currents, attenuated PM-mediated cardiac arrhythmias and shortened PM-elongated QT intervals in vivo. These observations provide mechanistic insights into the epidemiologic findings in susceptibility of human CHF populations to PM exposure. Our results suggest a multiorgan pathobiology inherent to the CHF phenotype that is exaggerated by PM exposure via heightened carotid body sensitivity and cardiac Na+ channel dysfunction. PMID:22108299

  7. Sodium Channel β2 Subunits Prevent Action Potential Propagation Failures at Axonal Branch Points.

    Science.gov (United States)

    Cho, In Ha; Panzera, Lauren C; Chin, Morven; Hoppa, Michael B

    2017-09-27

    Neurotransmitter release depends on voltage-gated Na + channels (Na v s) to propagate an action potential (AP) successfully from the axon hillock to a synaptic terminal. Unmyelinated sections of axon are very diverse structures encompassing branch points and numerous presynaptic terminals with undefined molecular partners of Na + channels. Using optical recordings of Ca 2+ and membrane voltage, we demonstrate here that Na + channel β2 subunits (Na v β2s) are required to prevent AP propagation failures across the axonal arborization of cultured rat hippocampal neurons (mixed male and female). When Na v β2 expression was reduced, we identified two specific phenotypes: (1) membrane excitability and AP-evoked Ca 2+ entry were impaired at synapses and (2) AP propagation was severely compromised with >40% of axonal branches no longer responding to AP-stimulation. We went on to show that a great deal of electrical signaling heterogeneity exists in AP waveforms across the axonal arborization independent of axon morphology. Therefore, Na v β2 is a critical regulator of axonal excitability and synaptic function in unmyelinated axons. SIGNIFICANCE STATEMENT Voltage-gated Ca 2+ channels are fulcrums of neurotransmission that convert electrical inputs into chemical outputs in the form of vesicle fusion at synaptic terminals. However, the role of the electrical signal, the presynaptic action potential (AP), in modulating synaptic transmission is less clear. What is the fidelity of a propagating AP waveform in the axon and what molecules shape it throughout the axonal arborization? Our work identifies several new features of AP propagation in unmyelinated axons: (1) branches of a single axonal arborization have variable AP waveforms independent of morphology, (2) Na + channel β2 subunits modulate AP-evoked Ca 2+ -influx, and (3) β2 subunits maintain successful AP propagation across the axonal arbor. These findings are relevant to understanding the flow of excitation in the

  8. Sodium flux ratio in Na/K pump-channels opened by palytoxin.

    Science.gov (United States)

    Rakowski, R F; Artigas, Pablo; Palma, Francisco; Holmgren, Miguel; De Weer, Paul; Gadsby, David C

    2007-07-01

    Palytoxin binds to Na(+)/K(+) pumps in the plasma membrane of animal cells and opens an electrodiffusive cation pathway through the pumps. We investigated properties of the palytoxin-opened channels by recording macroscopic and microscopic currents in cell bodies of neurons from the giant fiber lobe, and by simultaneously measuring net current and (22)Na(+) efflux in voltage-clamped, internally dialyzed giant axons of the squid Loligo pealei. The conductance of single palytoxin-bound "pump-channels" in outside-out patches was approximately 7 pS in symmetrical 500 mM [Na(+)], comparable to findings in other cells. In these high-[Na(+)], K(+)-free solutions, with 5 mM cytoplasmic [ATP], the K(0.5) for palytoxin action was approximately 70 pM. The pump-channels were approximately 40-50 times less permeable to N-methyl-d-glucamine (NMG(+)) than to Na(+). The reversal potential of palytoxin-elicited current under biionic conditions, with the same concentration of a different permeant cation on each side of the membrane, was independent of the concentration of those ions over the range 55-550 mM. In giant axons, the Ussing flux ratio exponent (n') for Na(+) movements through palytoxin-bound pump-channels, over a 100-400 mM range of external [Na(+)] and 0 to -40 mV range of membrane potentials, averaged 1.05 +/- 0.02 (n = 28). These findings are consistent with occupancy of palytoxin-bound Na(+)/K(+) pump-channels either by a single Na(+) ion or by two Na(+) ions as might be anticipated from other work; idiosyncratic constraints are needed if the two Na(+) ions occupy a single-file pore, but not if they occupy side-by-side binding sites, as observed in related structures, and if only one of the sites is readily accessible from both sides of the membrane.

  9. Contactin regulates the current density and axonal expression of tetrodotoxin-resistant but not tetrodotoxin-sensitive sodium channels in DRG neurons.

    Science.gov (United States)

    Rush, Anthony M; Craner, Matthew J; Kageyama, Takashi; Dib-Hajj, Sulayman D; Waxman, Stephen G; Ranscht, Barbara

    2005-07-01

    Contactin, a glycosyl-phosphatidylinositol (GPI)-anchored predominantly neuronal cell surface glycoprotein, associates with sodium channels Nav1.2, Nav1.3 and Nav1.9, and enhances the density of these channels on the plasma membrane in mammalian expression systems. However, a detailed functional analysis of these interactions and of untested putative interactions with other sodium channel isoforms in mammalian neuronal cells has not been carried out. We examined the expression and function of sodium channels in small-diameter dorsal root ganglion (DRG) neurons from contactin-deficient (CNTN-/-) mice, compared to CNTN+/+ litter mates. Nav1.9 is preferentially expressed in isolectin B4 (IB4)-positive neurons and thus we used this marker to subdivide small-diameter DRG neurons. Using whole-cell patch-clamp recording, we observed a greater than two-fold reduction of tetrodotoxin-resistant (TTX-R) Nav1.8 and Nav1.9 current densities in IB4+ DRG neurons cultured from CNTN-/- vs. CNTN+/+ mice. Current densities for TTX-sensitive (TTX-S) sodium channels were unaffected. Contactin's effect was selective for IB4+ neurons as current densities for both TTX-R and TTX-S channels were not significantly different in IB4- DRG neurons from the two genotypes. Consistent with these results, we have demonstrated a reduction in Nav1.8 and Nav1.9 immunostaining on peripherin-positive unmyelinated axons in sciatic nerves from CNTN-/- mice but detected no changes in the expression for the two major TTX-S channels Nav1.6 and Nav1.7. These data provide evidence of a role for contactin in selectively regulating the cell surface expression and current densities of TTX-R but not TTX-S Na+ channel isoforms in nociceptive DRG neurons; this regulation could modulate the membrane properties and excitability of these neurons.

  10. Problematic alcohol use associates with sodium channel and clathrin linker 1 (SCLT1) in trauma-exposed populations.

    Science.gov (United States)

    Almli, Lynn M; Lori, Adriana; Meyers, Jacquelyn L; Shin, Jaemin; Fani, Negar; Maihofer, Adam X; Nievergelt, Caroline M; Smith, Alicia K; Mercer, Kristina B; Kerley, Kimberly; Leveille, Jennifer M; Feng, Hao; Abu-Amara, Duna; Flory, Janine D; Yehuda, Rachel; Marmar, Charles R; Baker, Dewleen G; Bradley, Bekh; Koenen, Karestan C; Conneely, Karen N; Ressler, Kerry J

    2017-10-30

    Excessive alcohol use is extremely prevalent in the United States, particularly among trauma-exposed individuals. While several studies have examined genetic influences on alcohol use and related problems, this has not been studied in the context of trauma-exposed populations. We report results from a genome-wide association study of alcohol consumption and associated problems as measured by the alcohol use disorders identification test (AUDIT) in a trauma-exposed cohort. Results indicate a genome-wide significant association between total AUDIT score and rs1433375 [N = 1036, P = 2.61 × 10-8 (dominant model), P = 7.76 × 10-8 (additive model)], an intergenic single-nucleotide polymorphism located 323 kb upstream of the sodium channel and clathrin linker 1 (SCLT1) at 4q28. rs1433375 was also significant in a meta-analysis of two similar, but independent, cohorts (N = 1394, P = 0.0004), the Marine Resiliency Study and Systems Biology PTSD Biomarkers Consortium. Functional analysis indicated that rs1433375 was associated with SCLT1 gene expression and cortical-cerebellar functional connectivity measured via resting state functional magnetic resonance imaging. Together, findings suggest a role for sodium channel regulation and cerebellar functioning in alcohol use behavior. Identifying mechanisms underlying risk for problematic alcohol use in trauma-exposed populations is critical for future treatment and prevention efforts. © 2017 Society for the Study of Addiction.

  11. Action potential changes associated with impairment of functional properties of sodium channels in hippocampal neurons induced by melamine.

    Science.gov (United States)

    Yang, Jia-Jia; Yang, Zhuo; Zhang, Tao

    2010-10-05

    Since the melamine-contamination event happened in September 2008, there have been lots of studies about melamine toxicity, but very limited studies focused on central nervous system (CNS). In the present study, we investigated the effects of melamine (5x10(-4), 5x10(-5) and 5x10(-6)g/ml) on voltage-gated sodium channels (VGSCs) in hippocampal CA1 neurons using whole-cell patch-clamp recordings technique. The results showed that only 5x10(-4)g/ml melamine reduced the amplitude of voltage-gated sodium current (I(Na)). At the concentrations of 5x10(-5) and 5x10(-4)g/ml, melamine produced a hyperpolarizing shift in the steady-state activation curve of I(Na) and also enhanced the steady-state inactivate processing of I(Na). Action potential properties and the pattern of repetitive firing were examined using current-clamp recording, which indicated that peak amplitude and overshoot of the evoked single action potential were decreased. The half-width and the firing rate of repetitive firing were increased in a concentration-dependent manner. The data suggest that melamine alters the action potential of hippocampal CA1 neurons by impairing the functional properties of VGSCs, which may be the underlie mechanisms of neurotoxicity induced by melamine. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

  12. CC chemokine ligand 2 upregulates the current density and expression of TRPV1 channels and Nav1.8 sodium channels in dorsal root ganglion neurons

    Directory of Open Access Journals (Sweden)

    Kao Der-Jang

    2012-08-01

    Full Text Available Abstract Background Inflammation or nerve injury-induced upregulation and release of chemokine CC chemokine ligand 2 (CCL2 within the dorsal root ganglion (DRG is believed to enhance the activity of DRG nociceptive neurons and cause hyperalgesia. Transient receptor potential vanilloid receptor 1 (TRPV1 and tetrodotoxin (TTX-resistant Nav1.8 sodium channels play an essential role in regulating the excitability and pain transmission of DRG nociceptive neurons. We therefore tested the hypothesis that CCL2 causes peripheral sensitization of nociceptive DRG neurons by upregulating the function and expression of TRPV1 and Nav1.8 channels. Methods DRG neuronal culture was prepared from 3-week-old Sprague–Dawley rats and incubated with various concentrations of CCL2 for 24 to 36 hours. Whole-cell voltage-clamp recordings were performed to record TRPV1 agonist capsaicin-evoked inward currents or TTX-insensitive Na+ currents from control or CCL2-treated small DRG sensory neurons. The CCL2 effect on the mRNA expression of TRPV1 or Nav1.8 was measured by real-time quantitative RT-PCR assay. Results Pretreatment of CCL2 for 24 to 36 hours dose-dependently (EC50 value = 0.6 ± 0.05 nM increased the density of capsaicin-induced currents in small putative DRG nociceptive neurons. TRPV1 mRNA expression was greatly upregulated in DRG neurons preincubated with 5 nM CCL2. Pretreating small DRG sensory neurons with CCL2 also increased the density of TTX-resistant Na+ currents with a concentration-dependent manner (EC50 value = 0.7 ± 0.06 nM. The Nav1.8 mRNA level was significantly increased in DRG neurons pretreated with CCL2. In contrast, CCL2 preincubation failed to affect the mRNA level of TTX-resistant Nav1.9. In the presence of the specific phosphatidylinositol-3 kinase (PI3K inhibitor LY294002 or Akt inhibitor IV, CCL2 pretreatment failed to increase the current density of capsaicin-evoked inward currents or TTX-insensitive Na+ currents and

  13. Activation of the epithelial sodium channel by the metalloprotease meprin β subunit

    Science.gov (United States)

    Ishmael, Susan S; Dang, Yan; Gillie, Daniel; Bond, Judith S; Milgram, Sharon L; Stutts, M Jackson

    2011-01-01

    The Epithelial Na+ Channel (ENaC) is an apical heteromeric channel that mediates Na+ entry into epithelial cells from the luminal cell surface. ENaC is activated by proteases that interact with the channel during biosynthesis or at the extracellular surface. Meprins are cell surface and secreted metalloproteinases of the kidney and intestine. We discovered by affinity chromatography that meprins bind γ-ENaC, a subunit of the ENaC hetero-oligomer. The physical interaction involves NH2-terminal cytoplasmic residues 37–54 of γ-ENaC, containing a critical gating domain immediately before the first transmembrane domain, and the cytoplasmic COOH-terminal tail of meprin β (residues 679–704). This potential association was confirmed by co-expression and co-immunoprecipitation studies. Functional assays revealed that meprins stimulate ENaC expressed exogenously in Xenopus oocytes and endogenously in epithelial cells. Co-expression of ENaC subunits and meprin β or α/β in Xenopus oocytes increased amiloride-sensitive Na+ currents approximately two-fold. This increase was blocked by preincubation with an inhibitor of meprin activity, actinonin. The meprin-mediated increase in ENaC currents in oocytes and epithelial cell monolayers required meprin β, but not the α subunit. Meprin β promoted cleavage of α and γ-ENaC subunits at sites close to the second transmembrane domain in the extracellular domain of each channel subunit. Thus, meprin β regulates the activity of ENaC in a metalloprotease-dependent fashion. PMID:20953144

  14. The role of tetrodotoxin-resistant sodium channels in pain states: are they the next target for analgesic drugs?

    Science.gov (United States)

    Silos-Santiago, Inmaculada

    2008-01-01

    Neuropathic pain, a persistent chronic pain resulting from damage to the central or peripheral nervous system, is a condition that severely affects the quality-of-life of millions of individuals worldwide. The treatment of neuropathic pain is still an unmet medical need; however, recent advances in our understanding of mechanisms underlying the perception and transmission of painful stimuli offer significant potential for improvement of therapies directed to neuropathic pain. Ectopic activity in damaged and dysfunctional sensory afferents is believed to have a role in the generation and maintenance of neuropathic pain. One of the mechanisms underlying this ectopic firing involves abnormal modulation of voltage-gated sodium channels (NaVs) in the soma and axonal membranes of dorsal root ganglion (DRG) sensory neurons. In fact, NaV blockers have been clinically validated as treatments for neuropathic pain. However, current drugs are weak, non-selective inhibitors of NaVs with dose-limiting CNS and cardiovascular side effects that prevent their use in long-term therapy. Selective NaV tetrodotoxin-resistant channels (NaV 1.8 and NaV 1.9) are expressed exclusively in nociceptive neurons in the DRGs where they play a key role in normal and/or pathological pain sensation, providing an opportunity for the development of novel peripheral analgesics with a better safety profile.

  15. Structure and function of splice variants of the cardiac voltage-gated sodium channel Na(v)1.5.

    Science.gov (United States)

    Schroeter, Annett; Walzik, Stefan; Blechschmidt, Steve; Haufe, Volker; Benndorf, Klaus; Zimmer, Thomas

    2010-07-01

    Voltage-gated sodium channels mediate the rapid upstroke of the action potential in excitable tissues. The tetrodotoxin (TTX) resistant isoform Na(v)1.5, encoded by the SCN5A gene, is the predominant isoform in the heart. This channel plays a key role for excitability of atrial and ventricular cardiomyocytes and for rapid impulse propagation through the specific conduction system. During recent years, strong evidence has been accumulated in support of the expression of several Na(v)1.5 splice variants in the heart, and in various other tissues and cell lines including brain, dorsal root ganglia, breast cancer cells and neuronal stem cell lines. This review summarizes our knowledge on the structure and putative function of nine Na(v)1.5 splice variants detected so far. Attention will be paid to the distinct biophysical properties of the four functional splice variants, to the pronounced tissue- and species-specific expression, and to the developmental regulation of Na(v)1.5 splicing. The implications of alternative splicing for SCN5A channelopathies, and for a better understanding of genotype-phenotype correlations, are discussed. Copyright 2010 Elsevier Ltd. All rights reserved.

  16. Cell-autonomous axon growth of young motoneurons is triggered by a voltage-gated sodium channel.

    Science.gov (United States)

    Wetzel, Andrea; Jablonka, Sibylle; Blum, Robert

    2013-01-01

    Spontaneous electrical activity preceding synapse formation contributes to the precise regulation of neuronal development. Examining the origins of spontaneous activity revealed roles for neurotransmitters that depolarize neurons and activate ion channels. Recently, we identified a new molecular mechanism underlying fluctuations in spontaneous neuronal excitability. We found that embryonic motoneurons with a genetic loss of the low-threshold sodium channel NaV1.9 show fewer fluctuations in intracellular calcium in axonal compartments and growth cones than wild-type littermates. As a consequence, axon growth of NaV1.9-deficient motoneurons in cell culture is drastically reduced while dendritic growth and cell survival are not affected. Interestingly, NaV1.9 function is observed under conditions that would hardly allow a ligand- or neurotransmitter-dependent depolarization. Thus, NaV1.9 may serve as a cell-autonomous trigger for neuronal excitation. In this addendum, we discuss a model for the interplay between cell-autonomous local neuronal activity and local cytoskeleton dynamics in growth cone function.

  17. Direct interaction with contactin targets voltage-gated sodium channel Na(v)1.9/NaN to the cell membrane

    National Research Council Canada - National Science Library

    Liu, C J; Dib-Hajj, S D; Black, J A; Greenwood, J; Lian, Z; Waxman, S G

    2001-01-01

    .... To examine this issue we studied the voltage-gated sodium channel Na(v)1.9/NaN, which is preferentially expressed in small sensory neurons of dorsal root ganglia and trigeminal ganglia and the nonmyelinated axons that arise...

  18. [A computer simulation research for the effects of dragon's blood and its component loureirin B on sodium channel in dorsal root ganglion cells].

    Science.gov (United States)

    Chen, Su; Liu, Xiangming

    2006-12-01

    Using patch clamp technique the effects of dragon's blood and its component loureirin B on tetrodotoxin-sensitive sodium channel currents in dorsal root ganglion cells were observed. The experimental data were simulated with Hodgkin-Huxley model and the corresponding parameters were estimated. In addition, computer-simulated neuron action potentials in the absence and presence of drugs were produced using Hodgkin-Huxley model. The results show that the conductance of tetrodotoxin-sensitive sodium channel was fitted with m3h model well, the half-activated potentials of the sodium channel in the presence of drugs were shifted to the depolarizing direction and the threshold intensity of the cells in the presence of drugs was increased. These results demonstrate that dragon's blood and loureirin B did not resemble the tetrodotoxin which inhibited tetrodotoxin-sensitive sodium channel currents completely. Perhaps the analgesic effects of dragon's blood were partly caused by loureirin B affecting the activation, blocking the action potential generation and interfering with the transmission of painful signals into the central nervous system.

  19. The sodium channel activator Lu AE98134 normalizes the altered firing properties of fast spiking interneurons in Dlx5/6+/- mice

    DEFF Research Database (Denmark)

    von Schoubye, Nadia Lybøl; Frederiksen, Kristen; Kristiansen, Uffe

    2018-01-01

    Mental disorders such as schizophrenia are associated with impaired firing properties of fast spiking inhibitory interneurons (FSINs) causing reduced task-evoked gamma-oscillation in prefrontal cortex. The voltage-gated sodium channel NaV1.1 is highly expressed in PV-positive interneurons, but only...

  20. Investigations of the Navβ1b sodium channel subunit in human ventricle; functional characterization of the H162P Brugada Syndrome mutant

    DEFF Research Database (Denmark)

    Yuan, Lei; Koivumaki, Jussi; Liang, Bo

    2014-01-01

    Brugada Syndrome (BrS) is a rare inherited disease which can give rise to ventricular arrhythmia and ultimately sudden cardiac death. Numerous loss-of-function mutations in the cardiac sodium channel Nav1.5 have been associated with BrS. However, few mutations in the auxiliary Navβ1-4 subunits ha...

  1. A chimeric prokaryotic-eukaryotic pentameric ligand gated ion channel reveals interactions between the extracellular and transmembrane domains shape neurosteroid modulation.

    Science.gov (United States)

    Ghosh, Borna; Tsao, Tzu-Wei; Czajkowski, Cynthia

    2017-10-01

    Pentameric ligand-gated ion channels (pLGICs) are the targets of several clinical and endogenous allosteric modulators including anesthetics and neurosteroids. Molecular mechanisms underlying allosteric drug modulation are poorly understood. Here, we constructed a chimeric pLGIC by fusing the extracellular domain (ECD) of the proton-activated, cation-selective bacterial channel GLIC to the transmembrane domain (TMD) of the human ρ1 chloride-selective GABAAR, and tested the hypothesis that drug actions are regulated locally in the domain that houses its binding site. The chimeric channels were proton-gated and chloride-selective demonstrating the GLIC ECD was functionally coupled to the GABAρ TMD. Channels were blocked by picrotoxin and inhibited by pentobarbital, etomidate and propofol. The point mutation, ρ TMD W328M, conferred positive modulation and direct gating by pentobarbital. The data suggest that the structural machinery mediating general anesthetic modulation resides in the TMD. Proton-activation and neurosteroid modulation of the GLIC-ρ chimeric channels, however, did not simply mimic their respective actions on GLIC and GABAρ revealing that across domain interactions between the ECD and TMD play important roles in determining their actions. Proton-induced current responses were biphasic suggesting that the chimeric channels contain an additional proton sensor. Neurosteroid modulation of the GLIC-ρ chimeric channels by the stereoisomers, 5α-THDOC and 5β-THDOC, were swapped compared to their actions on GABAρ indicating that positive versus negative neurosteroid modulation is not encoded solely in the TMD nor by neurosteroid isomer structure but is dependent on specific interdomain connections between the ECD and TMD. Our data reveal a new mechanism for shaping neurosteroid modulation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Sodium channel NaV1.9 mutations associated with insensitivity to pain dampen neuronal excitability.

    Science.gov (United States)

    Huang, Jianying; Vanoye, Carlos G; Cutts, Alison; Goldberg, Y Paul; Dib-Hajj, Sulayman D; Cohen, Charles J; Waxman, Stephen G; George, Alfred L

    2017-06-30

    Voltage-gated sodium channel (NaV) mutations cause genetic pain disorders that range from severe paroxysmal pain to a congenital inability to sense pain. Previous studies on NaV1.7 and NaV1.8 established clear relationships between perturbations in channel function and divergent clinical phenotypes. By contrast, studies of NaV1.9 mutations have not revealed a clear relationship of channel dysfunction with the associated and contrasting clinical phenotypes. Here, we have elucidated the functional consequences of a NaV1.9 mutation (L1302F) that is associated with insensitivity to pain. We investigated the effects of L1302F and a previously reported mutation (L811P) on neuronal excitability. In transfected heterologous cells, the L1302F mutation caused a large hyperpolarizing shift in the voltage-dependence of activation, leading to substantially enhanced overlap between activation and steady-state inactivation relationships. In transfected small rat dorsal root ganglion neurons, expression of L1302F and L811P evoked large depolarizations of the resting membrane potential and impaired action potential generation. Therefore, our findings implicate a cellular loss of function as the basis for impaired pain sensation. We further demonstrated that a U-shaped relationship between the resting potential and the neuronal action potential threshold explains why NaV1.9 mutations that evoke small degrees of membrane depolarization cause hyperexcitability and familial episodic pain disorder or painful neuropathy, while mutations evoking larger membrane depolarizations cause hypoexcitability and insensitivity to pain.

  3. Increased nerve fiber expression of sensory sodium channels Nav1.7, Nav1.8, And Nav1.9 in rhinitis.

    Science.gov (United States)

    Keh, Siew M; Facer, Paul; Simpson, Karen D; Sandhu, Guri; Saleh, Hesham A; Anand, Praveen

    2008-04-01

    Voltage-gated sodium channels Nav1.7, Nav1.8, and Nav1.9 are involved in nerve action potentials and have been proposed to underlie neuronal hypersensitivity. We have therefore studied their levels in allergic and nonallergic rhinitis. Inferior turbinate biopsies from 50 patients (n = 18 controls, n = 20 allergic, and n = 12 nonallergic rhinitis) were studied by immunohistology using antibodies to Nav1.7, Nav1.8, and Nav1.9, the structural nerve marker (protein gene product [PGP]9.5), nerve growth factor (NGF), mast cells (c-kit), macrophages (CD68), and T cells (CD3). Sodium channel-positive nerve fibers were counted per millimeter length of subepithelium, and immunoreactivity for inflammatory cell markers PGP9.5 and NGF were image analyzed. All three sodium channel-immunoreactive nerve fiber numbers were significantly increased in allergic (Nav1.7, P = .0004; Nav1.8, P = .028; Nav1.9, P = .02) and nonallergic (Nav1.7, P = .006; Nav1.8, P = .019; Nav1.9, P = .0037) rhinitis. There was a significant increase of subepithelial innervation (PGP9.5, P = .01) and epithelial NGF immunoreactivity (P = .03) in nonallergic rhinitis, comparable with our previous report in allergic rhinitis. Inflammatory cell markers were significantly increased in allergic (mast cells, P = .06; macrophages, P = .044; T cells, P = .007) but not nonallergic rhinitis. The increased levels of sensory sodium channels in allergic and nonallergic rhinitis may contribute to the hypersensitive state, irrespective of the degree of active inflammation. Selective blockers of these sodium channels, administered topically, may have therapeutic potential in rhinitis.

  4. Metabolic Constraints on the Eukaryotic Transition

    Science.gov (United States)

    Wallace, Rodrick

    2009-04-01

    Mutualism, obligate mutualism, symbiosis, and the eukaryotic ‘fusion’ of Serial Endosymbiosis Theory represent progressively more rapid and less distorted real-time communication between biological structures instantiating information sources. Such progression in accurate information transmission requires, in turn, progressively greater channel capacity that, through the homology between information source uncertainty and free energy density, requires ever more energetic metabolism. The eukaryotic transition, according to this model, may have been entrained by an ecosystem resilience shift from anaerobic to aerobic metabolism.

  5. Regulation of sodium channel function by bilayer elasticity: the importance of hydrophobic coupling. Effects of Micelle-forming amphiphiles and cholesterol

    DEFF Research Database (Denmark)

    Lundbæk, Jens August; Birn, Pia; Hansen, Anker J

    2004-01-01

    and kinetics of the protein conformational changes therefore will be regulated by the bilayer elasticity, which is determined by the lipid composition. This hydrophobic coupling mechanism has been studied extensively in gramicidin channels, where the channel-bilayer hydrophobic interactions link...... a "conformational" change (the monomerdimer transition) to an elastic bilayer deformation. Gramicidin channels thus are regulated by the lipid bilayer elastic properties (thickness, monolayer equilibrium curvature, and compression and bending moduli). To investigate whether this hydrophobic coupling mechanism could...... be a general mechanism regulating membrane protein function, we examined whether voltage-dependent skeletal-muscle sodium channels, expressed in HEK293 cells, are regulated by bilayer elasticity, as monitored using gramicidin A (gA) channels. Nonphysiological amphiphiles (beta-octyl-glucoside, Genapol X-100...

  6. Sensitization of sodium channels by cystathionine β-synthetase activation in colon sensory neurons in adult rats with neonatal maternal deprivation.

    Science.gov (United States)

    Hu, Shufen; Xu, Weiyuan; Miao, Xiuhua; Gao, Yanping; Zhu, Liyan; Zhou, Youlang; Xiao, Ying; Xu, Guang-Yin

    2013-10-01

    The pathogenesis of pain in irritable bowel syndrome (IBS) is poorly understood and treatment remains difficult. We have previously reported that TTX-resistant (TTX-R) sodium channels in colon-specific dorsal root ganglion (DRG) neurons were sensitized and the expression of the endogenous hydrogen sulfide producing enzyme cystathionine β-synthetase (CBS) was upregulated in a rat model of visceral hypersensitivity induced by neonatal maternal deprivation (NMD). However, the detailed molecular mechanism for activation of sodium channels remains unknown. This study was designed to examine roles for CBS-H₂S signaling in sensitization of sodium channels in a previously validated rat model of IBS. Neonatal male rats (postnatal days 2-15) were exposed to a 3 hour period of daily maternal separation with temperature maintained at ~33 °C. Colon-specific dorsal root ganglion (DRG) neurons were labeled with DiI and acutely dissociated for measuring excitability and sodium channel current under whole-cell patch clamp configurations. The expression of Na(V)1.8 was analyzed by Western blot and Immunofluorescence study. The endogenous H₂S producing enzyme CBS antagonist was injected intraperitoneally. We showed that CBS was colocalized with Na(V)1.8 in colon-specific DRG neurons pre-labeled with DiI. Pretreatment of O-(Carboxymethyl) hydroxylamine hemihydrochloride (AOAA), an inhibitor of CBS, significantly reduced expression of Na(V)1.8 in NMD rats. AOAA treatment also inhibited the TTX-R sodium current density, right-shifted the V₁/₂ of activation curve, and reversed hyperexcitability of colon-specific DRG neurons in NMD rats. Conversely, addition of NaHS, a donor of H₂S, greatly enhanced TTX-R sodium current density, left shifted the activation curve and enhanced excitability of colon DRG neurons in age-matched healthy rats. Furthermore, application of H-89, an inhibitor of protein kinase A, markedly attenuated the potentiation of TTX-R sodium current density by Na

  7. Modification of sodium channels in myelinated nerve by Anemonia sulcata toxin II.

    Science.gov (United States)

    Ulbricht, W; Schmidtmayer, J

    1981-05-01

    1. Single myelinated nerve fibres of the frog, Rana esculenta, were investigated predominantly in voltage clamp experiments. 2. Sodium current (INa) inactivation was measured in the presence of 10 mM TEA to suppress IK. Inactivation was diphasic but complete in toxin-free solution; it was delayed and became incomplete in Anemonia sulcata toxin II (ATX II) leading to persistent INa flow even during long depolarizations. The effects were reversible. Activation was not affected. 3. The persistent INa component increased with increasing toxin concentration and saturated at ca. 15 microM. The lowest concentration yielding unequivocal effects in the voltage clamp was 0.5 microM. 4. The curve relating the steady-state inactivation parameter, h infinity to the conditioning potential V became non-monotonic in ATX II i.e. dh infinity/dV greater than 0 for V greater than 30 mV. 5. Inactivation could be formally described by a three-state model with two conducting (h2 and h2) and one closed state (x) in the sequence h1 in equilibrium x in equilibrium h2. 6. Ca2+ modifies h2(V) more than h1(V) whose reaction to Ca2+ is similar to h(V) in toxin-free solution. The Ca2+ effect is very rapid and reversible.

  8. NaN/Nav1.9: a sodium channel with unique properties.

    Science.gov (United States)

    Dib-Hajj, Sulayman; Black, Joel A; Cummins, Theodore R; Waxman, Stephen G

    2002-05-01

    The Na(v)1.9 Na(+) channel (also known as NaN) is preferentially expressed in nociceptive neurons of the dorsal root ganglia (DRG) and trigeminal ganglia. Na(v)1.9 produces a persistent, tetrodotoxin-resistant current with wide overlap between activation and steady-state inactivation, and appears to modulate resting potential and to amplify small depolarizations. These unique properties indicate that Na(v)1.9 has significant effects on the electroresponsive properties of primary nociceptive neurons. Downregulation of Na(v)1.9, which results from a lack of peripheral glial cell-derived neurotrophic factor following peripheral axotomy, might retune DRG neurons and contribute to their hyperexcitability after nerve injury. Thus, Na(v)1.9 appears to play a key role in nociception and is an attractive target in the search for more effective treatments for pain.

  9. Hypotonic regulation of mouse epithelial sodium channel in Xenopus laevis oocytes.

    Science.gov (United States)

    Galizia, Luciano; Marino, Gabriela I; Ojea, Alejandro; Kotsias, Basilio A

    2013-12-01

    The regulation of the epithelial Na⁺ channel (ENaC) during cell swelling is relevant in cellular processes in which cell volume changes occur, i.e., migration, proliferation and cell absorption. Its sensitivity to hypotonically induced swelling was investigated in the Xenopus oocyte expression system with the injection of the three subunits of mouse ENaC. We used voltage-clamp techniques to study the amiloride-sensitive Na⁺ currents (INa(amil)) and video microscopic methodologies to assess oocyte volume changes. Under conditions of mild swelling (25 % reduced hypotonicity) inward current amplitude decreased rapidly over 1.5 min. In contrast, there was no change in current amplitude of H₂O-injected oocytes to the osmotic insult. INa(amil) kinetics analysis revealed a decrease in the slower inactivation time constant during the hypotonic stimuli. Currents from ENaC-injected oocytes were not sensitive to external Cl⁻ reduction. Neither short- nor long-term cytochalasin D treatment affected the observed response. Oocytes expressing a DEG mutant β-ENaC subunit (β-S518K) with an open probability of 1 had reduced INa(amil) hypotonic response compared to oocytes injected with wild-type ENaC subunits. Finally, during the hypotonic response ENaC-injected oocytes did not show a cell volume difference compared with water-injected oocytes. On this basis we suggest that hypotonicity-dependent ENaC inhibition is principally mediated through an effect on open probability of channels in the membrane.

  10. Brevenal, a brevetoxin antagonist from Karenia brevis, binds to a previously unreported site on mammalian sodium channels.

    Science.gov (United States)

    Gold, Elena P; Jacocks, Henry M; Bourdelais, Andrea J; Baden, Daniel G

    2013-06-01

    Brevetoxins are a family of ladder-frame polyether toxins produced by the marine dinoflagellate Karenia brevis. During blooms of K. brevis, inhalation of brevetoxins aerosolized by wind and wave action can lead to asthma-like symptoms in persons at the beach. Consumption of either shellfish or finfish contaminated by K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to binding at a defined site on, and subsequent activation of, voltage-sensitive sodium channels (VSSCs) in cell membranes (site 5). In addition to brevetoxins, K. brevis produces several other ladder-frame compounds. One of these compounds, brevenal, has been shown to antagonize the effects of brevetoxin. In an effort to further characterize to effects of brevenal, a radioactive analog ([3H]-brevenol) was produced by reducing the side-chain terminal aldehyde moiety of brevenal to an alcohol using tritiated sodium borohydride. A KD of 67 nM and Bmax of 7.1 pmol/mg protein were obtained for [3H]-brevenol in rat brain synaptosomes, suggesting a 1:1 matching with VSSCs. Brevenal and brevenol competed for [3H]-brevenol binding with Ki values of 75 nM and 56 nM, respectively. However, although both brevenal and brevenol can inhibit brevetoxin binding, brevetoxin was completely ineffective at competition for [3H]-brevenol binding. After examining other site-specific compounds, it was determined that [3H]-brevenol binds to a site that is distinct from the other known sites including the brevetoxin site (site 5) although some interaction with site 5 is apparent.

  11. Ultrafast sodium channel block by dietary fish oil prevents dofetilide-induced ventricular arrhythmias in rabbit hearts.

    Science.gov (United States)

    Dujardin, K S; Dumotier, B; David, M; Guizy, M; Valenzuela, C; Hondeghem, L M

    2008-10-01

    Several epidemiologic and clinical studies show that following myocardial infarction, dietary supplements of omega-3 polyunsaturated fatty acids (omega3FA) reduce sudden death. Animal data show that omega3FA have antiarrhythmic properties, but their mechanisms of action require further elucidation. The effects of omega3FA supplementation were studied in female rabbits to analyze whether their antiarrhythmic effects are due to a reduction of triangulation, reverse use-dependence, instability, and dispersion (TRIaD) of the cardiac action potential (TRIaD as a measure of proarrhythmic effects). In Langendorff-perfused hearts challenged by a selective rapidly activating delayed rectifier potassium current inhibitor that has been shown to exhibit proarrhythmic effects (dofetilide; 1 to 100 nM), omega3FA pretreatment (30 days; n=6) prolonged the plateau phase of the monophasic action potential; did not slow the terminal fast repolarization; reduced the dofetilide-induced prolongation of the action potential duration; reduced dofetilide-induced triangulation; and reduced dofetilide-induced reverse use-dependence, instability of repolarization, and dispersion. Dofetilide reduced excitability in omega3FA-pretreated hearts but not in control hearts. Whereas torsades de pointes (TdP) were observed in five out of six in control hearts, none were observed in omega3FA-pretreated hearts. Docosahexaenoic acid (DHA) inhibited the sodium current with ultrafast kinetics. Dietary omega3FA supplementation markedly reduced dofetilide-induced TRIaD and abolished dofetilide-induced TdP. Ultrafast sodium channel block by DHA may account for the antiarrhythmic protection of the dietary supplements of omega3FA against dofetilide-induced proarrhythmia observed in this animal model.

  12. Block of inactivated sodium channels and of depolarization-induced automaticity in guinea pig papillary muscle by amiodarone.

    Science.gov (United States)

    Mason, J W; Hondeghem, L M; Katzung, B G

    1984-09-01

    The electrophysiological effects of amiodarone were studied in guinea pig papillary muscle by means of the single sucrose gap voltage clamp technique. The first time derivative of the upstroke of the action potential was measured as an indicator of the sodium current. The preparations were not voltage clamped during the action potential upstroke. Acute effects of amiodarone (4.4 X 10(-5) M and 8.8 X 10(-5) M; six experiments each) and effects of chronic administration at a single dose level (nine experimental vs. eight control animals) were studied. Results were qualitatively the same for all experimental conditions, and concentration dependent in the acute studies. Amiodarone caused marked use-dependent depression of the first time derivative of the upstroke of the action potential during stimulus trains. For example, at normal resting potential, chronic amiodarone treatment reduced the first time derivative of the upstroke of the action potential of the 16th beat of trains of cycle length 300 msec to 70 +/- 15% (mean +/- SD) of the initial value. This blocking effect was accentuated at more depolarized holding potentials and reduced at hyperpolarized holding potentials. Reduction of the first time derivative of the upstroke of the action potential was found to depend upon sodium channel inactivation. For all experiments, the mean normalized first time derivative of the upstroke of the action potential following a 1-second clamp in the -20 to +20 mV range was 0.92 +/- 0.08 in the control condition and 0.66 +/- 0.20 in the presence of amiodarone (less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

  13. The central mechanism underlying hypertension: a review of the roles of sodium ions, epithelial sodium channels, the renin–angiotensin–aldosterone system, oxidative stress and endogenous digitalis in the brain

    Science.gov (United States)

    Takahashi, Hakuo; Yoshika, Masamichi; Komiyama, Yutaka; Nishimura, Masato

    2011-01-01

    The central nervous system has a key role in regulating the circulatory system by modulating the sympathetic and parasympathetic nervous systems, pituitary hormone release, and the baroreceptor reflex. Digoxin- and ouabain-like immunoreactive materials were found >20 years ago in the hypothalamic nuclei. These factors appeared to localize to the paraventricular and supraoptic nuclei and the nerve fibers at the circumventricular organs and supposed to affect electrolyte balance and blood pressure. The turnover rate of these materials increases with increasing sodium intake. As intracerebroventricular injection of ouabain increases blood pressure via sympathetic activation, an endogenous digitalis-like factor (EDLF) was thought to regulate cardiovascular system-related functions in the brain, particularly after sodium loading. Experiments conducted mainly in rats revealed that the mechanism of action of ouabain in the brain involves sodium ions, epithelial sodium channels (ENaCs) and the renin–angiotensin–aldosterone system (RAAS), all of which are affected by sodium loading. Rats fed a high-sodium diet develop elevated sodium levels in their cerebrospinal fluid, which activates ENaCs. Activated ENaCs and/or increased intracellular sodium in neurons activate the RAAS; this releases EDLF in the brain, activating the sympathetic nervous system. The RAAS promotes oxidative stress in the brain, further activating the RAAS and augmenting sympathetic outflow. Angiotensin II and aldosterone of peripheral origin act in the brain to activate this cascade, increasing sympathetic outflow and leading to hypertension. Thus, the brain Na+–ENaC–RAAS–EDLF axis activates sympathetic outflow and has a crucial role in essential and secondary hypertension. This report provides an overview of the central mechanism underlying hypertension and discusses the use of antihypertensive agents. PMID:21814209

  14. Effect of insecticidal fusion proteins containing spider toxins targeting sodium and calcium ion channels on pyrethroid-resistant strains of peach-potato aphid (Myzus persicae).

    Science.gov (United States)

    Yang, Sheng; Fitches, Elaine; Pyati, Prashant; Gatehouse, John A

    2015-07-01

    The recombinant fusion proteins Pl1a/GNA and Hv1a/GNA contain the spider venom peptides δ-amaurobitoxin-PI1a or ω-hexatoxin-Hv1a respectively, linked to snowdrop lectin (GNA). Pl1a targets receptor site 4 of insect voltage-gated sodium channels (NaCh), while Hv1a targets voltage-gated calcium channels. Insecticide-resistant strains of peach-potato aphid (Myzus persicae) contain mutations in NaCh. The pyrethroid-resistant kdr (794J) and super-kdr (UKO) strains contain mutations at residues L1014 and M918 in the channel α-subunit respectively, while the kdr + super-kdr strain (4824J), insensitive to pyrethroids, contains mutations at both L1014 and M918. Pl1a/GNA and Hv1a/GNA fusion proteins have estimated LC50 values of 0.35 and 0.19 mg mL(-1) when fed to wild-type M. persicae. For insecticide-resistant aphids, LC50 for the Pl1a/GNA fusion protein increased by 2-6-fold, correlating with pyrethroid resistance (wild type < kdr < super-kdr < kdr + super-kdr strains). In contrast, LC50 for the Hv1a/GNA fusion protein showed limited correlation with pyrethroid resistance. Mutations in the sodium channel in pyrethroid-resistant aphids also protect against a fusion protein containing a sodium-channel-specific toxin, in spite of differences in ligand-channel interactions, but do not confer resistance to a fusion protein targeting calcium channels. The use of fusion proteins with differing targets could play a role in managing pesticide resistance. © 2014 Society of Chemical Industry.

  15. Rufinamide attenuates mechanical allodynia in a model of neuropathic pain in the mouse and stabilizes voltage-gated sodium channel inactivated state.

    Science.gov (United States)

    Suter, Marc R; Kirschmann, Guylène; Laedermann, Cedric J; Abriel, Hugues; Decosterd, Isabelle

    2013-01-01

    Voltage-gated sodium channels dysregulation is important for hyperexcitability leading to pain persistence. Sodium channel blockers currently used to treat neuropathic pain are poorly tolerated. Getting new molecules to clinical use is laborious. We here propose a drug already marketed as anticonvulsant, rufinamide. We compared the behavioral effect of rufinamide to amitriptyline using the Spared Nerve Injury neuropathic pain model in mice. We compared the effect of rufinamide on sodium currents using in vitro patch clamp in cells expressing the voltage-gated sodium channel Nav1.7 isoform and on dissociated dorsal root ganglion neurons to amitriptyline and mexiletine. In naive mice, amitriptyline (20 mg/kg) increased withdrawal threshold to mechanical stimulation from 1.3 (0.6-1.9) (median [95% CI]) to 2.3 g (2.2-2.5) and latency of withdrawal to heat stimulation from 13.1 (10.4-15.5) to 30.0 s (21.8-31.9), whereas rufinamide had no effect. Rufinamide and amitriptyline alleviated injury-induced mechanical allodynia for 4 h (maximal effect: 0.10 ± 0.03 g (mean ± SD) to 1.99 ± 0.26 g for rufinamide and 0.25 ± 0.22 g to 1.92 ± 0.85 g for amitriptyline). All drugs reduced peak current and stabilized the inactivated state of voltage-gated sodium channel Nav1.7, with similar effects in dorsal root ganglion neurons. At doses alleviating neuropathic pain, amitriptyline showed alteration of behavioral response possibly related to either alteration of basal pain sensitivity or sedative effect or both. Side-effects and drug tolerance/compliance are major problems with drugs such as amitriptyline. Rufinamide seems to have a better tolerability profile and could be a new alternative to explore for the treatment of neuropathic pain.

  16. Activation of tetrodotoxin-resistant sodium channel NaV1.9 in rat primary sensory neurons contributes to melittin-induced pain behavior.

    Science.gov (United States)

    Yu, Yao-Qing; Zhao, Zhen-Yu; Chen, Xue-Feng; Xie, Fang; Yang, Yan; Chen, Jun

    2013-03-01

    Tetrodotoxin-resistant (TTX-R) sodium channels NaV1.8 and NaV1.9 in dorsal root ganglion (DRG) neurons play important roles in pathological pain. We recently reported that melittin, the major toxin of whole bee venom, induced action potential firings in DRG neurons even in the presence of a high concentration (500 nM) of TTX, indicating the contribution of TTX-R sodium channels. This hypothesis is fully investigated in the present study. After subcutaneous injection of melittin, NaV1.8 and NaV1.9 significantly upregulate mRNA and protein expressions, and related sodium currents also increase. Double immunohistochemical results show that NaV1.8-positive neurons are mainly medium- and small-sized, whereas NaV1.9-positive ones are only small-sized. Antisense oligodeoxynucleotides (AS ODNs) targeting NaV1.8 and NaV1.9 are used to evaluate functional significance of the increased expressions of TTX-R sodium channels. Behavioral tests demonstrate that AS ODN targeting NaV1.9, but not NaV1.8, reverses melittin-induced heat hypersensitivity. Neither NaV1.8 AS ODN nor NaV1.9 AS ODN affects melittin-induced mechanical hypersensitivity. These results provide previously unknown evidence that upregulation of NaV1.9, but not NaV1.8, in small-sized DRG neurons contributes to melittin-induced heat hypersensitivity. Furthermore, melittin-induced biological effect indicates a potential strategy to study properties of TTX-R sodium channels.

  17. The Past, Present, and Potential Future of Sodium Channel Block as an Atrial Fibrillation Suppressing Strategy.

    Science.gov (United States)

    Aguilar, Martin; Nattel, Stanley

    2015-11-01

    Despite major advances in arrhythmia therapy, atrial fibrillation (AF) remains a challenge. A significant limitation in AF management is the lack of safe and effective drugs to restore and/or maintain sinus rhythm. The rational design of a new generation of AF-selective Na(+) channel blockers (NCBs) is emerging as a promising AF-suppressing strategy. Recent theoretical and experimental advances have generated insights into the mechanisms underlying AF maintenance and termination by antiarrhythmic drugs. Our understanding of antiarrhythmic drug-induced proarrhythmia has also grown in sophistication. These discoveries have created new possibilities in therapeutic targeting and renewed interest in improved NCB antiarrhythmic drugs. Recently described differences in atrial versus ventricular electrophysiology can be exploited in the prospective design of atrial-selective NCBs. Furthermore, state-dependent block has been shown to be an important modulator of NCB rate selectivity. Together, differential atrial-ventricular electrophysiological actions and state-dependent block form the backbone for the rational design of an AF-selective NCB. Synergistic combinations incorporating both NCB and block of K(+) currents may allow for further enhancement of AF selectivity. Future work on translating these basic research advances into the development of an optimized AF-selective NCB has the potential to provide safer and more effective pharmacotherapeutic options for AF, thereby fulfilling a major unmet clinical need.

  18. Ethanol alters alveolar fluid balance via Nadph oxidase (NOX signaling to epithelial sodium channels (ENaC in the lung.

    Directory of Open Access Journals (Sweden)

    Charles A Downs

    Full Text Available Chronic alcohol consumption is associated with increased incidence of ICU-related morbidity and mortality, primarily from acute respiratory distress syndrome (ARDS. However, the mechanisms involved are unknown. One explanation is that alcohol regulates epithelial sodium channels (ENaC via oxidant signaling to promote a pro- injury environment. We used small rodent models to mimic acute and chronic alcohol consumption and tested the hypothesis that ethanol (EtOH would affect lung fluid clearance by up-regulating ENaC activity in the lung. Fluorescence labeling of rat lung slices and in vivo mouse lung revealed an increase in ROS production in response to acute EtOH exposure. Using western blots and fluorescein-5-maleimide labeling, we conclude that EtOH exposure modifies cysteines of α-ENaC while data from single channel patch clamp analysis confirm that 0.16% EtOH increased ENaC activity in rat alveolar cells. In vivo lung fluid clearance demonstrated a latent increase in fluid clearance in mice receiving EtOH diet. Ethanol mice given a tracheal instillation of LPS demonstrated early lung fluid clearance compared to caloric control mice and C57Bl/6 mice. Standard biochemical techniques reveal that chronic EtOH consumption resulted in greater protein expression of the catalytic gp91(phox subunit and the obligate Rac1 protein. Collectively these data suggest that chronic EtOH consumption may lead to altered regulation of ENaC, contributing to a 'pro-injury' environment in the alcohol lung.

  19. Different pH-sensitivity patterns of 30 sodium channel inhibitors suggest chemically different pools along the access pathway.

    Directory of Open Access Journals (Sweden)

    Alexandra eLazar

    2015-09-01

    Full Text Available The major drug binding site of sodium channels is inaccessible from the extracellular side, drug molecules can only access it either from the membrane phase, or from the intracellular aqueous phase. For this reason, ligand-membrane interactions are as important determinants of inhibitor properties, as ligand-protein interactions. One way to probe this is to modify the pH of the extracellular fluid, which alters the ratio of charged vs. uncharged forms of some compounds, thereby changing their interaction with the membrane. In this electrophysiology study we used three different pH values: 6.0, 7.3 and 8.6 to test the significance of the protonation-deprotonation equilibrium in drug access and affinity. We investigated drugs of several different indications: carbamazepine, lamotrigine, phenytoin, lidocaine, bupivacaine, mexiletine, flecainide, ranolazine, riluzole, memantine, ritanserin, tolperisone, silperisone, ambroxol, haloperidol, chlorpromazine, clozapine, fluoxetine, sertraline, paroxetine, amitriptyline, imipramine, desipramine, maprotiline, nisoxetine, mianserin, mirtazapine, venlafaxine, nefazodone and trazodone. We recorded the pH-dependence of potency, reversibility, as well as onset/offset kinetics. As expected, we observed a strong correlation between the acidic dissociation constant (pKa of drugs and the pH-dependence of their potency. Unexpectedly, however, the pH-dependence of reversibility or kinetics showed diverse patterns, not simple correlation. Our data are best explained by a model where drug molecules can be trapped in at least two chemically different environments: A hydrophilic trap (which may be the aqueous cavity within the inner vestibule, which favors polar and less lipophilic compounds, and a lipophilic trap (which may be the membrane phase itself, and/or lipophilic binding sites on the channel. Rescue from the hydrophilic and lipophilic traps can be promoted by alkalic and acidic extracellular pH, respectively.

  20. Direct interaction with contactin targets voltage-gated sodium channel Na(v)1.9/NaN to the cell membrane.

    Science.gov (United States)

    Liu, C J; Dib-Hajj, S D; Black, J A; Greenwood, J; Lian, Z; Waxman, S G

    2001-12-07

    The mechanisms that target various sodium channels within different regions of the neuronal membrane, which they endow with different physiological properties, are not yet understood. To examine this issue we studied the voltage-gated sodium channel Na(v)1.9/NaN, which is preferentially expressed in small sensory neurons of dorsal root ganglia and trigeminal ganglia and the nonmyelinated axons that arise from them. Our results show that the cell adhesion molecule contactin binds directly to Na(v)1.9/NaN and recruits tenascin to the protein complex in vitro. Na(v)1.9/NaN and contactin co-immunoprecipitate from dorsal root ganglia and transfected Chinese hamster ovary cell line, and co-localize in the C-type neuron soma and along nonmyelinated C-fibers and at nerve endings in the skin. Co-transfection of Chinese hamster ovary cells with Na(v)1.9/NaN and contactin enhances the surface expression of the sodium channel over that of Na(v)1.9/NaN alone. Thus contactin binds directly to Na(v)1.9/NaN and participates in the surface localization of this channel along nonmyelinated axons.

  1. Tetrodotoxin-resistant voltage-gated sodium channels Na(v)1.8 and Na(v)1.9 are expressed in the retina.

    Science.gov (United States)

    O'Brien, Brendan J; Caldwell, John H; Ehring, George R; Bumsted O'Brien, Keely M; Luo, Songjiang; Levinson, S Rock

    2008-06-20

    Voltage-gated sodium channels (VGSCs) are one of the fundamental building blocks of electrically excitable cells in the nervous system. These channels are responsible for the generation of action potentials that are required for the communication of neuronal signals over long distances within a cell. VGSCs are encoded by a family of nine genes whose products have widely varying biophysical properties. In this study, we have detected the expression of two atypical VGSCs (Na(v)1.8 and Na(v)1.9) in the retina. Compared with more common VGSCs, Na(v)1.8 and Na(v)1.9 have unusual biophysical and pharmacological properties, including persistent sodium currents and resistance to the canonical sodium channel blocker tetrodotoxin (TTX). Our molecular biological and immunohistochemical data derived from mouse (Mus musculus) retina demonstrate expression of Na(v)1.8 by retinal amacrine and ganglion cells, whereas Na(v)1.9 is expressed by photoreceptors and Müller glia. The fact that these channels exist in the central nervous system (CNS) and exhibit robust TTX resistance requires a re-evaluation of prior physiological, pharmacological, and developmental data in the visual system, in which the diversity of VGSCs has been previously underestimated. (c) 2008 Wiley-Liss, Inc.

  2. Novel Mutations in the Voltage-Gated Sodium Channel of Pyrethroid-Resistant Varroa destructor Populations from the Southeastern USA.

    Science.gov (United States)

    González-Cabrera, Joel; Rodríguez-Vargas, Sonia; Davies, T G Emyr; Field, Linda M; Schmehl, Daniel; Ellis, James D; Krieger, Klemens; Williamson, Martin S

    2016-01-01

    The parasitic mite Varroa destructor has a significant worldwide impact on bee colony health. In the absence of control measures, parasitized colonies invariably collapse within 3 years. The synthetic pyrethroids tau-fluvalinate and flumethrin have proven very effective at managing this mite within apiaries, but intensive control programs based mainly on one active ingredient have led to many reports of pyrethroid resistance. In Europe, a modification of leucine to valine at position 925 (L925V) of the V. destructor voltage-gated sodium channel was correlated with resistance, the mutation being found at high frequency exclusively in hives with a recent history of pyrethroid treatment. Here, we identify two novel mutations, L925M and L925I, in tau-fluvalinate resistant V. destructor collected at seven sites across Florida and Georgia in the Southeastern region of the USA. Using a multiplexed TaqMan® allelic discrimination assay, these mutations were found to be present in 98% of the mites surviving tau-fluvalinate treatment. The mutations were also found in 45% of the non-treated mites, suggesting a high potential for resistance evolution if selection pressure is applied. The results from a more extensive monitoring programme, using the Taqman® assay described here, would clearly help beekeepers with their decision making as to when to include or exclude pyrethroid control products and thereby facilitate more effective mite management programmes.

  3. Novel Mutations in the Voltage-Gated Sodium Channel of Pyrethroid-Resistant Varroa destructor Populations from the Southeastern USA.

    Directory of Open Access Journals (Sweden)

    Joel González-Cabrera

    Full Text Available The parasitic mite Varroa destructor has a significant worldwide impact on bee colony health. In the absence of control measures, parasitized colonies invariably collapse within 3 years. The synthetic pyrethroids tau-fluvalinate and flumethrin have proven very effective at managing this mite within apiaries, but intensive control programs based mainly on one active ingredient have led to many reports of pyrethroid resistance. In Europe, a modification of leucine to valine at position 925 (L925V of the V. destructor voltage-gated sodium channel was correlated with resistance, the mutation being found at high frequency exclusively in hives with a recent history of pyrethroid treatment. Here, we identify two novel mutations, L925M and L925I, in tau-fluvalinate resistant V. destructor collected at seven sites across Florida and Georgia in the Southeastern region of the USA. Using a multiplexed TaqMan® allelic discrimination assay, these mutations were found to be present in 98% of the mites surviving tau-fluvalinate treatment. The mutations were also found in 45% of the non-treated mites, suggesting a high potential for resistance evolution if selection pressure is applied. The results from a more extensive monitoring programme, using the Taqman® assay described here, would clearly help beekeepers with their decision making as to when to include or exclude pyrethroid control products and thereby facilitate more effective mite management programmes.

  4. Inhibition of veratridine-induced delayed inactivation of the voltage-sensitive sodium channel by synthetic analogs of crambescin B.

    Science.gov (United States)

    Tsukamoto, Tadaaki; Chiba, Yukie; Nakazaki, Atsuo; Ishikawa, Yuki; Nakane, Yoshiki; Cho, Yuko; Yotsu-Yamashita, Mari; Nishikawa, Toshio; Wakamori, Minoru; Konoki, Keiichi

    2017-03-01

    Crambescin B carboxylic acid, a synthetic analog of crambescin B, was recently found to inhibit the voltage-sensitive sodium channels (VSSC) in a cell-based assay using neuroblastoma Neuro 2A cells. In the present study, whole-cell patch-clamp recordings were conducted with three heterologously expressed VSSC subtypes, Na v 1.2, Na v 1.6 and Na v 1.7, in a human embryonic kidney cell line HEK293T to further characterize the inhibition of VSSC by crambescin B carboxylic acid. Contrary to the previous observation, crambescin B carboxylic acid did not inhibit peak current evoked by depolarization from the holding potential of -100mV to the test potential of -10mV in the absence or presence of veratridine (VTD). In the presence of VTD, however, crambescin B carboxylic acid diminished VTD-induced sustained and tail currents through the three VSSC subtypes in a dose-dependent manner, whereas TTX inhibited both the peak current and the VTD-induced sustained and tail currents through all subtypes of VSSC tested. We thus concluded that crambescin B carboxylic acid does not block VSSC in a similar manner to TTX but modulate the action of VTD, thereby causing an apparent block of VSSC in the cell-based assay. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Inactivation properties of sodium channel Nav1.8 maintain action potential amplitude in small DRG neurons in the context of depolarization.

    Science.gov (United States)

    Patrick Harty, T; Waxman, Stephen G

    2007-05-31

    Small neurons of the dorsal root ganglion (DRG) express five of the nine known voltage-gated sodium channels. Each channel has unique biophysical characteristics which determine how it contributes to the generation of action potentials (AP). To better understand how AP amplitude is maintained in nociceptive DRG neurons and their centrally projecting axons, which are subjected to depolarization within the dorsal horn, we investigated the dependence of AP amplitude on membrane potential, and how that dependence is altered by the presence or absence of sodium channel Nav1.8. In small neurons cultured from wild type (WT) adult mouse DRG, AP amplitude decreases as the membrane potential is depolarized from -90 mV to -30 mV. The decrease in amplitude is best fit by two Boltzmann equations, having V1/2 values of -73 and -37 mV. These values are similar to the V1/2 values for steady-state fast inactivation of tetrodotoxin-sensitive (TTX-s) sodium channels, and the tetrodotoxin-resistant (TTX-r) Nav1.8 sodium channel, respectively. Addition of TTX eliminates the more hyperpolarized V1/2 component and leads to increasing AP amplitude for holding potentials of -90 to -60 mV. This increase is substantially reduced by the addition of potassium channel blockers. In neurons from Nav1.8(-/-) mice, the voltage-dependent decrease in AP amplitude is characterized by a single Boltzmann equation with a V1/2 value of -55 mV, suggesting a shift in the steady-state fast inactivation properties of TTX-s sodium channels. Transfection of Nav1.8(-/-) DRG neurons with DNA encoding Nav1.8 results in a membrane potential-dependent decrease in AP amplitude that recapitulates WT properties. We conclude that the presence of Nav1.8 allows AP amplitude to be maintained in DRG neurons and their centrally projecting axons even when depolarized within the dorsal horn.

  6. Correlation of Nav1.8 and Nav1.9 sodium channel expression with neuropathic pain in human subjects with lingual nerve neuromas

    OpenAIRE

    Bird, E.V.; Christmas, C.R.; Loescher, A.R.; Smith, K G; Robinson, P.P.; Black, J.A.; Waxman, S G; Boissonade, F.M.

    2013-01-01

    Background: Voltage-gated sodium channels Nav1.8 and Nav1.9 are expressed preferentially in small diameter sensory neurons, and are thought to play a role in the generation of ectopic activity in neuronal cell bodies and/or their axons following peripheral nerve injury. The expression of Nav1.8 and Nav1.9 has been quantified in human lingual nerves that have been previously injured inadvertently during lower third molar removal, and any correlation between the expression of these ion channels...

  7. Direct amplification of a single dissected chromosomal segment by polymerase chain reaction: a human brain sodium channel gene is on chromosome 2q22-q23.

    Science.gov (United States)

    Han, J A; Lu, C M; Brown, G B; Rado, T A

    1991-01-01

    We have devised a general strategy for gene mapping based upon the direct amplification of a target sequence within a single microdissected Giemsa-banded chromosomal segment using the polymerase chain reaction. The usefulness of this approach was demonstrated by mapping a cloned human brain sodium channel (alpha subunit) gene sequence to chromosome 2q22-q23. When DNA from single, dissected chromosome segments 2q21-qter and 2q24-pter were used as templates, a sodium channel-specific 172-base-pair polymerase chain reaction product was obtained. This product was not synthesized when segments 2q21-pter and 2q24-qter were used. Chromosome microdissection-polymerase chain reaction is not only a simple, fast, and accurate method for gene mapping but also may offer significant advantages for other applications, such as cancer cytogenetics and linkage analysis. Images PMID:1846440

  8. Antinociceptive Effects of AGAP, a Recombinant Neurotoxic Polypeptide: Possible Involvement of the Tetrodotoxin-Resistant Sodium Channels in Small Dorsal Root Ganglia Neurons

    Science.gov (United States)

    Li, Chun-Li; Liu, Xi-Fang; Li, Gui-Xia; Ban, Meng-qi; Chen, Jian-Zhao; Cui, Yong; Zhang, Jing-Hai; Wu, Chun-Fu

    2016-01-01

    Antitumor-analgesic peptide (AGAP) is a novel recombinant polypeptide. The primary study showed that AGAP 1.0 mg/kg exhibited strong analgesic and antitumor effects. The tail vein administration of AGAP potently reduced pain behaviors in mice induced by intraplantar injection of formalin or intraperitoneal injection of acetic acid, without affecting basal pain perception. To further assess the mechanisms of AGAP, the effects of AGAP on sodium channels were assessed using the whole-cell patch clamp recordings in dorsal root ganglia (DRG) neurons. The results showed that AGAP (3–1000 nM) inhibited the sodium currents in small-diameter DRG neurons in a dose-dependent manner. 1000 nM AGAP could inhibit the current density-voltage relationship curve of sodium channels in a voltage-dependent manner and negatively shift the activation curves. 1000 nM AGAP could reduce the tetrodotoxin-resistant (TTX-R) sodium currents by 42.8% in small-diameter DRG neurons. Further analysis revealed that AGAP potently inhibited NaV1.8 currents by 59.4%, and negatively shifted the activation and inactivation kinetics. 1000 nM AGAP also reduced the NaV1.9 currents by 33.7%, but had no significant effect on activation and inactivation kinetics. Thus, our results demonstrated that submicromolar concentrations of AGAP inhibited TTX-R sodium channel in rat small-diameter DRG neurons. It is concluded that these new results may better explain, at least in part, the analgesic properties of this polypeptide. PMID:28066245

  9. Antinociceptive effects of AGAP, a recombinant neurotoxic polypeptide: Possible involvement of the tetrodotoxin-resistant sodium channels in small dorsal root ganglia neurons

    Directory of Open Access Journals (Sweden)

    Li Chunli

    2016-12-01

    Full Text Available Antitumor-analgesic peptide(AGAP)is a novel recombinant polypeptide. The primary study showed that AGAP 1.0 mg/kg exhibited strong analgesic and antitumor effects. The tail vein administration of AGAP potently reduced pain behaviors in mice induced by intraplantar injection of formalin or intraperitoneal injection of acetic acid, without affecting basal pain perception. To further assess the mechanisms of AGAP, the effects of AGAP on sodium channels were assessed using the whole-cell patch clamp recordings in dorsal root ganglia (DRG neurons. The results showed that AGAP (3-1000 nM inhibited the sodium currents in small-diameter DRG neurons in a dose-dependent manner. 1000 nM AGAP could inhibit the current density-voltage relationship curve of sodium channels in a voltage-dependent manner and negatively shift the activation curves. 1000 nM AGAP could reduce the tetrodotoxin-resistant (TTX-R sodium currents by 42.8% in small-diameter DRG neurons. Further analysis revealed that AGAP potently inhibited NaV1.8 currents by 59.4%, and negatively shifted the activation and inactivation kinetics. 1000 nM AGAP also reduced the NaV1.9 currents by 33.7%, but had no significant effect on activation and inactivation kinetics. Thus, our results demonstrated that submicromolar concentrations of AGAP inhibited TTX-R sodium channel in rat small-diameter DRG neurons. It is concluded that these new results may better explain, at least in part, the analgesic properties of this polypeptide.

  10. Evidence for Dual Binding Sites for 1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) in Insect Sodium Channels*

    Science.gov (United States)

    Du, Yuzhe; Nomura, Yoshiko; Zhorov, Boris S.; Dong, Ke

    2016-01-01

    1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), the first organochlorine insecticide, and pyrethroid insecticides are sodium channel agonists. Although the use of DDT is banned in most of the world due to its detrimental impact on the ecosystem, indoor residual spraying of DDT is still recommended for malaria control in Africa. Development of resistance to DDT and pyrethroids is a serious global obstacle for managing disease vectors. Mapping DDT binding sites is necessary for understanding mechanisms of resistance and modulation of sodium channels by structurally different ligands. The pioneering model of the housefly sodium channel visualized the first receptor for pyrethroids, PyR1, in the II/III domain interface and suggested that DDT binds within PyR1. Previously, we proposed the second pyrethroid receptor, PyR2, at the I/II domain interface. However, whether DDT binds to both pyrethroid receptor sites remains unknown. Here, using computational docking of DDT into the Kv1.2-based mosquito sodium channel model, we predict that two DDT molecules can bind simultaneously within PyR1 and PyR2. The bulky trichloromethyl group of each DDT molecule fits snugly between four helices in the bent domain interface, whereas two p-chlorophenyl rings extend into two wings of the interface. Model-driven mutagenesis and electrophysiological analysis confirmed these propositions and revealed 10 previously unknown DDT-sensing residues within PyR1 and PyR2. Our study proposes a dual DDT-receptor model and provides a structural background for rational development of new insecticides. PMID:26637352

  11. Contribution of the tetrodotoxin-resistant voltage-gated sodium channel NaV1.9 to sensory transmission and nociceptive behavior

    OpenAIRE

    Priest, Birgit T.; Murphy, Beth A.; Lindia, Jill A.; Diaz, Carmen; Abbadie, Catherine; Ritter, Amy M.; Liberator, Paul; Iyer, Leslie M.; Kash, Shera F.; Kohler, Martin G.; Kaczorowski, Gregory J.; MacIntyre, D. Euan; Martin, William J.

    2005-01-01

    The transmission of pain signals after injury or inflammation depends in part on increased excitability of primary sensory neurons. Nociceptive neurons express multiple subtypes of voltage-gated sodium channels (NaV1s), each of which possesses unique features that may influence primary afferent excitability. Here, we examined the contribution of NaV1.9 to nociceptive signaling by studying the electrophysiological and behavioral phenotypes of mice with a disruption of the SCN11A gene, which en...

  12. No evidence for induction or selection of mutant sodium channel expression in the copepod Acartia husdsonica challenged with the toxic dinoflagellate Alexandrium fundyense.

    Science.gov (United States)

    Finiguerra, Michael; Avery, David E; Dam, Hans G

    2014-09-01

    Some species in the dinoflagellate genus Alexandrium spp. produce a suite of neurotoxins that block sodium channels, known as paralytic shellfish toxins (PST), which have deleterious effects on grazers. Populations of the ubiquitous copepod grazer Acartia hudsonica that have co-occurred with toxic Alexandrium spp. are better adapted than naïve populations. The mechanism of adaptation is currently unknown. We hypothesized that a mutation in the sodium channel could account for the grazer adaptation. We tested two hypotheses: (1) Expression of the mutant sodium channel could be induced by exposure to toxic Alexandrium fundyense; (2) in the absence of induction, selection exerted by toxic A. fundyense would favor copepods that predominantly express the mutant isoform. In the copepod A. hudsonica, both isoforms are expressed in all individuals in varying proportions. Thus, in addition to comparing expression ratios of wild-type to mutant isoforms for individual copepods, we also partitioned copepods into three groups: those that predominantly express the mutant (PMI) isoform, the wild-type (PWI) isoform, or both isoforms approximately equally (EI). There were no differences in isoform expression between individuals that were fed toxic and nontoxic food after three and 6 days; induction of mutant isoform expression did not occur. Furthermore, the hypothesis that mutant isoform expression responds to toxic food was also rejected. That is, no consistent evidence showed that the wild-type to mutant isoform ratios decreased, or that the relative proportion of PMI individuals increased, due to the consumption of toxic food over four generations. However, in the selected line that was continuously exposed to toxic food sources, egg production rate increased, which suggested that adaptation occurred but was unrelated to sodium channel isoform expression.

  13. Determining the Advantages, Costs, and Trade-Offs of a Novel Sodium Channel Mutation in the Copepod Acartia hudsonica to Paralytic Shellfish Toxins (PST)

    Science.gov (United States)

    Finiguerra, Michael; Avery, David E.; Dam, Hans G.

    2015-01-01

    The marine copepod Acartia hudsonica was shown to be adapted to dinoflagellate prey, Alexandrium fundyense, which produce paralytic shellfish toxins (PST). Adaptation to PSTs in other organisms is caused by a mutation in the sodium channel. Recently, a mutation in the sodium channel in A. hudsonica was found. In this study, we rigorously tested for advantages, costs, and trade-offs associated with the mutant isoform of A. hudsonica under toxic and non-toxic conditions. We combined fitness with wild-type: mutant isoform ratio measurements on the same individual copepod to test our hypotheses. All A. hudsonica copepods express both the wild-type and mutant sodium channel isoforms, but in different proportions; some individuals express predominantly mutant (PMI) or wild-type isoforms (PWI), while most individuals express relatively equal amounts of each (EI). There was no consistent pattern of improved performance as a function of toxin dose for egg production rate (EPR), ingestion rate (I), and gross growth efficiency (GGE) for individuals in the PMI group relative to individuals in the PWI expression group. Neither was there any evidence to indicate a fitness benefit to the mutant isoform at intermediate toxin doses. No clear advantage under toxic conditions was associated with the mutation. Using a mixed-diet approach, there was also no observed relationship between individual wild-type: mutant isoform ratios and among expression groups, on both toxic and non-toxic diets, for eggs produced over three days. Lastly, expression of the mutant isoform did not mitigate the negative effects of the toxin. That is, the reductions in EPR from a toxic to non-toxic diet for copepods were independent of expression groups. Overall, the results did not support our hypotheses; the mutant sodium channel isoform does not appear to be related to adaptation to PST in A. hudsonica. Other potential mechanisms responsible for the adaptation are discussed. PMID:26075900

  14. Determining the Advantages, Costs, and Trade-Offs of a Novel Sodium Channel Mutation in the Copepod Acartia hudsonica to Paralytic Shellfish Toxins (PST.

    Directory of Open Access Journals (Sweden)

    Michael Finiguerra

    Full Text Available The marine copepod Acartia hudsonica was shown to be adapted to dinoflagellate prey, Alexandrium fundyense, which produce paralytic shellfish toxins (PST. Adaptation to PSTs in other organisms is caused by a mutation in the sodium channel. Recently, a mutation in the sodium channel in A. hudsonica was found. In this study, we rigorously tested for advantages, costs, and trade-offs associated with the mutant isoform of A. hudsonica under toxic and non-toxic conditions. We combined fitness with wild-type: mutant isoform ratio measurements on the same individual copepod to test our hypotheses. All A. hudsonica copepods express both the wild-type and mutant sodium channel isoforms, but in different proportions; some individuals express predominantly mutant (PMI or wild-type isoforms (PWI, while most individuals express relatively equal amounts of each (EI. There was no consistent pattern of improved performance as a function of toxin dose for egg production rate (EPR, ingestion rate (I, and gross growth efficiency (GGE for individuals in the PMI group relative to individuals in the PWI expression group. Neither was there any evidence to indicate a fitness benefit to the mutant isoform at intermediate toxin doses. No clear advantage under toxic conditions was associated with the mutation. Using a mixed-diet approach, there was also no observed relationship between individual wild-type: mutant isoform ratios and among expression groups, on both toxic and non-toxic diets, for eggs produced over three days. Lastly, expression of the mutant isoform did not mitigate the negative effects of the toxin. That is, the reductions in EPR from a toxic to non-toxic diet for copepods were independent of expression groups. Overall, the results did not support our hypotheses; the mutant sodium channel isoform does not appear to be related to adaptation to PST in A. hudsonica. Other potential mechanisms responsible for the adaptation are discussed.

  15. TNF-α enhances the currents of voltage gated sodium channels in uninjured dorsal root ganglion neurons following motor nerve injury.

    Science.gov (United States)

    Chen, Xi; Pang, Rui-Ping; Shen, Kai-Feng; Zimmermann, Manfred; Xin, Wen-Jun; Li, Yong-Yong; Liu, Xian-Guo

    2011-02-01

    The ectopic discharges observed in uninjured dorsal root ganglion (DRG) neurons following various lesions of spinal nerves have been attributed to functional alterations of voltage-gated sodium channels (VGSCs). Such mechanisms may be important for the development of neuropathic pain. However, the pathophysiology underlying the functional modulation of VGSCs following nerve injury is largely unknown. Here, we studied this issue with use of a selective lumbar 5 ventral root transection (L5-VRT) model, in which dorsal root ganglion (DRG) neurons remain intact. We found that the L5-VRT increased the current densities of TTX-sensitive Na channels as well as currents in Nav1.8, but not Nav1.9 channels in uninjured DRG neurons. The thresholds of action potentials decreased and firing rates increased in DRG neurons following L5-VRT. As we found that levels of tumor necrosis factor-alpha (TNF-α) increased in cerebrospinal fluid (CSF) and in DRG tissue after L5-VRT, we tested whether the increased TNF-α might result in the changes in sodium channels. Indeed, recombinant rat TNF (rrTNF) enhanced the current densities of TTX-S and Nav1.8 in cultured DRG neurons dose-dependently. Furthermore, genetic deletion of TNF receptor 1 (TNFR-1) in mice attenuated the mechanical allodynia and prevented the increase in sodium currents in DRG neurons induced by L5-VRT. These data suggest that the increase in sodium currents in uninjured DRG neurons following nerve injury might be mediated by over-production of TNF-α. Copyright © 2010 Elsevier Inc. All rights reserved.

  16. Bromodomain-containing Protein 4 Activates Voltage-gated Sodium Channel 1.7 Transcription in Dorsal Root Ganglia Neurons to Mediate Thermal Hyperalgesia in Rats.

    Science.gov (United States)

    Hsieh, Ming-Chun; Ho, Yu-Cheng; Lai, Cheng-Yuan; Wang, Hsueh-Hsiao; Lee, An-Sheng; Cheng, Jen-Kun; Chau, Yat-Pang; Peng, Hsien-Yu

    2017-11-01

    Bromodomain-containing protein 4 binds acetylated promoter histones and promotes transcription; however, the role of bromodomain-containing protein 4 in inflammatory hyperalgesia remains unclear. Male Sprague-Dawley rats received hind paw injections of complete Freund's adjuvant to induce hyperalgesia. The dorsal root ganglia were examined to detect changes in bromodomain-containing protein 4 expression and the activation of genes involved in the expression of voltage-gated sodium channel 1.7, which is a key pain-related ion channel. The intraplantar complete Freund's adjuvant injections resulted in thermal hyperalgesia (4.0 ± 1.5 s; n = 7). The immunohistochemistry and immunoblotting results demonstrated an increase in the bromodomain-containing protein 4-expressing dorsal root ganglia neurons (3.78 ± 0.38 fold; n = 7) and bromodomain-containing protein 4 protein levels (2.62 ± 0.39 fold; n = 6). After the complete Freund's adjuvant injection, histone H3 protein acetylation was enhanced in the voltage-gated sodium channel 1.7 promoter, and cyclin-dependent kinase 9 and phosphorylation of RNA polymerase II were recruited to this area. Furthermore, the voltage-gated sodium channel 1.7-mediated currents were enhanced in neurons of the complete Freund's adjuvant rats (55 ± 11 vs. 19 ± 9 pA/pF; n = 4 to 6 neurons). Using bromodomain-containing protein 4-targeted antisense small interfering RNA to the complete Freund's adjuvant-treated rats, the authors demonstrated a reduction in the expression of bromodomain-containing protein 4 (0.68 ± 0.16 fold; n = 7), a reduction in thermal hyperalgesia (7.5 ± 1.5 s; n = 7), and a reduction in the increased voltage-gated sodium channel 1.7 currents (21 ± 4 pA/pF; n = 4 to 6 neurons). Complete Freund's adjuvant triggers enhanced bromodomain-containing protein 4 expression, ultimately leading to the enhanced excitability of nociceptive neurons and thermal hyperalgesia. This effect is

  17. Molecular Surface of JZTX-V (β-Theraphotoxin-Cj2a Interacting with Voltage-Gated Sodium Channel Subtype NaV1.4

    Directory of Open Access Journals (Sweden)

    Ji Luo

    2014-07-01

    Full Text Available Voltage-gated sodium channels (VGSCs; NaV1.1–NaV1.9 have been proven to be critical in controlling the function of excitable cells, and human genetic evidence shows that aberrant function of these channels causes channelopathies, including epilepsy, arrhythmia, paralytic myotonia, and pain. The effects of peptide toxins, especially those isolated from spider venom, have shed light on the structure–function relationship of these channels. However, most of these toxins have not been analyzed in detail. In particular, the bioactive faces of these toxins have not been determined. Jingzhaotoxin (JZTX-V (also known as β-theraphotoxin-Cj2a is a 29-amino acid peptide toxin isolated from the venom of the spider Chilobrachys jingzhao. JZTX-V adopts an inhibitory cysteine knot (ICK motif and has an inhibitory effect on voltage-gated sodium and potassium channels. Previous experiments have shown that JZTX-V has an inhibitory effect on TTX-S and TTX-R sodium currents on rat DRG cells with IC50 values of 27.6 and 30.2 nM, respectively, and is able to shift the activation and inactivation curves to the depolarizing and the hyperpolarizing direction, respectively. Here, we show that JZTX-V has a much stronger inhibitory effect on NaV1.4, the isoform of voltage-gated sodium channels predominantly expressed in skeletal muscle cells, with an IC50 value of 5.12 nM, compared with IC50 values of 61.7–2700 nM for other heterologously expressed NaV1 subtypes. Furthermore, we investigated the bioactive surface of JZTX-V by alanine-scanning the effect of toxin on NaV1.4 and demonstrate that the bioactive face of JZTX-V is composed of three hydrophobic (W5, M6, and W7 and two cationic (R20 and K22 residues. Our results establish that, consistent with previous assumptions, JZTX-V is a Janus-faced toxin which may be a useful tool for the further investigation of the structure and function of sodium channels.

  18. Downregulation of the sodium channel Nav1.6 by potential transcriptomic deregulation may explain sensory deficits in critical illness neuropathy.

    Science.gov (United States)

    Li, Nan; Liu, Zhongmin; Wang, Guang; Wang, Shiji

    2015-12-15

    Sepsis patients and other patients in the critical care settings are at very high risk of mortality due to the primary illness. However, a fraction of patients, even after showing initial clinical improvement, deteriorates relentlessly at later stages. Increasingly, it is being identified that this is mostly due to dysfunction of the neurological system. We obtained peripheral nerve biopsies from the sural nerve from ICU patients. Nav1.6 expression was significantly diminished. The expression of cellular membrane anchoring protein for Nav1.6, ankyrin, remained unaffected, suggesting that genomic repression may be responsible for the diminished expression of the sodium channels. We examined the expression of two regulatory transcription factors: (a) a positive regulator YY1 that binds to the promoter region of sodium channels and (b) an upstream negative neuronal regulator REST. REST expression was significantly elevated, while YY1 expression was diminished. Finally, we also observed that the cholinergic synthetic enzyme acyltransferase was also significantly diminished in sensory nerve lysates. Finally, circulating antibodies was detected in the peripheral blood against all the major sodium channels Nav1.6, 1.8 and 1.9, which contribute to the development and propagation of action potentials. This may potentially explain why its dysfunction affects neurological functions across all systems of the body during critical illness. The underlying mechanism of why the expression of the REST transcriptional factor is affected in critical illnesses remains our future goals of investigation. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Molecular Characterization of Voltage-Gated Sodium Channels and Their Relations with Paralytic Shellfish Toxin Bioaccumulation in the Pacific Oyster Crassostrea gigas

    Directory of Open Access Journals (Sweden)

    Floriane Boullot

    2017-01-01

    Full Text Available Paralytic shellfish toxins (PST bind to voltage-gated sodium channels (Nav and block conduction of action potential in excitable cells. This study aimed to (i characterize Nav sequences in Crassostrea gigas and (ii investigate a putative relation between Nav and PST-bioaccumulation in oysters. The phylogenetic analysis highlighted two types of Nav in C. gigas: a Nav1 (CgNav1 and a Nav2 (CgNav2 with sequence properties of sodium-selective and sodium/calcium-selective channels, respectively. Three alternative splice transcripts of CgNav1 named A, B and C, were characterized. The expression of CgNav1, analyzed by in situ hybridization, is specific to nervous cells and to structures corresponding to neuromuscular junctions. Real-time PCR analyses showed a strong expression of CgNav1A in the striated muscle while CgNav1B is mainly expressed in visceral ganglia. CgNav1C expression is ubiquitous. The PST binding site (domain II of CgNav1 variants possess an amino acid Q that could potentially confer a partial saxitoxin (STX-resistance to the channel. The CgNav1 genotype or alternative splicing would not be the key point determining PST bioaccumulation level in oysters.

  20. Effects of intracellular sodium and hydrogen ion on the sodium activated potassium channel in isolated patches from guinea pig ventricular myocytes

    NARCIS (Netherlands)

    Veldkamp, M. W.; Vereecke, J.; Carmeliet, E.

    1994-01-01

    The Na+ activated K+ channel (IK(Na)) may be activated during ischaemia when the intracellular Na+ concentration is raised. As ischaemia is also associated with intracellular acidification, the influence of intracellular pH on this K+ channel was investigated. The effects of intracellular Na+ and H+

  1. Sodium channel Na(v)1.7 is essential for lowering heat pain threshold after burn injury.

    Science.gov (United States)

    Shields, Shannon D; Cheng, Xiaoyang; Uçeyler, Nurcan; Sommer, Claudia; Dib-Hajj, Sulayman D; Waxman, Stephen G

    2012-08-08

    Marked hypersensitivity to heat and mechanical (pressure) stimuli develop after a burn injury, but the neural mechanisms underlying these effects are poorly understood. In this study, we establish a new mouse model of focal second-degree burn injury to investigate the molecular and cellular basis for burn injury-induced pain. This model features robust injury-induced behavioral effects and tissue-specific altered cytokine profile, but absence of glial activation in spinal dorsal horn. Three voltage-gated sodium channels, Na(v)1.7, Na(v)1.8, and Na(v)1.9, are preferentially expressed in peripheral somatosensory neurons of the dorsal root ganglia (DRGs) and have been implicated in injury-induced neuronal hyperexcitability. Using knock-out mice, we provide evidence that Na(v)1.7 selectively contributes to burn-induced hypersensitivity to heat, but not mechanical, stimuli. After burn model injury, wild-type mice display increased sensitivity to heat stimuli, and a normally non-noxious warm stimulus induces activity-dependent Fos expression in spinal dorsal horn neurons. Strikingly, both effects are absent in Na(v)1.7 conditional knock-out (cKO) mice. Furthermore, burn injury increases density and shifts activation of tetrodotoxin-sensitive currents in a hyperpolarized direction, both pro-excitatory properties, in DRG neurons from wild-type but not Na(v)1.7 cKO mice. We propose that, in sensory neurons damaged by burn injury to the hindpaw, Na(v)1.7 currents contribute to the hyperexcitability of sensory neurons, their communication with postsynaptic spinal pain pathways, and behavioral thresholds to heat stimuli. Our results offer insights into the molecular and cellular mechanisms of modality-specific pain signaling, and suggest Na(v)1.7-blocking drugs may be effective in burn patients.

  2. Sodium channel-inhibiting drugs and cancer survival: protocol for a cohort study using the CPRD primary care database.

    Science.gov (United States)

    Fairhurst, Caroline; Martin, Fabiola; Watt, Ian; Doran, Tim; Bland, Martin; Brackenbury, William J

    2016-09-06

    Voltage-gated sodium channel (VGSC)-inhibiting drugs are commonly used to treat epilepsy and cardiac arrhythmia. VGSCs are also widely expressed in various cancers, including those of the breast, bowel and prostate. A number of VGSC-inhibiting drugs have been shown to inhibit cancer cell proliferation, invasion, tumour growth and metastasis in preclinical models, suggesting that VGSCs may be novel molecular targets for cancer treatment. Surprisingly, we previously found that prior exposure to VGSC-inhibiting drugs may be associated with reduced overall survival in patients with cancer, but we were unable to control for the cause of death or indication for prescription. The purpose of the present study is to interrogate a different database to further investigate the relationship between VGSC-inhibiting drugs and cancer-specific survival. A cohort study using primary care data from the Clinical Practice Research Datalink database will include patients with diagnosis of breast, bowel and prostate cancer (13 000). The primary outcome will be cancer-specific survival from the date of cancer diagnosis. Cox proportional hazards regression will be used to compare survival of patients taking VGSC-inhibiting drugs (including antiepileptic drugs and class I antiarrhythmic agents) with patients with cancer not taking these drugs, adjusting for cancer type, age and sex. Drug exposure will be treated as a time-varying covariate to account for potential immortal time bias. Various sensitivity and secondary analyses will be performed. The project has been reviewed and approved by the University of York Ethical Review Process. Results will be presented at an international conference and published in open access peer-reviewed journals according to the STROBE and RECORD guidelines. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  3. Epithelial sodium channel (ENaC) family: Phylogeny, structure-function, tissue distribution, and associated inherited diseases.

    Science.gov (United States)

    Hanukoglu, Israel; Hanukoglu, Aaron

    2016-04-01

    The epithelial sodium channel (ENaC) is composed of three homologous subunits and allows the flow of Na(+) ions across high resistance epithelia, maintaining body salt and water homeostasis. ENaC dependent reabsorption of Na(+) in the kidney tubules regulates extracellular fluid (ECF) volume and blood pressure by modulating osmolarity. In multi-ciliated cells, ENaC is located in cilia and plays an essential role in the regulation of epithelial surface liquid volume necessary for cilial transport of mucus and gametes in the respiratory and reproductive tracts respectively. The subunits that form ENaC (named as alpha, beta, gamma and delta, encoded by genes SCNN1A, SCNN1B, SCNN1G, and SCNN1D) are members of the ENaC/Degenerin superfamily. The earliest appearance of ENaC orthologs is in the genomes of the most ancient vertebrate taxon, Cyclostomata (jawless vertebrates) including lampreys, followed by earliest representatives of Gnathostomata (jawed vertebrates) including cartilaginous sharks. Among Euteleostomi (bony vertebrates), Actinopterygii (ray finned-fishes) branch has lost ENaC genes. Yet, most animals in the Sarcopterygii (lobe-finned fish) branch including Tetrapoda, amphibians and amniotes (lizards, crocodiles, birds, and mammals), have four ENaC paralogs. We compared the sequences of ENaC orthologs from 20 species and established criteria for the identification of ENaC orthologs and paralogs, and their distinction from other members of the ENaC/Degenerin superfamily, especially ASIC family. Differences between ENaCs and ASICs are summarized in view of their physiological functions and tissue distributions. Structural motifs that are conserved throughout vertebrate ENaCs are highlighted. We also present a comparative overview of the genotype-phenotype relationships in inherited diseases associated with ENaC mutations, including multisystem pseudohypoaldosteronism (PHA1B), Liddle syndrome, cystic fibrosis-like disease and essential hypertension. Copyright

  4. Mutation in the Sodium Channel Gene Corresponds With Phenotypic Resistance of Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) to Pyrethroids.

    Science.gov (United States)

    Klafke, G M; Miller, R J; Tidwell, J; Barreto, R; Guerrero, F D; Kaufman, P E; Pérez de León, A A

    2017-07-13

    The brown dog tick, Rhipicephalus sanguineus sensu lato (Latreille), is a cosmopolitan ectoparasite and vector of pathogens that kill humans and animals. Pyrethroids represent a class of synthetic acaricides that have been used intensely to try to control the brown dog tick and mitigate the risk of tick-borne disease transmission. However, acaricide resistance is an emerging problem in the management of the brown dog tick. Understanding the mechanism of resistance to acaricides, including pyrethroids, is important to adapt brown dog tick control strategies. The main objective of this study was to determine if target-site mutations associated with pyrethroid resistance in other pests could be associated with phenotypic resistance detected in a brown dog tick population from Florida. We amplified segment 6 of the domain III of the voltage-sensitive sodium channel protein, using cDNAs synthesized from pyrethroid-susceptible and pyrethroid-resistant tick strains. A single nucleotide point mutation (SNP) identified in a highly conserved region of domain III S6 in the resistant ticks resulted in an amino acid change from phenylalanine to leucine. This mutation is characteristic of resistance phenotypes in other tick species, and is the first report of this mutation in R. sanguineus. Molecular assays based on this knowledge could be developed to diagnose the risk for pyrethroid resistance, and to inform decisions on integrated brown dog tick management practices. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  5. NVP-QBE170: an inhaled blocker of the epithelial sodium channel with a reduced potential to induce hyperkalaemia.

    Science.gov (United States)

    Coote, K J; Paisley, D; Czarnecki, S; Tweed, M; Watson, H; Young, A; Sugar, R; Vyas, M; Smith, N J; Baettig, U; Groot-Kormelink, P J; Gosling, M; Lock, R; Ethell, B; Williams, G; Schumacher, A; Harris, J; Abraham, W M; Sabater, J; Poll, C T; Faller, T; Collingwood, S P; Danahay, H

    2015-06-01

    Inhaled amiloride, a blocker of the epithelial sodium channel (ENaC), enhances mucociliary clearance (MCC) in cystic fibrosis (CF) patients. However, the dose of amiloride is limited by the mechanism-based side effect of hyperkalaemia resulting from renal ENaC blockade. Inhaled ENaC blockers with a reduced potential to induce hyperkalaemia provide a therapeutic strategy to improve mucosal hydration and MCC in the lungs of CF patients. The present study describes the preclinical profile of a novel ENaC blocker, NVP-QBE170, designed for inhaled delivery, with a reduced potential to induce hyperkalaemia. The in vitro potency and duration of action of NVP-QBE170 were compared with amiloride and a newer ENaC blocker, P552-02, in primary human bronchial epithelial cells (HBECs) by short-circuit current. In vivo efficacy and safety were assessed in guinea pig (tracheal potential difference/hyperkalaemia), rat (hyperkalaemia) and sheep (MCC). In vitro, NVP-QBE170 potently inhibited ENaC function in HBEC and showed a longer duration of action to comparator molecules. In vivo, intratracheal (i.t.) instillation of NVP-QBE170 attenuated ENaC activity in the guinea pig airways with greater potency and duration of action than that of amiloride without inducing hyperkalaemia in either guinea pig or rat. Dry powder inhalation of NVP-QBE170 by conscious sheep increased MCC and was better than inhaled hypertonic saline in terms of efficacy and duration of action. NVP-QBE170 highlights the potential for inhaled ENaC blockers to exhibit efficacy in the airways with a reduced risk of hyperkalaemia, relative to existing compounds. © 2015 The British Pharmacological Society.

  6. Electrophysiological and Pharmacological Analyses of Nav1.9 Voltage-Gated Sodium Channel by Establishing a Heterologous Expression System.

    Science.gov (United States)

    Zhou, Xi; Xiao, Zhen; Xu, Yan; Zhang, Yunxiao; Tang, Dongfang; Wu, Xinzhou; Tang, Cheng; Chen, Minzhi; Shi, Xiaoliu; Chen, Ping; Liang, Songping; Liu, Zhonghua

    2017-01-01

    Nav1. 9 voltage-gated sodium channel is preferentially expressed in peripheral nociceptive neurons. Recent progresses have proved its role in pain sensation, but our understanding of Nav1.9, in general, has lagged behind because of limitations in heterologous expression in mammal cells. In this work, functional expression of human Nav1.9 (hNav1.9) was achieved by fusing GFP to the C-terminal of hNav1.9 in ND7/23 cells, which has been proved to be a reliable method to the electrophysiological and pharmacological studies of hNav1.9. By using the hNav1.9 expression system, we investigated the electrophysiological properties of four mutations of hNav1.9 (K419N, A582T, A842P, and F1689L), whose electrophysiological functions have not been determined yet. The four mutations significantly caused positive shift of the steady-state fast inactivation and therefore increased hNav1.9 activity, consistent with the phenotype of painful peripheral neuropathy. Meanwhile, the effects of inflammatory mediators on hNav1.9 were also investigated. Impressively, histamine was found for the first time to enhance hNav1.9 activity, indicating its vital role in hNav1.9 modulating inflammatory pain. Taken together, our research provided a useful platform for hNav1.9 studies and new insight into mechanism of hNav1.9 linking to pain.

  7. Electrophysiological and Pharmacological Analyses of Nav1.9 Voltage-Gated Sodium Channel by Establishing a Heterologous Expression System

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    Xi Zhou

    2017-11-01

    Full Text Available Nav1. 9 voltage-gated sodium channel is preferentially expressed in peripheral nociceptive neurons. Recent progresses have proved its role in pain sensation, but our understanding of Nav1.9, in general, has lagged behind because of limitations in heterologous expression in mammal cells. In this work, functional expression of human Nav1.9 (hNav1.9 was achieved by fusing GFP to the C-terminal of hNav1.9 in ND7/23 cells, which has been proved to be a reliable method to the electrophysiological and pharmacological studies of hNav1.9. By using the hNav1.9 expression system, we investigated the electrophysiological properties of four mutations of hNav1.9 (K419N, A582T, A842P, and F1689L, whose electrophysiological functions have not been determined yet. The four mutations significantly caused positive shift of the steady-state fast inactivation and therefore increased hNav1.9 activity, consistent with the phenotype of painful peripheral neuropathy. Meanwhile, the effects of inflammatory mediators on hNav1.9 were also investigated. Impressively, histamine was found for the first time to enhance hNav1.9 activity, indicating its vital role in hNav1.9 modulating inflammatory pain. Taken together, our research provided a useful platform for hNav1.9 studies and new insight into mechanism of hNav1.9 linking to pain.

  8. Epithelial sodium channel (ENaC) family: Phylogeny, structure-function, tissue distribution, and associated inherited diseases

    Science.gov (United States)

    Hanukoglu, Israel; Hanukoglu, Aaron

    2016-01-01

    The epithelial sodium channel (ENaC) is composed of three homologous subunits and allows the flow of Na+ ions across high resistance epithelia, maintaining body salt and water homeostasis. ENaC dependent reabsorption of Na+ in the kidney tubules regulates extracellular fluid (ECF) volume and blood pressure by modulating osmolarity. In multi-ciliated cells, ENaC is located in cilia and plays an essential role in the regulation of epithelial surface liquid volume necessary for cilial transport of mucus and gametes in the respiratory and reproductive tracts respectively. The subunits that form ENaC (named as alpha, beta, gamma and delta, encoded by genes SCNN1A, SCNN1B, SCNN1G, and SCNN1D) are members of the ENaC/Degenerin superfamily. The earliest appearance of ENaC orthologs is in the genomes of the most ancient vertebrate taxon, Cyclostomata (jawless vertebrates) including lampreys, followed by earliest representatives of Gnathostomata (jawed vertebrates) including cartilaginous sharks. Among Euteleostomi (bony vertebrates), Actinopterygii (ray finned-fishes) branch has lost ENaC genes. Yet, most animals in the Sarcopterygii (lobe-finned fish) branch including Tetrapoda, amphibians and amniotes (lizards, crocodiles, birds, and mammals), have four ENaC paralogs. We compared the sequences of ENaC orthologs from 20 species and established criteria for the identification of ENaC orthologs and paralogs, and their distinction from other members of the ENaC/Degenerin superfamily, especially ASIC family. Differences between ENaCs and ASICs are summarized in view of their physiological functions and tissue distributions. Structural motifs that are conserved throughout vertebrate ENaCs are highlighted. We also present a comparative overview of the genotype-phenotype relationships in inherited diseases associated with ENaC mutations, including multisystem pseudohypoaldosteronism (PHA1B), Liddle syndrome, cystic fibrosis-like disease and essential hypertension. PMID:26772908

  9. Spontaneous excitation patterns computed for axons with injury-like impairments of sodium channels and Na/K pumps.

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    Na Yu

    Full Text Available In injured neurons, "leaky" voltage-gated sodium channels (Nav underlie dysfunctional excitability that ranges from spontaneous subthreshold oscillations (STO, to ectopic (sometimes paroxysmal excitation, to depolarizing block. In recombinant systems, mechanical injury to Nav1.6-rich membranes causes cytoplasmic Na(+-loading and "Nav-CLS", i.e., coupled left-(hyperpolarizing-shift of Nav activation and availability. Metabolic injury of hippocampal neurons (epileptic discharge results in comparable impairment: left-shifted activation and availability and hence left-shifted I(Na-window. A recent computation study revealed that CLS-based I(Na-window left-shift dissipates ion gradients and impairs excitability. Here, via dynamical analyses, we focus on sustained excitability patterns in mildly damaged nodes, in particular with more realistic Gaussian-distributed Nav-CLS to mimic "smeared" injury intensity. Since our interest is axons that might survive injury, pumps (sine qua non for live axons are included. In some simulations, pump efficacy and system volumes are varied. Impacts of current noise inputs are also characterized. The diverse modes of spontaneous rhythmic activity evident in these scenarios are studied using bifurcation analysis. For "mild CLS injury", a prominent feature is slow pump/leak-mediated E(Ion oscillations. These slow oscillations yield dynamic firing thresholds that underlie complex voltage STO and bursting behaviors. Thus, Nav-CLS, a biophysically justified mode of injury, in parallel with functioning pumps, robustly engenders an emergent slow process that triggers a plethora of pathological excitability patterns. This minimalist "device" could have physiological analogs. At first nodes of Ranvier and at nociceptors, e.g., localized lipid-tuning that modulated Nav midpoints could produce Nav-CLS, as could co-expression of appropriately differing Nav isoforms.

  10. Gene expression profile of sodium channel subunits in the anterior cingulate cortex during experimental paclitaxel-induced neuropathic pain in mice.

    Science.gov (United States)

    Masocha, Willias

    2016-01-01

    Paclitaxel, a chemotherapeutic agent, causes neuropathic pain whose supraspinal pathophysiology is not fully understood. Dysregulation of sodium channel expression, studied mainly in the periphery and spinal cord level, contributes to the pathogenesis of neuropathic pain. We examined gene expression of sodium channel (Nav) subunits by real time polymerase chain reaction (PCR) in the anterior cingulate cortex (ACC) at day 7 post first administration of paclitaxel, when mice had developed paclitaxel-induced thermal hyperalgesia. The ACC was chosen because increased activity in the ACC has been observed during neuropathic pain. In the ACC of vehicle-treated animals the threshold cycle (Ct) values for Nav1.4, Nav1.5, Nav1.7, Nav1.8 and Nav1.9 were above 30 and/or not detectable in some samples. Thus, comparison in mRNA expression between untreated control, vehicle-treated and paclitaxel treated animals was done for Nav1.1, Nav1.2, Nav1.3, Nav1.6, Nax as well as Navβ1-Navβ4. There were no differences in the transcript levels of Nav1.1-Nav1.3, Nav1.6, Nax, Navβ1-Navβ3 between untreated and vehicle-treated mice, however, vehicle treatment increased Navβ4 expression. Paclitaxel treatment significantly increased the mRNA expression of Nav1.1, Nav1.2, Nav1.6 and Nax, but not Nav1.3, sodium channel alpha subunits compared to vehicle-treated animals. Treatment with paclitaxel significantly increased the expression of Navβ1 and Navβ3, but not Navβ2 and Navβ4, sodium channel beta subunits compared to vehicle-treated animals. These findings suggest that during paclitaxel-induced neuropathic pain (PINP) there is differential upregulation of sodium channels in the ACC, which might contribute to the increased neuronal activity observed in the area during neuropathic pain.

  11. Gene expression profile of sodium channel subunits in the anterior cingulate cortex during experimental paclitaxel-induced neuropathic pain in mice

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    Willias Masocha

    2016-11-01

    Full Text Available Paclitaxel, a chemotherapeutic agent, causes neuropathic pain whose supraspinal pathophysiology is not fully understood. Dysregulation of sodium channel expression, studied mainly in the periphery and spinal cord level, contributes to the pathogenesis of neuropathic pain. We examined gene expression of sodium channel (Nav subunits by real time polymerase chain reaction (PCR in the anterior cingulate cortex (ACC at day 7 post first administration of paclitaxel, when mice had developed paclitaxel-induced thermal hyperalgesia. The ACC was chosen because increased activity in the ACC has been observed during neuropathic pain. In the ACC of vehicle-treated animals the threshold cycle (Ct values for Nav1.4, Nav1.5, Nav1.7, Nav1.8 and Nav1.9 were above 30 and/or not detectable in some samples. Thus, comparison in mRNA expression between untreated control, vehicle-treated and paclitaxel treated animals was done for Nav1.1, Nav1.2, Nav1.3, Nav1.6, Nax as well as Navβ1–Navβ4. There were no differences in the transcript levels of Nav1.1–Nav1.3, Nav1.6, Nax, Navβ1–Navβ3 between untreated and vehicle-treated mice, however, vehicle treatment increased Navβ4 expression. Paclitaxel treatment significantly increased the mRNA expression of Nav1.1, Nav1.2, Nav1.6 and Nax, but not Nav1.3, sodium channel alpha subunits compared to vehicle-treated animals. Treatment with paclitaxel significantly increased the expression of Navβ1 and Navβ3, but not Navβ2 and Navβ4, sodium channel beta subunits compared to vehicle-treated animals. These findings suggest that during paclitaxel-induced neuropathic pain (PINP there is differential upregulation of sodium channels in the ACC, which might contribute to the increased neuronal activity observed in the area during neuropathic pain.

  12. Activity-dependent formation and location of voltage-gated sodium channel clusters at a CNS nerve terminal during postnatal development.

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    Xu, Jie; Berret, Emmanuelle; Kim, Jun Hee

    2017-02-01

    In auditory pathways, the precision of action potential (AP) propagation depends on axon myelination and high densities of voltage-gated Na (Nav) channels clustered at nodes of Ranvier. Changes in Nav channel expression at the heminode, the final node before the nerve terminal, can alter AP invasion into the presynaptic terminal. We studied the activity-dependent formation of Nav channel clusters before and after hearing onset at postnatal day 12 in the rat and mouse auditory brain stem. In rats, the Nav channel cluster at the heminode formed progressively during the second postnatal week, around hearing onset, whereas the Nav channel cluster at the nodes was present before hearing onset. Initiation of heminodal Nav channel clustering was correlated with the expression of scaffolding protein ankyrinG and paranodal protein Caspr. However, in whirler mice with congenital deafness, heminodal Nav channels did not form clusters and maintained broad expression, but Nav channel clustering was normal at the nodes. In addition, a clear difference in the distance from the heminodal Nav channel to the calyx across the mediolateral axis of the medial nucleus of the trapezoid body (MNTB) developed after hearing onset. In the medial MNTB, where neurons respond best to high-frequency sounds, the heminodal Nav channel cluster was located closer to the terminal than in the lateral MNTB, where neurons respond best to low-frequency sounds. Thus sound-mediated neuronal activities are potentially associated with the refinement of the heminode adjacent to the presynaptic terminal in the auditory brain stem. Clustering of voltage-gated sodium (Nav) channels and their distribution along the axon, specifically at the unmyelinated axon segment next to the nerve terminal, are essential for tuning propagated action potentials. Nav channel clusters near the nerve terminal and their location as a function of neuronal position along the mediolateral axis are controlled by auditory inputs after

  13. Elevated neuronal excitability due to modulation of the voltage-gated sodium channel Nav1.6 by Aβ1-42

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    Xi eWang

    2016-03-01

    Full Text Available Aberrant increases in neuronal network excitability may contribute to the cognitive deficits in Alzheimer’s disease (AD. However, the mechanisms underlying hyperexcitability are not fully understood. Such overexcitation of neuronal networks has been detected in the brains of APP/PS1 mice. In the present study, using current-clamp recording techniques, we observed that 12 days in vitro (DIV primary cultured pyramidal neurons from P0 APP/PS1 mice exhibited a more prominent action potential burst and a lower threshold than WT littermates. Moreover, after treatment with Aβ1-42 peptide, 12 DIV primary cultured neurons showed similar changes, to a greater degree than in controls. Voltage-clamp recordings revealed that the voltage-dependent sodium current density of neurons incubated with Aβ1-42 was significantly increased, without change in the voltage-dependent sodium channel kinetic characteristics. Immunohistochemistry and western blot results showed that, after treatment with Aβ1-42, expressions of Nav and Nav1.6 subtype increased in cultured neurons or APP/PS1 brains compared to control groups. The intrinsic neuronal hyperexcitability of APP/PS1 mice might thus be due to an increased expression of voltage-dependent sodium channels induced by Aβ1-42. These results may illuminate the mechanism of aberrant neuronal networks in AD.

  14. Sensory renal innervation: a kidney-specific firing activity due to a unique expression pattern of voltage-gated sodium channels?

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    Freisinger, Wolfgang; Schatz, Johannes; Ditting, Tilmann; Lampert, Angelika; Heinlein, Sonja; Lale, Nena; Schmieder, Roland; Veelken, Roland

    2013-03-01

    Sensory neurons with afferent axons from the kidney are extraordinary in their response to electrical stimulation. More than 50% exhibit a tonic firing pattern, i.e., sustained action potential firing throughout depolarizing, pointing to an increased excitability, whereas nonrenal neurons show mainly a phasic response, i.e., less than five action potentials. Here we investigated whether these peculiar firing characteristics of renal afferent neurons are due to differences in the expression of voltage-gated sodium channels (Navs). Dorsal root ganglion (DRG) neurons from rats (Th11-L2) were recorded by the current-clamp technique and distinguished as "tonic" or "phasic." In voltage-clamp recordings, Navs were characterized by their tetrodotoxoxin (TTX) sensitivity, and their molecular identity was revealed by RT-PCR. The firing pattern of 66 DRG neurons (41 renal and 25 nonrenal) was investigated. Renal neurons exhibited more often a tonic firing pattern (56.1 vs. 12%). Tonic neurons showed a more positive threshold (-21.75 ± 1.43 vs.-29.33 ± 1.63 mV; P sodium currents. Interestingly, mRNA expression of TTX-resistant sodium channels was significantly increased in renal, predominantly tonic, DRG neurons. Hence, under physiological conditions, renal sensory neurons exhibit predominantly a firing pattern associated with higher excitability. Our findings support that this is due to an increased expression and activation of TTX-resistant Navs.

  15. Inhibition of late sodium current suppresses calcium-related ventricular arrhythmias by reducing the phosphorylation of CaMK-II and sodium channel expressions

    OpenAIRE

    Xiao-Hong Wei; Shan-Dong Yu; Lu Ren; Si-Hui Huang; Qiao-Mei Yang; Ping Wang; Yan-Peng Chu; Wei Yang; Yan-Sheng Ding; Yong Huo; Lin Wu

    2017-01-01

    Cardiac arrhythmias associated with intracellular calcium inhomeostasis are refractory to antiarrhythmic therapy. We hypothesized that late sodium current (I Na) contributed to the calcium-related arrhythmias. Monophasic action potential duration at 90% completion of repolarization (MAPD90) was significantly increased and ventricular arrhythmias were observed in hearts with increased intracellular calcium concentration ([Ca2+]i) by using Bay K 8644, and the increase became greater in hearts t...

  16. Autophagy in unicellular eukaryotes

    NARCIS (Netherlands)

    Kiel, J.A.K.W.

    2010-01-01

    Cells need a constant supply of precursors to enable the production of macromolecules to sustain growth and survival. Unlike metazoans, unicellular eukaryotes depend exclusively on the extracellular medium for this supply. When environmental nutrients become depleted, existing cytoplasmic components

  17. Tumor necrosis factor α modulates sodium-activated potassium channel SLICK in rat dorsal horn neurons via p38 MAPK activation pathway

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    Wang K

    2017-05-01

    Full Text Available Kun Wang,1 Feng Wang,1 Jun-Ping Bao,2 Zhi-Yang Xie,1 Lu Chen,1 Bao-Yi Zhou,1 Xin-Hui Xie,2 Xiao-Tao Wu1,2 1Medical School of Southeast University, 2Department of Orthopaedics, Zhongda Hospital, Southeast University, Nanjing, People’s Republic of China Abstract: The dorsal horn (DH of the spinal cord is the integrative center that processes and transmits pain sensation. Abnormal changes in ion channel expression can enhance the excitability of pain-related DH neurons. Sodium-activated potassium (KNa channels are highly expressed particularly in the central nervous system; however, information about whether rat DH neurons express the SLICK channel protein is lacking, and the direct effects on SLICK in response to inflammation and the potential signaling pathway mediating such effects are yet to be elucidated. Here, using cultured DH neurons, we have shown that tumor necrosis factor-α inhibits the total outward potassium current IK and the KNa current predominantly as well as induces a progressive loss of firing accommodation. However, we found that this change in channel activity is offset by the p38 inhibitor SB202190, thereby suggesting the modulation of SLICK channel activity via the p38 MAPK pathway. Furthermore, we have demonstrated that the tumor necrosis factor-α modulation of KNa channels does not occur at the level of SLICK channel gating but arises from possible posttranslational modification. Keywords: p38 MAPK, SLICK channel, neuropathic pain, dorsal horn, TNF-α

  18. Functional up-regulation of Nav1.8 sodium channel in Aβ afferent fibers subjected to chronic peripheral inflammation

    Science.gov (United States)

    2014-01-01

    Background Functional alterations in the properties of Aβ afferent fibers may account for the increased pain sensitivity observed under peripheral chronic inflammation. Among the voltage-gated sodium channels involved in the pathophysiology of pain, Nav1.8 has been shown to participate in the peripheral sensitization of nociceptors. However, to date, there is no evidence for a role of Nav1.8 in controlling Aβ-fiber excitability following persistent inflammation. Methods Distribution and expression of Nav1.8 in dorsal root ganglia and sciatic nerves were qualitatively or quantitatively assessed by immunohistochemical staining and by real time-polymerase chain reaction at different time points following complete Freund’s adjuvant (CFA) administration. Using a whole-cell patch-clamp configuration, we further determined both total INa and TTX-R Nav1.8 currents in large-soma dorsal root ganglia (DRG) neurons isolated from sham or CFA-treated rats. Finally, we analyzed the effects of ambroxol, a Nav1.8-preferring blocker on the electrophysiological properties of Nav1.8 currents and on the mechanical sensitivity and inflammation of the hind paw in CFA-treated rats. Results Our findings revealed that Nav1.8 is up-regulated in NF200-positive large sensory neurons and is subsequently anterogradely transported from the DRG cell bodies along the axons toward the periphery after CFA-induced inflammation. We also demonstrated that both total INa and Nav1.8 peak current densities are enhanced in inflamed large myelinated Aβ-fiber neurons. Persistent inflammation leading to nociception also induced time-dependent changes in Aβ-fiber neuron excitability by shifting the voltage-dependent activation of Nav1.8 in the hyperpolarizing direction, thus decreasing the current threshold for triggering action potentials. Finally, we found that ambroxol significantly reduces the potentiation of Nav1.8 currents in Aβ-fiber neurons observed following intraplantar CFA injection and

  19. Mutations in the voltage-gated sodium channel gene of anophelines and their association with resistance to pyrethroids - a review.

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    Silva, Ana Paula B; Santos, Joselita Maria M; Martins, Ademir J

    2014-10-07

    Constant and extensive use of chemical insecticides has created a selection pressure and favored resistance development in many insect species worldwide. One of the most important pyrethroid resistance mechanisms is classified as target site insensitivity, due to conformational changes in the target site that impair a proper binding of the insecticide molecule. The voltage-gated sodium channel (NaV) is the target of pyrethroids and DDT insecticides, used to control insects of medical, agricultural and veterinary importance, such as anophelines. It has been reported that the presence of a few non-silent point mutations in the NaV gene are associated with pyrethroid resistance, termed as 'kdr' (knockdown resistance) for preventing the knockdown effect of these insecticides. The presence of these mutations, as well as their effects, has been thoroughly studied in Anopheles mosquitoes. So far, kdr mutations have already been detected in at least 13 species (Anopheles gambiae, Anopheles arabiensis, Anopheles sinensis, Anopheles stephensi, Anopheles subpictus, Anopheles sacharovi, Anopheles culicifacies, Anopheles sundaicus, Anopheles aconitus, Anopheles vagus, Anopheles paraliae, Anopheles peditaeniatus and Anopheles albimanus) from populations of African, Asian and, more recently, American continents. Seven mutational variants (L1014F, L1014S, L1014C, L1014W, N1013S, N1575Y and V1010L) were described, with the highest prevalence of L1014F, which occurs at the 1014 site in NaV IIS6 domain. The increase of frequency and distribution of kdr mutations clearly shows the importance of this mechanism in the process of pyrethroid resistance. In this sense, several species-specific and highly sensitive methods have been designed in order to genotype individual mosquitoes for kdr in large scale, which may serve as important tolls for monitoring the dynamics of pyrethroid resistance in natural populations. We also briefly discuss investigations concerning the course of Plasmodium

  20. [Expression of voltage gated sodium channel Nav1.9 in experimental pulpal lesions in the rats].

    Science.gov (United States)

    Xu, Le; Zhu, Xinwei; Chen, Qingfeng; Hu, Yuanping; Zhu, Ling; Jiang, Yong

    2014-02-01

    To investigate the relationship between pulpitis pain and voltage-gated sodium channel (Nav1.9) by detecting the expression of Nav1.9 at different time points of the rat pulpal lesion model. Thirty-six SD pulpal lesions rat models were divided into three experimental groups, 1 d (n = 12), 3 d (n = 12) and 5 d group(n = 12).Normal SD rats served as control(n = 12). Tumor necrosis factor-α (TNF-α) and Nav1.9 mRNA expressions were evaluated by reverse transcription PCR (RT-PCR) .Nav1.9 protein expressions were analyzed by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The expression of TNF-α in the experimental group (1 d:0.514 ± 0.098, 3 d:0.739 ± 0.104, 5 d:1.238 ± 0.082) was higher than those in the control group (0.147 ± 0.016) (P Nav1.9 mRNA was up-regulated markedly in experimental groups (1 d: 0.296 ± 0.038, 3 d:0.409 ± 0.013, 5 d: 0.487 ± 0.028) , compare with control group (0.223 ± 0.020) (P Nav1.9 in control pulp tissue was (4.013 ± 0.292) µg/L, in painful pulp tissue of 1 d group was (5.143 ± 0.101) µg/L, in painful pulp tissue of 3 d group was (5.835 ± 0.088) µg/L and in painful pulp tissue of 5 d group was (6.307 ± 0.137) µg/L (P Nav1.9 in experimental groups (1 d: 0.106 ± 0.007, 3 d:0.170 ± 0.013, 5 d:0.238 ± 0.004) was up-regulated significantly compared with control group (0.073 ± 0.004)(P Nav1.9 had a significant increase in painful pulp tissue. Moreover, with the degree of pain aggravation, the expression of Nav1.9 increased in pulp tissue.It suggests that Nav1.9 may play an important role in the development of pulpitis pain.

  1. Structural disorder in eukaryotes.

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    Rita Pancsa

    Full Text Available Based on early bioinformatic studies on a handful of species, the frequency of structural disorder of proteins is generally thought to be much higher in eukaryotes than in prokaryotes. To refine this view, we present here a comparative prediction study and analysis of 194 fully described eukaryotic proteomes and 87 reference prokaryotes for structural disorder. We found that structural disorder does distinguish eukaryotes from prokaryotes, but its frequency spans a very wide range in the two superkingdoms that largely overlap. The number of disordered binding regions and different Pfam domain types also contribute to distinguish eukaryotes from prokaryotes. Unexpectedly, the highest levels--and highest variability--of predicted disorder is found in protists, i.e. single-celled eukaryotes, often surpassing more complex eukaryote organisms, plants and animals. This trend contrasts with that of the number of domain types, which increases rather monotonously toward more complex organisms. The level of structural disorder appears to be strongly correlated with lifestyle, because some obligate intracellular parasites and endosymbionts have the lowest levels, whereas host-changing parasites have the highest level of predicted disorder. We conclude that protists have been the evolutionary hot-bed of experimentation with structural disorder, in a period when structural disorder was actively invented and the major functional classes of disordered proteins established.

  2. Antillatoxin is a sodium channel activator that displays unique efficacy in heterologously expressed rNav1.2, rNav1.4 and rNav1.5 alpha subunits

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    Gerwick William H

    2010-12-01

    Full Text Available Abstract Background Antillatoxin (ATX is a structurally unique lipopeptide produced by the marine cyanobacterium Lyngbya majuscula. ATX activates voltage-gated sodium channel α-subunits at an undefined recognition site and stimulates sodium influx in neurons. However, the pharmacological properties and selectivity of ATX on the sodium channel α-subunits were not fully characterized. Results In this study, we characterized the pharmacological properties and selectivity of ATX in cells heterologously expressing rNav1.2, rNav1.4 or rNav1.5 α-subunits by using the Na+ selective fluorescent dye, sodium-binding benzofuran isophthalate. ATX produced sodium influx in cells expressing each sodium channel α-subunit, whereas two other sodium channel activators, veratridine and brevetoxin-2, were without effect. The ATX potency at rNav1.2, rNav1.4 and rNav1.5 did not differ significantly. Similarly, there were no significant differences in the efficacy for ATX-induced sodium influx between rNav1.2, rNav1.4 and rNav1.5 α-subunits. ATX also produced robust Ca2+ influx relative to other sodium channel activators in the calcium-permeable DEAA mutant of rNav1.4 α-subunit. Finally, we demonstrated that the 8-demethyl-8,9-dihydro-antillatoxin analog was less efficacious and less potent in stimulating sodium influx. Conclusions ATX displayed a unique efficacy with respect to stimulation of sodium influx in cells expressing rNav1.2, rNav1.4 and rNav1.5 α-subunits. The efficacy of ATX was distinctive inasmuch as it was not shared by activators of neurotoxin sites 2 and 5 on VGSC α-subunits. Given the unique pharmacological properties of ATX interaction with sodium channel α-subunits, decoding the molecular determinants and mechanism of action of antillatoxin may provide further insight into sodium channel gating mechanisms.

  3. Antillatoxin is a sodium channel activator that displays unique efficacy in heterologously expressed rNav1.2, rNav1.4 and rNav1.5 α subunits.

    Science.gov (United States)

    Cao, Zhengyu; Gerwick, William H; Murray, Thomas F

    2010-12-14

    Antillatoxin (ATX) is a structurally unique lipopeptide produced by the marine cyanobacterium Lyngbya majuscula. ATX activates voltage-gated sodium channel α-subunits at an undefined recognition site and stimulates sodium influx in neurons. However, the pharmacological properties and selectivity of ATX on the sodium channel α-subunits were not fully characterized. In this study, we characterized the pharmacological properties and selectivity of ATX in cells heterologously expressing rNa(v)1.2, rNa(v)1.4 or rNa(v)1.5 α-subunits by using the Na(+) selective fluorescent dye, sodium-binding benzofuran isophthalate. ATX produced sodium influx in cells expressing each sodium channel α-subunit, whereas two other sodium channel activators, veratridine and brevetoxin-2, were without effect. The ATX potency at rNa(v)1.2, rNa(v)1.4 and rNa(v)1.5 did not differ significantly. Similarly, there were no significant differences in the efficacy for ATX-induced sodium influx between rNa(v)1.2, rNa(v)1.4 and rNa(v)1.5 α-subunits. ATX also produced robust Ca²(+) influx relative to other sodium channel activators in the calcium-permeable DEAA mutant of rNa(v)1.4 α-subunit. Finally, we demonstrated that the 8-demethyl-8,9-dihydro-antillatoxin analog was less efficacious and less potent in stimulating sodium influx. ATX displayed a unique efficacy with respect to stimulation of sodium influx in cells expressing rNa(v)1.2, rNa(v)1.4 and rNa(v)1.5 α-subunits. The efficacy of ATX was distinctive inasmuch as it was not shared by activators of neurotoxin sites 2 and 5 on VGSC α-subunits. Given the unique pharmacological properties of ATX interaction with sodium channel α-subunits, decoding the molecular determinants and mechanism of action of antillatoxin may provide further insight into sodium channel gating mechanisms.

  4. TNF Lectin-Like Domain Restores Epithelial Sodium Channel Function in Frameshift Mutants Associated with Pseudohypoaldosteronism Type 1B

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    Anita Willam

    2017-05-01

    Full Text Available Previous in vitro studies have indicated that tumor necrosis factor (TNF activates amiloride-sensitive epithelial sodium channel (ENaC current through its lectin-like (TIP domain, since cyclic peptides mimicking the TIP domain (e.g., solnatide, showed ENaC-activating properties. In the current study, the effects of TNF and solnatide on individual ENaC subunits or ENaC carrying mutated glycosylation sites in the α-ENaC subunit were compared, revealing a similar mode of action for TNF and solnatide and corroborating the previous assumption that the lectin-like domain of TNF is the relevant molecular structure for ENaC activation. Accordingly, TNF enhanced ENaC current by increasing open probability of the glycosylated channel, position N511 in the α-ENaC subunit being identified as the most important glycosylation site. TNF significantly increased Na+ current through ENaC comprising only the pore forming subunits α or δ, was less active in ENaC comprising only β-subunits, and showed no effect on ENaC comprising γ-subunits. TNF did not increase the membrane abundance of ENaC subunits to the extent observed with solnatide. Since the α-subunit is believed to play a prominent role in the ENaC current activating effect of TNF and TIP, we investigated whether TNF and solnatide can enhance αβγ-ENaC current in α-ENaC loss-of-function frameshift mutants. The efficacy of solnatide has been already proven in pathological conditions involving ENaC in phase II clinical trials. The frameshift mutations αI68fs, αT169fs, αP197fs, αE272fs, αF435fs, αR438fs, αY447fs, αR448fs, αS452fs, and αT482fs have been reported to cause pseudohypoaldosteronism type 1B (PHA1B, a rare, life-threatening, salt-wasting disease, which hitherto has been treated only symptomatically. In a heterologous expression system, all frameshift mutants showed significantly reduced amiloride-sensitive whole-cell current compared to wild type αβγ-ENaC, whereas membrane

  5. TNF Lectin-Like Domain Restores Epithelial Sodium Channel Function in Frameshift Mutants Associated with Pseudohypoaldosteronism Type 1B.

    Science.gov (United States)

    Willam, Anita; Aufy, Mohammed; Tzotzos, Susan; El-Malazi, Dina; Poser, Franziska; Wagner, Alina; Unterköfler, Birgit; Gurmani, Didja; Martan, David; Iqbal, Shahid Muhammad; Fischer, Bernhard; Fischer, Hendrik; Pietschmann, Helmut; Czikora, Istvan; Lucas, Rudolf; Lemmens-Gruber, Rosa; Shabbir, Waheed

    2017-01-01

    Previous in vitro studies have indicated that tumor necrosis factor (TNF) activates amiloride-sensitive epithelial sodium channel (ENaC) current through its lectin-like (TIP) domain, since cyclic peptides mimicking the TIP domain (e.g., solnatide), showed ENaC-activating properties. In the current study, the effects of TNF and solnatide on individual ENaC subunits or ENaC carrying mutated glycosylation sites in the α-ENaC subunit were compared, revealing a similar mode of action for TNF and solnatide and corroborating the previous assumption that the lectin-like domain of TNF is the relevant molecular structure for ENaC activation. Accordingly, TNF enhanced ENaC current by increasing open probability of the glycosylated channel, position N511 in the α-ENaC subunit being identified as the most important glycosylation site. TNF significantly increased Na+ current through ENaC comprising only the pore forming subunits α or δ, was less active in ENaC comprising only β-subunits, and showed no effect on ENaC comprising γ-subunits. TNF did not increase the membrane abundance of ENaC subunits to the extent observed with solnatide. Since the α-subunit is believed to play a prominent role in the ENaC current activating effect of TNF and TIP, we investigated whether TNF and solnatide can enhance αβγ-ENaC current in α-ENaC loss-of-function frameshift mutants. The efficacy of solnatide has been already proven in pathological conditions involving ENaC in phase II clinical trials. The frameshift mutations αI68fs, αT169fs, αP197fs, αE272fs, αF435fs, αR438fs, αY447fs, αR448fs, αS452fs, and αT482fs have been reported to cause pseudohypoaldosteronism type 1B (PHA1B), a rare, life-threatening, salt-wasting disease, which hitherto has been treated only symptomatically. In a heterologous expression system, all frameshift mutants showed significantly reduced amiloride-sensitive whole-cell current compared to wild type αβγ-ENaC, whereas membrane abundance varied

  6. The AMPK Activator A769662 Blocks Voltage-Gated Sodium Channels: Discovery of a Novel Pharmacophore with Potential Utility for Analgesic Development.

    Science.gov (United States)

    Asiedu, Marina N; Han, Chongyang; Dib-Hajj, Sulayman D; Waxman, Stephen G; Price, Theodore J; Dussor, Gregory

    2017-01-01

    Voltage-gated sodium channels (VGSC) regulate neuronal excitability by governing action potential (AP) generation and propagation. Recent studies have revealed that AMP-activated protein kinase (AMPK) activators decrease sensory neuron excitability, potentially by preventing sodium (Na+) channel phosphorylation by kinases such as ERK or via modulation of translation regulation pathways. The direct positive allosteric modulator A769662 displays substantially greater efficacy than other AMPK activators in decreasing sensory neuron excitability suggesting additional mechanisms of action. Here, we show that A769662 acutely inhibits AP firing stimulated by ramp current injection in rat trigeminal ganglion (TG) neurons. PT1, a structurally dissimilar AMPK activator that reduces nerve growth factor (NGF) -induced hyperexcitability, has no influence on AP firing in TG neurons upon acute application. In voltage-clamp recordings, application of A769662 reduces VGSC current amplitudes. These findings, based on acute A769662 application, suggest a direct channel blocking effect. Indeed, A769662 dose-dependently blocks VGSC in rat TG neurons and in Nav1.7-transfected cells with an IC50 of ~ 10 μM. A769662 neither displayed use-dependent inhibition nor interacted with the local anesthetic (LA) binding site. Popliteal fossa administration of A769662 decreased noxious thermal responses with a peak effect at 5 mins demonstrating an analgesic effect. These data indicate that in addition to AMPK activation, A769662 acts as a direct blocker/modulator of VGSCs, a potential mechanism enhancing the analgesic property of this compound.

  7. Effects of a new potent analog of tocainide on hNav1.7 sodium channels and in vivo neuropathic pain models.

    Science.gov (United States)

    Ghelardini, C; Desaphy, J-F; Muraglia, M; Corbo, F; Matucci, R; Dipalma, A; Bertucci, C; Pistolozzi, M; Nesi, M; Norcini, M; Franchini, C; Camerino, D Conte

    2010-08-25

    The role of voltage-gated sodium channels in the transmission of neuropathic pain is well recognized. For instance, genetic evidence recently indicate that the human Nav1.7 sodium channel subtype plays a crucial role in the ability to perceive pain sensation and may represent an important target for analgesic/anti-hyperalgesic drugs. In this study a newly synthesized tocainide congener, named NeP1, was tested in vitro on recombinant hNav1.4 and hNav1.7 channels using patch-clamp technique and, in vivo, in two rat models of persistent neuropathic pain obtained either by chronic constriction injury of the sciatic nerve or by oxaliplatin treatment. NeP1 efficiently blocked hNav1.4 and hNav1.7 channels in a dose- and use-dependent manner, being by far more potent than tocainide. Importantly, the new compound displayed a remarkable use-dependent effect, which likely resulted from a very high affinity for inactivated compared to closed channels. In both models of neuropathic pain, NeP1 was greatly more potent than tocainide in reverting the reduction of pain threshold in vivo. In oxaliplatin-treated rats, NeP1 even produced greater and more durable anti-hyperalgesia than the reference drug tramadol. In addition, in vivo and in vitro studies suggest a better toxicological and pharmacokinetic profile for NeP1 compared to tocainide. Overall, these results indicate NeP1 as a new promising lead compound for further development in the treatment of chronic pain of neuropathic origin. Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

  8. Effect of mitochondrial potassium channel on the renal protection mediated by sodium thiosulfate against ethylene glycol induced nephrolithiasis in rat model

    Directory of Open Access Journals (Sweden)

    N. Baldev

    2015-12-01

    Full Text Available Purpose: Sodium thiosulfate (STS is clinically reported to be a promising drug in preventing nephrolithiasis. However, its mechanism of action remains unclear. In the present study, we investigated the role of mitochondrial KATP channel in the renal protection mediated by STS. Materials and Methods: Nephrolithiasis was induced in Wistar rats by administrating 0.4% ethylene glycol (EG along with 1% ammonium chloride for one week in drinking water followed by only 0.75% EG for two weeks. Treatment groups received STS, mitochondrial KATP channel opener and closer exclusively or in combination with STS for two weeks. Results: Animals treated with STS showed normal renal tissue architecture, supported by near normal serum creatinine, urea and ALP activity. Diazoxide (mitochondria KATP channel opening treatment to the animal also showed normal renal tissue histology and improved serum chemistry. However, an opposite result was shown by glibenclamide (mitochondria KATP channel closer treated rats. STS administered along with diazoxide negated the renal protection rendered by diazoxide alone, while it imparted protection to the glibenclamide treated rats, formulating a mitochondria modulated STS action. Conclusion: The present study confirmed that STS render renal protection not only through chelation and antioxidant effect but also by modulating the mitochondrial KATP channel for preventing urolithiasis.

  9. The plant-derived glucocorticoid receptor agonist Endiandrin A acts as co-stimulator of colonic epithelial sodium channels (ENaC via SGK-1 and MAPKs.

    Directory of Open Access Journals (Sweden)

    Dana Kuntzsch

    Full Text Available In a search for secondary plant compounds that bind to the glucocorticoid receptor (GR, the cyclobutane lignan endiandrin A was discovered from the rainforest tree Endiandra anthropophagorum Domin. Our present study aims to characterize the effect of endiandrin A on GR-dependent induction of colonic sodium transport. The effect of endiandrin A was analyzed in GR-expressing colonic HT-29/B6 cells (HT-29/B6-GR. GR transactivation and subcellular localization were investigated by reporter gene assay and immunofluorescence. Epithelial sodium channel (ENaC was analyzed by qRT-PCR and by measuring amiloride-sensitive short-circuit current (I(sc in Ussing chambers. Endiandrin A (End A has been identified as GR receptor binder. However, it did not cause significant GR transactivation as pGRE-luciferase activity was only 7% of that of the maximum effect of dexamethasone. Interestingly, endiandrin A had a significant impact on dexamethasone-dependent sodium absorption in cells co-exposed to tumor necrosis factor (TNF-α. This was in part due to up-regulation of β- and γ-ENaC subunit expression. Endiandrin A potentiated GR-mediated transcription by increasing GR protein expression and phosphorylation. It inhibited c-Jun N-terminal kinase (JNK activation induced by dexamethasone and/or TNF-α and increased levels of GR localized to the nucleus. Additionally, endiandrin A increased the serum- and glucocorticoid-induced kinase (sgk-1 via activation of p38. Finally, the regulation of ENaC function by endiandrin A was confirmed in rat native colon. In conclusion, endiandrin A potentiates glucocorticoid-driven activation of colonic epithelial sodium channels via JNK inhibition and p38 activation due to transcriptional up-regulation of β- and γ-ENaC-subunits along with induction of sgk-1.

  10. Sodium Channel Nav1.8 Underlies TTX-Resistant Axonal Action Potential Conduction in Somatosensory C-Fibers of Distal Cutaneous Nerves.

    Science.gov (United States)

    Klein, Amanda H; Vyshnevska, Alina; Hartke, Timothy V; De Col, Roberto; Mankowski, Joseph L; Turnquist, Brian; Bosmans, Frank; Reeh, Peter W; Schmelz, Martin; Carr, Richard W; Ringkamp, Matthias

    2017-05-17

    Voltage-gated sodium (Na V ) channels are responsible for the initiation and conduction of action potentials within primary afferents. The nine Na V channel isoforms recognized in mammals are often functionally divided into tetrodotoxin (TTX)-sensitive (TTX-s) channels (Na V 1.1-Na V 1.4, Na V 1.6-Na V 1.7) that are blocked by nanomolar concentrations and TTX-resistant (TTX-r) channels (Na V 1.8 and Na V 1.9) inhibited by millimolar concentrations, with Na V 1.5 having an intermediate toxin sensitivity. For small-diameter primary afferent neurons, it is unclear to what extent different Na V channel isoforms are distributed along the peripheral and central branches of their bifurcated axons. To determine the relative contribution of TTX-s and TTX-r channels to action potential conduction in different axonal compartments, we investigated the effects of TTX on C-fiber-mediated compound action potentials (C-CAPs) of proximal and distal peripheral nerve segments and dorsal roots from mice and pigtail monkeys ( Macaca nemestrina ). In the dorsal roots and proximal peripheral nerves of mice and nonhuman primates, TTX reduced the C-CAP amplitude to 16% of the baseline. In contrast, >30% of the C-CAP was resistant to TTX in distal peripheral branches of monkeys and WT and Na V 1.9 -/- mice. In nerves from Na V 1.8 -/- mice, TTX-r C-CAPs could not be detected. These data indicate that Na V 1.8 is the primary isoform underlying TTX-r conduction in distal axons of somatosensory C-fibers. Furthermore, there is a differential spatial distribution of Na V 1.8 within C-fiber axons, being functionally more prominent in the most distal axons and terminal regions. The enrichment of Na V 1.8 in distal axons may provide a useful target in the treatment of pain of peripheral origin. SIGNIFICANCE STATEMENT It is unclear whether individual sodium channel isoforms exert differential roles in action potential conduction along the axonal membrane of nociceptive, unmyelinated peripheral nerve

  11. Odontoblasts in developing, mature and ageing rat teeth have multiple phenotypes that variably express all nine voltage-gated sodium channels.

    Science.gov (United States)

    Byers, Margaret R; Westenbroek, Ruth E

    2011-11-01

    Our goal was to evaluate the expression patterns for voltage gated sodium channels in odontoblasts of developing and mature rat teeth. We analysed immunoreactivity (IR) of the alpha subunit for all nine voltage gated sodium channels (Nav1.1-1.9) in teeth of immature (4 weeks), young adult (7 weeks), fully mature adult (3 months), and old rats (6-12 months). We were interested in developmental changes, crown/root differences, tetrodotoxin sensitivity or resistance, co-localization with nerve regions, occurrence in periodontium, and coincidence with other expression patterns by odontoblasts such as for transient receptor potential A1 (TRPA1). We found that Nav1.1-1.9-IR each had unique odontoblast patterns in mature molars that all differed from developmental stages and from incisors. Nav1.4- and Nav1.7-IR were intense in immature odontoblasts, becoming limited to specific zones in adults. Crown odontoblasts lost Nav1.7-IR and gained Nav1.8-IR where dentine became innervated. Odontoblast staining for Nav1.1- and Nav1.5-IR increased in crown with age but decreased in roots. Nav1.9-IR was especially intense in regularly scattered odontoblasts. Two tetrodotoxin-resistant isoforms (Nav1.5, Nav1.8) had strong expression in odontoblasts near dentinal innervation zones. Nav1.6-IR was concentrated at intercusp and cervical odontoblasts in adults as was TRPA1-IR. Nav1.3-IR gradually became intense in all odontoblasts during development except where dentinal innervation was dense. All nine voltage-gated sodium channels could be expressed by odontoblasts, depending on intradental location and tooth maturity. Our data reveal much greater complexity and niche-specific specialization for odontoblasts than previously demonstrated, with implications for tooth sensitivity. Copyright © 2011 Elsevier Ltd. All rights reserved.

  12. Antimetastatic potential of amide-linked local anesthetics: inhibition of lung adenocarcinoma cell migration and inflammatory Src signaling independent of sodium channel blockade.

    Science.gov (United States)

    Piegeler, Tobias; Votta-Velis, E Gina; Liu, Guoquan; Place, Aaron T; Schwartz, David E; Beck-Schimmer, Beatrice; Minshall, Richard D; Borgeat, Alain

    2012-09-01

    Retrospective analysis of patients undergoing cancer surgery suggests the use of regional anesthesia may reduce cancer recurrence and improve survival. Amide-linked local anesthetics have antiinflammatory properties, although the mechanism of action in this regard is unclear. As inflammatory processes involving Src tyrosine protein kinase and intercellular adhesion molecule-1 are important in tumor growth and metastasis, we hypothesized that amide-linked local anesthetics may inhibit inflammatory Src-signaling involved in migration of adenocarcinoma cells. NCI-H838 lung cancer cells were incubated with tumor necrosis factor-α in absence/presence of ropivacaine, lidocaine, or chloroprocaine (1 nM-100 μM). Cell migration and total cell lysate Src-activation and intercellular adhesion molecule-1 phosphorylation were assessed. The role of voltage-gated sodium-channels in the mechanism of local anesthetic effects was also evaluated. Ropivacaine treatment (100 μM) of H838 cells for 20 min decreased basal Src activity by 62% (P=0.003), and both ropivacaine and lidocaine coadministered with tumor necrosis factor-α statistically significantly decreased Src-activation and intercellular adhesion molecule-1 phosphorylation, whereas chloroprocaine had no such effect. Migration of these cells at 4 h was inhibited by 26% (P=0.005) in presence of 1 μM ropivacaine and 21% by 1 μM lidocaine (P=0.004). These effects of ropivacaine and lidocaine were independent of voltage-gated sodium-channel inhibition. This study indicates that amide-, but not ester-linked, local anesthetics may provide beneficial antimetastatic effects. The observed inhibition of NCI-H838 cell migration by lidocaine and ropivacaine was associated with the inhibition of tumor necrosis factor-α-induced Src-activation and intercellular adhesion molecule-1 phosphorylation, providing the first evidence of a molecular mechanism that appears to be independent of their known role as sodium-channel blockers.

  13. System of closing relations of a two-fluid model for the HYDRA-IBRAE/LM/V1 code for calculation of sodium boiling in channels of power equipment

    Science.gov (United States)

    Usov, E. V.; Butov, A. A.; Dugarov, G. A.; Kudasov, I. G.; Lezhnin, S. I.; Mosunova, N. A.; Pribaturin, N. A.

    2017-07-01

    The system of equations from a two-fluid model is widely used in modeling thermohydraulic processes during accidents in nuclear reactors. The model includes conservation equations governing the balance of mass, momentum, and energy in each phase of the coolant. The features of heat and mass transfer, as well as of mechanical interaction between phases or with the channel wall, are described by a system of closing relations. Properly verified foreign and Russian codes with a comprehensive system of closing relations are available to predict processes in water coolant. As to the sodium coolant, only a few open publications on this subject are known. A complete system of closing relations used in the HYDRA-IBRAE/LM/V1 thermohydraulic code for calculation of sodium boiling in channels of power equipment is presented. The selection of these relations is corroborated on the basis of results of analysis of available publications with an account taken of the processes occurring in liquid sodium. A comparison with approaches outlined in foreign publications is presented. Particular attention has been given to the calculation of the sodium two-phase flow boiling. The flow regime map and a procedure for the calculation of interfacial friction and heat transfer in a sodium flow with account taken of high conductivity of sodium are described in sufficient detail. Correlations are presented for calculation of heat transfer for a single-phase sodium flow, sodium flow boiling, and sodium flow boiling crisis. A method is proposed for prediction of flow boiling crisis initiation.

  14. Targeting voltage gated sodium channels NaV1.7, Na V1.8, and Na V1.9 for treatment of pathological cough.

    Science.gov (United States)

    Muroi, Yukiko; Undem, Bradley J

    2014-02-01

    Recent advances in our understanding of voltage-gated sodium channels (NaVs) lead to the rational hypothesis that drugs capable of selective blockade of NaV subtypes may be a safe and effective strategy for the treatment of unwanted cough. Among the nine NaV subtypes (NaV1.1-NaV1.9), the afferent nerves involved in initiating cough, in common with nociceptive neurons in the somatosensory system, express mainly NaV1.7, NaV1.8, and NaV1.9. Although knowledge about the effect of selectively blocking these channels on the cough reflex is limited, their biophysical properties indicate that each may contribute to the hypertussive and allotussive state that typifies subacute and chronic nonproductive cough.

  15. Comparative study of voltage-gated sodium channel α-subunits in non-overlapping four neuronal populations in the rat dorsal root ganglion.

    Science.gov (United States)

    Fukuoka, Tetsuo; Noguchi, Koichi

    2011-06-01

    Voltage-gated sodium channel α-subunit (Nav) is the major determinant of neuronal electrophysiological characters. In order to compare the composition of Navs among neurochemically different neurons in the rat dorsal root ganglion (DRG), we examined the expression of Nav transcripts in four non-overlapping neuronal populations, with (+) or without (-) N52 immunoreactivity, a marker of neurons with myelinated axons, and TrkA mRNA identified by in situ hybridization histochemistry. Both N52-/TrkA+ and N52-/TrkA- populations had high levels of signals for Nav1.7, Nav1.8, and Nav1.9 mRNAs, but rarely expressed Nav1.1 or Nav1.6. There was no significant difference in these signals, suggesting that C-fiber peptidergic and non-peptidergic neurons have similar electrophysiological characters with regard to sodium currents. N52+/TrkA+ neurons (putative A-fiber nociceptors) had similar high levels of signals for Nav1.7 and Nav1.8, but a significantly lower level of Nav1.9 signals, as compared to N52- neurons. Although, almost no N52+/TrkA- neurons had Nav1.8 or Nav1.9, half of this population expressed Nav1.7 at similar levels to other three populations and the other half completely lacked this channel. These data suggest that Nav1.8 is a common channel for both C- and A-fiber nociceptors, and Nav1.9 is rather selective for C-fiber nociceptors. Nav1.7 is the most universal channel while some functionally unknown N52+/TrkA- subpopulation selectively lacks it. Copyright © 2011 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

  16. Estradiol upregulates voltage-gated sodium channel 1.7 in trigeminal ganglion contributing to hyperalgesia of inflamed TMJ

    Science.gov (United States)

    Bi, Rui-Yun; Meng, Zhen; Zhang, Peng; Wang, Xue-Dong

    2017-01-01

    Background Temporomandibular disorders (TMDs) have the highest prevalence in women of reproductive age. The role of estrogen in TMDs and especially in TMDs related pain is not fully elucidated. Voltage-gated sodium channel 1.7 (Nav1.7) plays a prominent role in pain perception and Nav1.7 in trigeminal ganglion (TG) is involved in the hyperalgesia of inflamed Temporomandibular joint (TMJ). Whether estrogen could upregulate trigeminal ganglionic Nav1.7 expression to enhance hyperalgesia of inflamed TMJ remains to be explored. Methods Estrous cycle and plasma levels of 17β-estradiol in female rats were evaluated with vaginal smear and enzyme linked immunosorbent assay, respectively. Female rats were ovariectomized and treated with 17β-estradiol at 0 μg, 20 μg and 80 μg, respectively, for 10 days. TMJ inflammation was induced using complete Freund’s adjuvant. Head withdrawal thresholds and food intake were measured to evaluate the TMJ nociceptive responses. The expression of Nav1.7 in TG was examined using real-time PCR and western blot. The activity of Nav1.7 promoter was examined using luciferase reporter assay. The locations of estrogen receptors (ERα and ERβ), the G protein coupled estrogen receptor (GPR30), and Nav1.7 in TG were examined using immunohistofluorescence. Results Upregulation of Nav1.7 in TG and decrease in head withdrawal threshold were observed with the highest plasma 17β-estradiol in the proestrus of female rats. Ovariectomized rats treated with 80 μg 17β-estradiol showed upregulation of Nav1.7 in TG and decrease in head withdrawal threshold as compared with that of the control or ovariectomized rats treated with 0 μg or 20 μg. Moreover, 17β-estradiol dose-dependently potentiated TMJ inflammation-induced upregulation of Nav1.7 in TG and also enhanced TMJ inflammation-induced decrease of head withdrawal threshold in ovariectomized rats. In addition, the estrogen receptor antagonist, ICI 182,780, partially blocked the 17

  17. Abnormal Conduction in the Diseased Heart - Enhanced fibrosis due to reduced gap junction and sodium channel expression

    NARCIS (Netherlands)

    Jansen, J.A.

    2011-01-01

    The velocity with which the impulse is conducted is dependent on three different factors: excitability, cell-to-cell coupling and tissue characteristics. Excitability is mainly determined by Nav1.5 channels, determining the upstroke-velocity of the action potential, but also by Kir2.1 channels. Gap

  18. Exploring the role of nociceptor-specific sodium channels in pain transmission using Nav1.8 and Nav1.9 knockout mice.

    Science.gov (United States)

    Leo, Sandra; D'Hooge, Rudi; Meert, Theo

    2010-03-17

    Two voltage gated sodium channels, Na(v)1.8 and Na(v)1.9, are exclusively expressed in primary sensory neurons and are suggested to play a role in different pain conditions, including chronic inflammatory and neuropathic pain states. Since no selective pharmacological tools are available, we investigated the involvement of Na(v)1.8 and Na(v)1.9 in pain transmission by the phenotypic characterization of Na(v)1.8 and Na(v)1.9 knockout mice and their wild-type littermates in models of acute nociception, peripheral inflammation and neuropathic pain. The present study provides evidence for a modulatory role of Na(v)1.9, and to a lesser extent Na(v)1.8 in the development of cold, but not mechanical allodynia in neuropathic pain conditions. Moreover, our results also indicate that Na(v)1.9 signaling might be involved in visceral pain. In contrast, the presumed critical role of these two sodium channel subtypes to inflammatory pain hypersensitivity seem, according to our results, to be limited and temporarily. Copyright 2009 Elsevier B.V. All rights reserved.

  19. GAPDH-mediated posttranscriptional regulations of sodium channel Scn1a and Scn3a genes under seizure and ketogenic diet conditions.

    Science.gov (United States)

    Lin, Guo-Wang; Lu, Ping; Zeng, Tao; Tang, Hui-Ling; Chen, Yong-Hong; Liu, Shu-Jing; Gao, Mei-Mei; Zhao, Qi-Hua; Yi, Yong-Hong; Long, Yue-Sheng

    2017-02-01

    Abnormal expressions of sodium channel SCN1A and SCN3A genes alter neural excitability that are believed to contribute to the pathogenesis of epilepsy, a long-term risk of recurrent seizures. Ketogenic diet (KD), a high-fat and low-carbohydrate treatment for difficult-to-control (refractory) epilepsy in children, has been suggested to reverse gene expression patterns. Here, we reveal a novel role of GAPDH on the posttranscriptional regulation of mouse Scn1a and Scn3a expressions under seizure and KD conditions. We show that GAPDH binds to a conserved region in the 3' UTRs of human and mouse SCN1A and SCN3A genes, which decreases and increases genes' expressions by affecting mRNA stability through SCN1A 3' UTR and SCN3A 3' UTR, respectively. In seizure mice, the upregulation and phosphorylation of GAPDH enhance its binding to the 3' UTR, which lead to downregulation of Scn1a and upregulation of Scn3a. Furthermore, administration of KD generates β-hydroxybutyric acid which rescues the abnormal expressions of Scn1a and Scn3a by weakening the GAPDH's binding to the element. Taken together, these data suggest that GAPDH-mediated expression regulation of sodium channel genes may be associated with epilepsy and the anticonvulsant action of KD. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. A null mutation of the neuronal sodium channel NaV1.6 disrupts action potential propagation and excitation-contraction coupling in the mouse heart.

    Science.gov (United States)

    Noujaim, Sami F; Kaur, Kuljeet; Milstein, Michelle; Jones, Julie M; Furspan, Philip; Jiang, Daniel; Auerbach, David S; Herron, Todd; Meisler, Miriam H; Jalife, José

    2012-01-01

    Evidence supports the expression of brain-type sodium channels in the heart. Their functional role, however, remains controversial. We used global Na(V)1.6-null mice to test the hypothesis that Na(V)1.6 contributes to the maintenance of propagation in the myocardium and to excitation-contraction (EC) coupling. We demonstrated expression of transcripts encoding full-length Na(V)1.6 in isolated ventricular myocytes and confirmed the striated pattern of Na(V)1.6 fluorescence in myocytes. On the ECG, the PR and QRS intervals were prolonged in the null mice, and the Ca(2+) transients were longer in the null cells. Under patch clamping, at holding potential (HP) = -120 mV, the peak I(Na) was similar in both phenotypes. However, at HP = -70 mV, the peak I(Na) was smaller in the nulls. In optical mapping, at 4 mM [K(+)](o), 17 null hearts showed slight (7%) reduction of ventricular conduction velocity (CV) compared to 16 wild-type hearts. At 12 mM [K(+)](o), CV was 25% slower in a subset of 9 null vs. 9 wild-type hearts. These results highlight the importance of neuronal sodium channels in the heart, whereby Na(V)1.6 participates in EC coupling, and represents an intrinsic depolarizing reserve that contributes to excitation.

  1. A De Novo Mutation in the Sodium-Activated Potassium Channel KCNT2 Alters Ion Selectivity and Causes Epileptic Encephalopathy

    Directory of Open Access Journals (Sweden)

    Sushmitha Gururaj

    2017-10-01

    Full Text Available Early infantile epileptic encephalopathies (EOEE are a debilitating spectrum of disorders associated with cognitive impairments. We present a clinical report of a KCNT2 mutation in an EOEE patient. The de novo heterozygous variant Phe240Leu SLICK was identified by exome sequencing and confirmed by Sanger sequencing. Phe240Leu rSlick and hSLICK channels were electrophysiologically, heterologously characterized to reveal three significant alterations to channel function. First, [Cl−]i sensitivity was reversed in Phe240Leu channels. Second, predominantly K+-selective WT channels were made to favor Na+ over K+ by Phe240Leu. Third, and consequent to altered ion selectivity, Phe240Leu channels had larger inward conductance. Further, rSlick channels induced membrane hyperexcitability when expressed in primary neurons, resembling the cellular seizure phenotype. Taken together, our results confirm that Phe240Leu is a “change-of-function” KCNT2 mutation, demonstrating unusual altered selectivity in KNa channels. These findings establish pathogenicity of the Phe240Leu KCNT2 mutation in the reported EOEE patient.

  2. Comparative genomics of Eukaryotes

    NARCIS (Netherlands)

    Noort, Vera van

    2007-01-01

    This thesis focuses on developing comparative genomics methods in eukaryotes, with an emphasis on applications for gene function prediction and regulatory element detection. In the past, methods have been developed to predict functional associations between gene pairs in prokaryotes. The challenge

  3. Α- and β-subunit composition of voltage-gated sodium channels investigated with μ-conotoxins and the recently discovered μO§-conotoxin GVIIJ.

    Science.gov (United States)

    Wilson, Michael J; Zhang, Min-Min; Gajewiak, Joanna; Azam, Layla; Rivier, Jean E; Olivera, Baldomero M; Yoshikami, Doju

    2015-04-01

    We investigated the identities of the isoforms of the α (NaV1)- and β (NaVβ)-subunits of voltage-gated sodium channels, including those responsible for action potentials in rodent sciatic nerves. To examine α-subunits, we used seven μ-conotoxins, which target site 1 of the channel. With the use of exogenously expressed channels, we show that two of the μ-conotoxins, μ-BuIIIB and μ-SxIIIA, are 50-fold more potent in blocking NaV1.6 from mouse than that from rat. Furthermore, we observed that μ-BuIIIB and μ-SxIIIA are potent blockers of large, myelinated A-fiber compound action potentials (A-CAPs) [but not small, unmyelinated C-fiber CAPs (C-CAPs)] in the sciatic nerve of the mouse (unlike A-CAPs of the rat, previously shown to be insensitive to these toxins). To investigate β-subunits, we used two synthetic derivatives of the recently discovered μO§-conotoxin GVIIJ that define site 8 of the channel, as previously characterized with cloned rat NaV1- and NaVβ-subunits expressed in Xenopus laevis oocytes, where it was shown that μO§-GVIIJ is a potent inhibitor of several NaV1-isoforms and that coexpression of NaVβ2 or -β4 (but not NaVβ1 or -β3) totally protects against block by μO§-GVIIJ. We report here the effects of μO§-GVIIJ on 1) sodium currents of mouse NaV1.6 coexpressed with various combinations of NaVβ-subunits in oocytes; 2) A- and C-CAPs of mouse and rat sciatic nerves; and 3) sodium currents of small and large neurons dissociated from rat dorsal root ganglia. Our overall results lead us to conclude that action potentials in A-fibers of the rodent sciatic nerve are mediated primarily by NaV1.6 associated with NaVβ2 or NaVβ4. Copyright © 2015 the American Physiological Society.

  4. Inhibition of late sodium current suppresses calcium-related ventricular arrhythmias by reducing the phosphorylation of CaMK-II and sodium channel expressions.

    Science.gov (United States)

    Wei, Xiao-Hong; Yu, Shan-Dong; Ren, Lu; Huang, Si-Hui; Yang, Qiao-Mei; Wang, Ping; Chu, Yan-Peng; Yang, Wei; Ding, Yan-Sheng; Huo, Yong; Wu, Lin

    2017-04-20

    Cardiac arrhythmias associated with intracellular calcium inhomeostasis are refractory to antiarrhythmic therapy. We hypothesized that late sodium current (I Na) contributed to the calcium-related arrhythmias. Monophasic action potential duration at 90% completion of repolarization (MAPD90) was significantly increased and ventricular arrhythmias were observed in hearts with increased intracellular calcium concentration ([Ca(2+)]i) by using Bay K 8644, and the increase became greater in hearts treated with a combination of ATX-II and Bay K 8644 compared to Bay K 8644 alone. The prolongations caused by Bay K 8644 and frequent episodes of ventricular tachycardias, both in absence and presence of ATX-II, were significantly attenuated or abolished by late I Na inhibitors TTX and eleclazine. In rabbit ventricular myocytes, Bay K 8644 increased I CaL density, calcium transient and myocyte contraction. TTX and eleclazine decreased the amplitude of late I Na, the reverse use dependence of MAPD90 at slower heart rate, and attenuated the increase of intracellular calcium transient and myocyte contraction. TTX diminished the phosphorylation of CaMKII-δ and Nav 1.5 in hearts treated with Bay K 8644 and ATX-II. In conclusion, late I Na contributes to ventricular arrhythmias and its inhibition is plausible to treat arrhythmias in hearts with increased [Ca(2+)]i.

  5. Voltage-gated sodium channel expression and action potential generation in differentiated NG108-15 cells.

    Science.gov (United States)

    Liu, Jinxu; Tu, Huiyin; Zhang, Dongze; Zheng, Hong; Li, Yu-Long

    2012-10-25

    The generation of action potential is required for stimulus-evoked neurotransmitter release in most neurons. Although various voltage-gated ion channels are involved in action potential production, the initiation of the action potential is mainly mediated by voltage-gated Na+ channels. In the present study, differentiation-induced changes of mRNA and protein expression of Na+ channels, Na+ currents, and cell membrane excitability were investigated in NG108-15 cells. Whole-cell patch-clamp results showed that differentiation (9 days) didn't change cell membrane excitability, compared to undifferentiated state. But differentiation (21 days) induced the action potential generation in 45.5% of NG108-15 cells (25/55 cells). In 9-day-differentiated cells, Na+ currents were mildly increased, which was also found in 21-day differentiated cells without action potential. In 21-day differentiated cells with action potential, Na+ currents were significantly enhanced. Western blot data showed that the expression of Na+ channels was increased with differentiated-time dependent manner. Single-cell real-time PCR data demonstrated that the expression of Na+ channel mRNA was increased by 21 days of differentiation in NG108-15 cells. More importantly, the mRNA level of Na+ channels in cells with action potential was higher than that in cells without action potential. Differentiation induces expression of voltage-gated Na+ channels and action potential generation in NG108-15 cells. A high level of the Na+ channel density is required for differentiation-triggered action potential generation.

  6. The tarantula toxin jingzhaotoxin-XI (κ-theraphotoxin-Cj1a) regulates the activation and inactivation of the voltage-gated sodium channel Nav1.5.

    Science.gov (United States)

    Tang, Cheng; Zhou, Xi; Huang, Yin; Zhang, Yunxiao; Hu, Zhaotun; Wang, Meichi; Chen, Ping; Liu, Zhonghua; Liang, Songping

    2014-12-15

    Specific peptide toxins interact with voltage-gated sodium channels by regulating the activation or inactivation of targeted channels. However, few toxins possessing dual effects have been identified. In the present study, we showed that jingzhaotoxin-XI/κ-theraphotoxin-Cj1a (JZTX-XI), a 34-residue peptide from the venom of the Chinese spider Chilobrachys jingzhao, inhibits the sodium conductance (IC50 = 124 ± 26 nM) and slows the fast inactivation (EC50 = 1.18 ± 0.2 μM) of Nav1.5 expressed in Chinese hamster ovary (CHO-K1) cells. JZTX-XI significantly shifted the activation to more depolarized voltages and decreased the deactivation of Nav1.5 currents upon extreme depolarization, but only slightly affected voltage-dependence of steady-state inactivation. In addition, JZTX-XI caused an approximately five-fold decrease in the rate of recovery from inactivation and an approximately 1.9-fold reduction in the closed-state inactivation rate. Our data suggest that JZTX-XI integrates the functions of site 3 toxins (α-scorpion toxins) with site 4 toxins (β-scorpion and spider toxins) by targeting multiple sites on Nav1.5. The unique properties displayed by JZTX-XI in its inhibitory activity on Nav1.5 suggest that its mechanism of action is distinct from those of site 3 and site 4 toxins, making JZTX-XI a useful probe for investigating the gating mechanism of Nav1.5 and toxin-channel interactions. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. The human Nav1.5 F1486 deletion associated with long QT syndrome leads to impaired sodium channel inactivation and reduced lidocaine sensitivity

    Science.gov (United States)

    Song, Weihua; Xiao, Yucheng; Chen, Hanying; Ashpole, Nicole M; Piekarz, Andrew D; Ma, Peilin; Hudmon, Andy; Cummins, Theodore R; Shou, Weinian

    2012-01-01

    The deletion of phenylalanine 1486 (F1486del) in the human cardiac voltage-gated sodium channel (hNav1.5) is associated with fatal long QT (LQT) syndrome. In this study we determined how F1486del impairs the functional properties of hNav1.5 and alters action potential firing in heterologous expression systems (human embryonic kidney (HEK) 293 cells) and their native cardiomyocyte background. Cells expressing hNav1.5-F1486del exhibited a loss-of-function alteration, reflected by an 80% reduction of peak current density, and several gain-of-function alterations, including reduced channel inactivation, enlarged window current, substantial augmentation of persistent late sodium current and an increase in ramp current. We also observed substantial action potential duration (APD) prolongation and prominent early afterdepolarizations (EADs) in neonatal cardiomyocytes expressing the F1486del channels, as well as in computer simulations of myocyte activity. In addition, lidocaine sensitivity was dramatically reduced, which probably contributed to the poor therapeutic outcome observed in the patient carrying the hNav1.5-F1486del mutation. Therefore, despite the significant reduction in peak current density, the F1486del mutation also leads to substantial gain-of-function alterations that are sufficient to cause APD prolongation and EADs, the predominant characteristic of LQTs. These data demonstrate that hNav1.5 mutations can have complex functional consequences and highlight the importance of identifying the specific molecular defect when evaluating potential treatments for individuals with prolonged QT intervals. PMID:22826127

  8. Induction of pseudo-periodic oscillation in voltage-gated sodium channel properties is dependent on the duration of prolonged depolarization.

    Science.gov (United States)

    Majumdar, Sriparna; Foster, Grant; Sikdar, Sujit K

    2004-07-01

    The neuronal voltage-gated sodium channels play a vital role in the action potential waveform shaping and propagation. Here, we report the effects of prolonged depolarization (1-160 s) on the detailed kinetics of activation, fast inactivation and recovery from slow inactivation in the rNa(v)1.2a voltage-gated sodium channel alpha-subunit expressed in Chinese hamster ovary (CHO) cells. Wavelet analysis revealed that the duration and amplitude of a prolonged sustained depolarization altered all the steady state and kinetic parameters of the channel in a pseudo-oscillatory fashion with time-variable period and amplitude, often superimposed on a linear trend. The half steady state activation potential showed a reversible depolarizing shift of 5-10 mV with duration of prolonged depolarization, while half steady state inactivation potential showed a hyperpolarizing shift of 43-55 mV. The time periods for most of the parameters relating to activation and fast and slow inactivation, lie close to 28-30 s, suggesting coupling of these kinetic processes through an oscillatory mechanism. Co-expression of the beta1-subunit affected the time periods of oscillation (close to 22 s for alpha + beta1) in steady state activation parameters. Application of a pulse protocol that mimicked paroxysmal depolarizing shift (PDS), a kind of depolarization seen in epileptic discharges, instead of a sustained depolarization, also caused oscillatory behaviour in the rNav1.2a alpha-subunit. This inherent pseudo-oscillatory mechanism may regulate excitability of the neurons, account for the epileptic discharges and subthreshold membrane potential oscillation and offer a molecular memory mechanism intrinsic to the neurons, independent of synaptic plasticity.

  9. Precambrian Skeletonized Microbial Eukaryotes

    Science.gov (United States)

    Lipps, Jere H.

    2017-04-01

    Skeletal heterotrophic eukaryotes are mostly absent from the Precambrian, although algal eukaryotes appear about 2.2 billion years ago. Tintinnids, radiolaria and foraminifera have molecular origins well back into the Precambrian yet no representatives of these groups are known with certainty in that time. These data infer times of the last common ancestors, not the appearance of true representatives of these groups which may well have diversified or not been preserved since those splits. Previous reports of these groups in the Precambrian are misinterpretations of other objects in the fossil record. Reported tintinnids at 1600 mya from China are metamorphic shards or mineral artifacts, the many specimens from 635-715 mya in Mongolia may be eukaryotes but they are not tintinnids, and the putative tintinnids at 580 mya in the Doushantou formation of China are diagenetic alterations of well-known acritarchs. The oldest supposed foraminiferan is Titanotheca from 550 to 565 mya rocks in South America and Africa is based on the occurrence of rutile in the tests and in a few modern agglutinated foraminifera, as well as the agglutinated tests. Neither of these nor the morphology are characteristic of foraminifera; hence these fossils remain as indeterminate microfossils. Platysolenites, an agglutinated tube identical to the modern foraminiferan Bathysiphon, occurs in the latest Neoproterozoic in Russia, Canada, and the USA (California). Some of the larger fossils occurring in typical Ediacaran (late Neoproterozoic) assemblages may be xenophyophorids (very large foraminifera), but the comparison is disputed and flawed. Radiolaria, on occasion, have been reported in the Precambrian, but the earliest known clearly identifiable ones are in the Cambrian. The only certain Precambrian heterotrophic skeletal eukaryotes (thecamoebians) occur in fresh-water rocks at about 750 mya. Skeletonized radiolaria and foraminifera appear sparsely in the Cambrian and radiate in the Ordovician

  10. Multi-country Survey Revealed Prevalent and Novel F1534S Mutation in Voltage-Gated Sodium Channel (VGSC) Gene in Aedes albopictus.

    Science.gov (United States)

    Xu, Jiabao; Bonizzoni, Mariangela; Zhong, Daibin; Zhou, Guofa; Cai, Songwu; Li, Yiji; Wang, Xiaoming; Lo, Eugenia; Lee, Rebecca; Sheen, Roger; Duan, Jinhua; Yan, Guiyun; Chen, Xiao-Guang

    2016-05-01

    Aedes albopictus is an important dengue vector because of its aggressive biting behavior and rapid spread out of its native home range in Southeast Asia. Pyrethroids are widely used for adult mosquito control, and resistance to pyrethroids should be carefully monitored because vector control is the only effective method currently available to prevent dengue transmission. The voltage-gated sodium channel gene is the target site of pyrethroids, and mutations in this gene cause knockdown resistance (kdr). Previous studies reported various mutations in the voltage-gated sodium channel (VGSC) gene, but the spatial distribution of kdr mutations in Ae. albopictus has not been systematically examined, and the association between kdr mutation and phenotypic resistance has not been established. A total of 597 Ae. albopictus individuals from 12 populations across Asia, Africa, America and Europe were examined for mutations in the voltage-gated sodium channel gene. Three domains for a total of 1,107 bp were sequenced for every individual. Two populations from southern China were examined for pyrethroid resistance using the World Health Organization standard tube bioassay, and the association between kdr mutations and phenotypic resistance was tested. A total of 29 synonymous mutations were found across domain II, III and IV of the VGSC gene. Non-synonymous mutations in two codons of the VGSC gene were detected in 5 populations from 4 countries. A novel mutation at 1532 codon (I1532T) was found in Rome, Italy with a frequency of 19.7%. The second novel mutation at codon 1534 (F1534S) was detected in southern China and Florida, USA with a frequency ranging from 9.5-22.6%. The WHO insecticide susceptibility bioassay found 90.1% and 96.1% mortality in the two populations from southern China, suggesting resistance and probable resistance. Positive association between kdr mutations with deltamethrin resistance was established in these two populations. Two novel kdr mutations, I1532T

  11. Multi-country Survey Revealed Prevalent and Novel F1534S Mutation in Voltage-Gated Sodium Channel (VGSC Gene in Aedes albopictus.

    Directory of Open Access Journals (Sweden)

    Jiabao Xu

    2016-05-01

    Full Text Available Aedes albopictus is an important dengue vector because of its aggressive biting behavior and rapid spread out of its native home range in Southeast Asia. Pyrethroids are widely used for adult mosquito control, and resistance to pyrethroids should be carefully monitored because vector control is the only effective method currently available to prevent dengue transmission. The voltage-gated sodium channel gene is the target site of pyrethroids, and mutations in this gene cause knockdown resistance (kdr. Previous studies reported various mutations in the voltage-gated sodium channel (VGSC gene, but the spatial distribution of kdr mutations in Ae. albopictus has not been systematically examined, and the association between kdr mutation and phenotypic resistance has not been established.A total of 597 Ae. albopictus individuals from 12 populations across Asia, Africa, America and Europe were examined for mutations in the voltage-gated sodium channel gene. Three domains for a total of 1,107 bp were sequenced for every individual. Two populations from southern China were examined for pyrethroid resistance using the World Health Organization standard tube bioassay, and the association between kdr mutations and phenotypic resistance was tested.A total of 29 synonymous mutations were found across domain II, III and IV of the VGSC gene. Non-synonymous mutations in two codons of the VGSC gene were detected in 5 populations from 4 countries. A novel mutation at 1532 codon (I1532T was found in Rome, Italy with a frequency of 19.7%. The second novel mutation at codon 1534 (F1534S was detected in southern China and Florida, USA with a frequency ranging from 9.5-22.6%. The WHO insecticide susceptibility bioassay found 90.1% and 96.1% mortality in the two populations from southern China, suggesting resistance and probable resistance. Positive association between kdr mutations with deltamethrin resistance was established in these two populations.Two novel kdr

  12. Anti-metastatic Potential of Amide-linked Local Anesthetics: Inhibition of Lung Adenocarcinoma Cell Migration and Inflammatory Src Signaling Independent of Sodium Channel Blockade

    Science.gov (United States)

    Piegeler, Tobias; Votta-Velis, E. Gina; Liu, Guoquan; Place, Aaron T.; Schwartz, David E.; Beck-Schimmer, Beatrice; Minshall, Richard D.; Borgeat, Alain

    2012-01-01

    Background Retrospective analysis of patients undergoing cancer surgery suggests the use of regional anesthesia may reduce cancer recurrence and improve survival. Amide-linked local anesthetics have anti-inflammatory properties, although the mechanism of action in this regard is unclear. As inflammatory processes involving Src tyrosine protein kinase and intercellular adhesion molecule-1 are important in tumor growth and metastasis, we hypothesized that amide-linked local anesthetics may inhibit inflammatory Src-signaling involved in migration of adenocarcinoma cells. Methods NCI-H838 lung cancer cells were incubated with Tumor Necrosis Factor-α in absence/presence of ropivacaine, lidocaine, or chloroprocaine (1nM-100μM). Cell migration and total cell lysate Src-activation and Intercellular Adhesion Molecule-1 phosphorylation were assessed. The role of voltage-gated sodium-channels in the mechanism of local anesthetic effects was also evaluated. Results Ropivacaine treatment (100μM) of H838 cells for 20 minutes decreased basal Src activity by 62% (p=0.003), and both ropivacaine and lidocaine co-administered with Tumor Necrosis Factor-α statistically significantly decreased Src-activation and Intercellular Adhesion Molecule-1 phosphorylation, whereas chloroprocaine had no such effect. Migration of these cells at 4 hours was inhibited by 26% (p=0.005) in presence of 1μM ropivacaine and 21% by 1μM lidocaine (p=0.004). These effects of ropivacaine and lidocaine were independent of voltage-gated sodium-channel inhibition. Conclusions This study indicates that amide-, but not ester-linked local anesthetics may provide beneficial anti-metastatic effects. The observed inhibition of NCI-H838 cell migration by lidocaine and ropivacaine was associated with the inhibition of Tumor Necrosis Factor-α-induced Src-activation and Intercellular Adhesion Molecule-1 phosphorylation, providing the first evidence of a molecular mechanism which appears to be independent of their

  13. Inhibition of sodium currents by local anesthetics in chloramine-T- treated squid axons. The role of channel activation

    Science.gov (United States)

    1987-01-01

    In order to test the requirement of Na channel inactivation for the action of local anesthetics, we investigated the inhibitory effects of quaternary and tertiary amine anesthetics on normally inactivating and noninactivating Na currents in squid axons under voltage clamp. Either the enzymatic mixture pronase, or chloramine-T (CT), a noncleaving, oxidizing reagent, was used to abolish Na channel inactivation. We found that both the local anesthetics QX-314 and etidocaine, when perfused internally at 1 mM, elicited a "tonic" (resting) block of Na currents, a "time-dependent" block that increased during single depolarizations, and a "use-dependent" (phasic) block that accumulated as a result of repetitive depolarizations. All three effects occurred in both control and CT-treated axons. As in previous reports, little time-dependent or phasic block by QX-314 appeared in pronase-treated axons, although tonic block remained. Time-dependent block was greatest and fastest at large depolarizations (Em greater than +60 mV) for both the control and CT-treated axons. The recovery kinetics from phasic block were the same in control and CT-modified axons. The voltage dependence of the steady state phasic block in CT-treated axons differed from that in the controls; an 8-10% reduction of the maximum phasic block and a steepening and shift of the voltage dependence in the hyperpolarizing direction resulted from CT treatment. The results show that these anesthetics can bind rapidly to open Na channels in a voltage-dependent manner, with no requirement for fast inactivation. We propose that the rapid phasic blocking reactions in nerve are consequences primarily of channel activation, mediated by binding of anesthetics to open channels, and that the voltage dependence of phasic block arises directly from that of channel activation. PMID:2438374

  14. Molecular diversity of voltage-gated sodium channel alpha and beta subunit mRNAs in human tissues.

    Science.gov (United States)

    Candenas, Luz; Seda, Marian; Noheda, Pedro; Buschmann, Helmut; Cintado, Cristina G; Martin, Julio D; Pinto, Francisco M

    2006-07-10

    Voltage-gated Na+ channels are composed of one alpha subunit and one or more auxiliary beta subunits. A reverse transcription-polymerase chain reaction assay was used to analyse the expression of the nine known alpha subunits (Na(v)1.1-Na(v)1.9) in 20 different human tissues. The mRNA expression of the currently known beta subunits (beta1, beta2, beta3 and beta4) was also assessed. The mRNAs of voltage-gated Na+ channel alpha and beta subunits were found in a wide variety of human tissues assayed and were present in neuronal and non-neuronal types of cells. These data suggest that, in addition to its well-established role in skeletal muscle, cardiac cells and neurons, voltage-gated Na+ channels might play important, still undetermined local roles in the regulation of cellular functions. These channels could emerge in the next future as potential, new therapeutic targets in the treatment of visceral diseases.

  15. Oligosaccharide composition of the neurotoxin-responsive sodium channel of mouse neuroblastoma and requirement of sialic acid for biological activity

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Negishi, M.; Kuik, J.A. van; Glick, M.C.

    1992-01-01

    A glycoprotein, Mr, 200000, which has the biological activity of the neurotoxin-responsive Na+ channel, was isolated from a clonal line of mouse neuroblastoma cells, N-18. The glycoprotein was purified to homogeneity in 18% yield by methods used to purify glycoproteins, which included metabolic

  16. Potential-dependent action of Anemonia sulcata toxins III and IV on sodium channels in crayfish giant axons.

    Science.gov (United States)

    Warashina, A; Jiang, Z Y; Ogura, T

    1988-01-01

    Effects of toxins III and IV (ATX III and IV) from the sea anemone Anemonia sulcata on the Na current of crayfish giant axons were studied. Both toxins slowed the inactivation of Na channels, producing a maintained Na current during a depolarizing voltage pulse. Using the intensity of the toxin-induced maintained current as an index for the fraction of Na channels to which toxin is bound, the toxin association and dissociation kinetics were analyzed. The dissociation rate of ATX III was increased by two orders of magnitudes by depolarizing the membrane from -70 to -40 mV. This increase of the dissociation rate caused a marked decrease in the binding rate of ATX III to Na channels in the same potential range. ATX IV exhibited association and dissociation kinetics that had a potential dependency quite similar to that of ATX III in spite of different ionic charge distribution in these two toxins. The results support the view that the potential-dependent kinetics of these toxins are not due to an electrostatic interaction between the ionic charges of toxins and the membrane potential but result from a modulation of the binding energy depending on the gate configuration of the Na channel.

  17. 17β-Estradiol regulates the gene expression of voltage-gated sodium channels: role of estrogen receptor α and estrogen receptor β.

    Science.gov (United States)

    Hu, Fang; Wang, Qiang; Wang, Peizhi; Wang, Wenjuan; Qian, Wenyi; Xiao, Hang; Wang, Lin

    2012-04-01

    Estradiol (E2) plays a key role in pain modulation, and the biological effects of E2 are transduced by binding estrogen receptors (ERs). Voltage-gated sodium (Nav) channels are responsible for the generation and propagation of action potentials in the membranes of most neurons and excitable cells. Adult dorsal root ganglion (DRG) neurons can express the ERs (ERα and ERβ), and Nav channels (TTX-S: Nav1.1, Nav1.6, and Nav1.7; and TTX-R: Nav1.8, and Nav1.9). Although E2 modulates Nav channel currents, little is known about the molecular mechanisms involved. In this study, we investigate the mRNA expressions of Nav channel subtypes mediated differentially by the ERs in the DRGs of wild-type (WT) and estrogen receptor knockout (αERKO and βERKO) mice. By means of quantitative real-time PCR, we found that the expressions of Nav1.1, Nav1.7, Nav1.8, and Nav1.9 subtypes were elevated in αERKO and βERKO mice, whereas Nav1.6 mRNA decreased in αERKO, but not in βERKO mice. The mRNA expressions of Nav subtypes were increased in E2-treated WT ovariectomized animals. We also found that E2-regulation of Nav1.1 and Nav1.9 mRNA expressions is dependent on ERα, ERβ, and another ER, whereas E2-regulation of Nav1.8 appears to be in an ERβ-dependent manner.

  18. A proton leak current through the cardiac sodium channel is linked to mixed arrhythmia and the dilated cardiomyopathy phenotype.

    Directory of Open Access Journals (Sweden)

    Pascal Gosselin-Badaroudine

    Full Text Available Cardiac Na(+ channels encoded by the SCN5A gene are essential for initiating heart beats and maintaining a regular heart rhythm. Mutations in these channels have recently been associated with atrial fibrillation, ventricular arrhythmias, conduction disorders, and dilated cardiomyopathy (DCM.We investigated a young male patient with a mixed phenotype composed of documented conduction disorder, atrial flutter, and ventricular tachycardia associated with DCM. Further family screening revealed DCM in the patient's mother and sister and in three of the mother's sisters. Because of the complex clinical phenotypes, we screened SCN5A and identified a novel mutation, R219H, which is located on a highly conserved region on the fourth helix of the voltage sensor domain of Na(v1.5. Three family members with DCM carried the R219H mutation.The wild-type (WT and mutant Na(+ channels were expressed in a heterologous expression system, and intracellular pH (pHi was measured using a pH-sensitive electrode. The biophysical characterization of the mutant channel revealed an unexpected selective proton leak with no effect on its biophysical properties. The H(+ leak through the mutated Na(v1.5 channel was not related to the Na(+ permeation pathway but occurred through an alternative pore, most probably a proton wire on the voltage sensor domain.We propose that acidification of cardiac myocytes and/or downstream events may cause the DCM phenotype and other electrical problems in affected family members. The identification of this clinically significant H(+ leak may lead to the development of more targeted treatments.

  19. A comparison of 15 Hz sine on-line and off-line magnetic stimulation affecting the voltage-gated sodium channel currents of prefrontal cortex pyramidal neurons

    Science.gov (United States)

    Zheng, Yu; Dong, Lei; Gao, Yang; Dou, Jun-Rong; Li, Ze-yan

    2016-10-01

    Combined with the use of patch-clamp techniques, repetitive transcranial magnetic stimulation (rTMS) has proven to be a noninvasive neuromodulation tool that can inhibit or facilitate excitability of neurons after extensive research. The studies generally focused on the method: the neurons are first stimulated in an external standard magnetic exposure device, and then moved to the patch-clamp to record electrophysiological characteristics (off-line magnetic exposure). Despite its universality, real-time observation of the effects of magnetic stimulation on the neurons is more effective (on-line magnetic stimulation). In this study, we selected a standard exposure device for magnetic fields acting on mouse prefrontal cortex pyramidal neurons, and described a new method that a patch-clamp setup was modified to allow on-line magnetic stimulation. By comparing the off-line exposure and on-line stimulation of the same magnetic field intensity and frequency affecting the voltage-gated sodium channel currents, we succeeded in proving the feasibility of the new on-line stimulation device. We also demonstrated that the sodium channel currents of prefrontal cortex pyramidal neurons increased significantly under the 15 Hz sine 1 mT, and 2 mT off-line magnetic field exposure and under the 1 mT and 2 mT on-line magnetic stimulation, and the rate of acceleration was most significant on 2 mT on-line magnetic stimulation. This study described the development of a new on-line magnetic stimulator and successfully demonstrated its practicability for scientific stimulation of neurons.

  20. Characterization of a Novel BmαTX47 Toxin Modulating Sodium Channels: The Crucial Role of Expression Vectors in Toxin Pharmacological Activity

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    Tian Li

    2014-02-01

    Full Text Available Long-chain scorpion toxins with four disulfide bridges exhibit various pharmacological features towards the different voltage-gated sodium channel subtypes. However, the toxin production still remains a huge challenge. Here, we reported the effects of different expression vectors on the pharmacological properties of a novel toxin BmαTX47 from the scorpion Buthus martensii Karsch. The recombinant BmαTX47 was obtained using the expression vector pET-14b and pET-28a, respectively. Pharmacological experiments showed that the recombinant BmαTX47 was a new α-scorpion toxin which could inhibit the fast inactivation of rNav1.2, mNav1.4 and hNav1.5 channels. Importantly, the different expression vectors were found to strongly affect BmαTX47 pharmacological activities while toxins were obtained by the same expression and purification procedures. When 10 µM recombinant BmαTX47 from the pET-28a vector was applied, the values of I5ms/Ipeak for rNav1.2, mNav1.4 and hNav1.5 channels were 44.12% ± 3.17%, 25.40% ± 4.89% and 65.34% ± 3.86%, respectively, which were better than those values of 11.33% ± 1.46%, 15.96% ± 1.87% and 5.24% ± 2.38% for rNav1.2, mNav1.4 and hNav1.5 channels delayed by 10 µM recombinant BmαTX47 from the pET-14b vector. The dose-response experiments further indicated the EC50 values of recombinant BmαTX47 from the pET-28a vector were 7262.9 ± 755.9 nM for rNav1.2 channel and 1005.8 ± 118.6 nM for hNav1.5 channel, respectively. Together, these findings highlighted the important role of expression vectors in scorpion toxin pharmacological properties, which would accelerate the understanding of the structure-function relationships of scorpion toxins and promote the potential application of toxins in the near future.

  1. A single Markov-type kinetic model accounting for the macroscopic currents of all human voltage-gated sodium channel isoforms

    Science.gov (United States)

    Hellgren Kotaleski, Jeanette

    2017-01-01

    Modelling ionic channels represents a fundamental step towards developing biologically detailed neuron models. Until recently, the voltage-gated ion channels have been mainly modelled according to the formalism introduced by the seminal works of Hodgkin and Huxley (HH). However, following the continuing achievements in the biophysical and molecular comprehension of these pore-forming transmembrane proteins, the HH formalism turned out to carry limitations and inconsistencies in reproducing the ion-channels electrophysiological behaviour. At the same time, Markov-type kinetic models have been increasingly proven to successfully replicate both the electrophysiological and biophysical features of different ion channels. However, in order to model even the finest non-conducting molecular conformational change, they are often equipped with a considerable number of states and related transitions, which make them computationally heavy and less suitable for implementation in conductance-based neurons and large networks of those. In this purely modelling study we develop a Markov-type kinetic model for all human voltage-gated sodium channels (VGSCs). The model framework is detailed, unifying (i.e., it accounts for all ion-channel isoforms) and computationally efficient (i.e. with a minimal set of states and transitions). The electrophysiological data to be modelled are gathered from previously published studies on whole-cell patch-clamp experiments in mammalian cell lines heterologously expressing the human VGSC subtypes (from NaV1.1 to NaV1.9). By adopting a minimum sequence of states, and using the same state diagram for all the distinct isoforms, the model ensures the lightest computational load when used in neuron models and neural networks of increasing complexity. The transitions between the states are described by original ordinary differential equations, which represent the rate of the state transitions as a function of voltage (i.e., membrane potential). The

  2. A single Markov-type kinetic model accounting for the macroscopic currents of all human voltage-gated sodium channel isoforms.

    Directory of Open Access Journals (Sweden)

    Pietro Balbi

    2017-09-01

    Full Text Available Modelling ionic channels represents a fundamental step towards developing biologically detailed neuron models. Until recently, the voltage-gated ion channels have been mainly modelled according to the formalism introduced by the seminal works of Hodgkin and Huxley (HH. However, following the continuing achievements in the biophysical and molecular comprehension of these pore-forming transmembrane proteins, the HH formalism turned out to carry limitations and inconsistencies in reproducing the ion-channels electrophysiological behaviour. At the same time, Markov-type kinetic models have been increasingly proven to successfully replicate both the electrophysiological and biophysical features of different ion channels. However, in order to model even the finest non-conducting molecular conformational change, they are often equipped with a considerable number of states and related transitions, which make them computationally heavy and less suitable for implementation in conductance-based neurons and large networks of those. In this purely modelling study we develop a Markov-type kinetic model for all human voltage-gated sodium channels (VGSCs. The model framework is detailed, unifying (i.e., it accounts for all ion-channel isoforms and computationally efficient (i.e. with a minimal set of states and transitions. The electrophysiological data to be modelled are gathered from previously published studies on whole-cell patch-clamp experiments in mammalian cell lines heterologously expressing the human VGSC subtypes (from NaV1.1 to NaV1.9. By adopting a minimum sequence of states, and using the same state diagram for all the distinct isoforms, the model ensures the lightest computational load when used in neuron models and neural networks of increasing complexity. The transitions between the states are described by original ordinary differential equations, which represent the rate of the state transitions as a function of voltage (i.e., membrane

  3. Potassium uptake supporting plant growth in the absence of AKT1 channel activity: Inhibition by ammonium and stimulation by sodium

    Science.gov (United States)

    Spalding, E. P.; Hirsch, R. E.; Lewis, D. R.; Qi, Z.; Sussman, M. R.; Lewis, B. D.

    1999-01-01

    A transferred-DNA insertion mutant of Arabidopsis that lacks AKT1 inward-rectifying K+ channel activity in root cells was obtained previously by a reverse-genetic strategy, enabling a dissection of the K+-uptake apparatus of the root into AKT1 and non-AKT1 components. Membrane potential measurements in root cells demonstrated that the AKT1 component of the wild-type K+ permeability was between 55 and 63% when external [K+] was between 10 and 1,000 microM, and NH4+ was absent. NH4+ specifically inhibited the non-AKT1 component, apparently by competing for K+ binding sites on the transporter(s). This inhibition by NH4+ had significant consequences for akt1 plants: K+ permeability, 86Rb+ fluxes into roots, seed germination, and seedling growth rate of the mutant were each similarly inhibited by NH4+. Wild-type plants were much more resistant to NH4+. Thus, AKT1 channels conduct the K+ influx necessary for the growth of Arabidopsis embryos and seedlings in conditions that block the non-AKT1 mechanism. In contrast to the effects of NH4+, Na+ and H+ significantly stimulated the non-AKT1 portion of the K+ permeability. Stimulation of akt1 growth rate by Na+, a predicted consequence of the previous result, was observed when external [K+] was 10 microM. Collectively, these results indicate that the AKT1 channel is an important component of the K+ uptake apparatus supporting growth, even in the "high-affinity" range of K+ concentrations. In the absence of AKT1 channel activity, an NH4+-sensitive, Na+/H+-stimulated mechanism can suffice.

  4. Voltage-Gated Sodium Channel Phosphorylation at Ser571 Regulates Late Current, Arrhythmia, and Cardiac Function In Vivo.

    Science.gov (United States)

    Glynn, Patric; Musa, Hassan; Wu, Xiangqiong; Unudurthi, Sathya D; Little, Sean; Qian, Lan; Wright, Patrick J; Radwanski, Przemyslaw B; Gyorke, Sandor; Mohler, Peter J; Hund, Thomas J

    2015-08-18

    Voltage-gated Na(+) channels (Nav) are essential for myocyte membrane excitability and cardiac function. Nav current (INa) is a large-amplitude, short-duration spike generated by rapid channel activation followed immediately by inactivation. However, even under normal conditions, a small late component of INa (INa,L) persists because of incomplete/failed inactivation of a subpopulation of channels. Notably, INa,L is directly linked with both congenital and acquired disease states. The multifunctional Ca(2+)/calmodulin-dependent kinase II (CaMKII) has been identified as an important activator of INa,L in disease. Several potential CaMKII phosphorylation sites have been discovered, including Ser571 in the Nav1.5 DI-DII linker, but the molecular mechanism underlying CaMKII-dependent regulation of INa,L in vivo remains unknown. To determine the in vivo role of Ser571, 2 Scn5a knock-in mouse models were generated expressing either: (1) Nav1.5 with a phosphomimetic mutation at Ser571 (S571E), or (2) Nav1.5 with the phosphorylation site ablated (S571A). Electrophysiology studies revealed that Ser571 regulates INa,L but not other channel properties previously linked to CaMKII. Ser571-mediated increases in INa,L promote abnormal repolarization and intracellular Ca(2+) handling and increase susceptibility to arrhythmia at the cellular and animal level. Importantly, Ser571 is required for maladaptive remodeling and arrhythmias in response to pressure overload. Our data provide the first in vivo evidence for the molecular mechanism underlying CaMKII activation of the pathogenic INa,L. Relevant for improved rational design of potential therapies, our findings demonstrate that Ser571-dependent regulation of Nav1.5 specifically tunes INa,L without altering critical physiological components of the current. © 2015 American Heart Association, Inc.

  5. Dynamic excitation states and firing patterns are controlled by sodium channel kinetics in myenteric neurons: a simulation study.

    Science.gov (United States)

    Korogod, Sergiy M; Osorio, Nancy; Kulagina, Iryna B; Delmas, Patrick

    2014-01-01

    Enteric neurons located in the gastro-intestinal tract are of particular importance to control digestive functions such as motility and secretion. In our recent publication, we showed that mouse myenteric neurons exhibit 2 types of tetrodotoxin-resistant Na(+) currents: a fast inactivating Na(+) current produced by Nav1.5 channels, present in nearly all myenteric neurons, and a persistent Na(+) current attributed to Nav1.9 channels, restricted to the intrinsic primary afferent neurons (IPANs). By combination of experimental recording and computer simulation we found that Nav1.5 contributed to the upstroke velocity of action potentials (APs), whereas Nav1.9 opposed AP repolarization. Here, we detailed the Na(+), Ca(2+) and K(+) currents used in our computational model of IPAN. We refined the prototype cell to reproduce the sustained firing pattern recorded in situ. As shown in experimental conditions we demonstrated that Nav1.9 channels critically determine the up-state life-time and thus, are essential to sustain tonic firing.

  6. Epidermal growth factor potentiates in vitro metastatic behaviour of human prostate cancer PC-3M cells: involvement of voltage-gated sodium channel

    Directory of Open Access Journals (Sweden)

    Uysal-Onganer Pinar

    2007-11-01

    Full Text Available Abstract Background Although a high level of functional voltage-gated sodium channel (VGSC expression has been found in strongly metastatic human and rat prostate cancer (PCa cells, the mechanism(s responsible for the upregulation is unknown. The concentration of epidermal growth factor (EGF, a modulator of ion channels, in the body is highest in prostatic fluid. Thus, EGF could be involved in the VGSC upregulation in PCa. The effects of EGF on VGSC expression in the highly metastatic human PCa PC-3M cell line, which was shown previously to express both functional VGSCs and EGF receptors, were investigated. A quantitative approach, from gene level to cell behaviour, was used. mRNA levels were determined by real-time PCR. Protein expression was studied by Western blots and immunocytochemistry and digital image analysis. Functional assays involved measurements of transverse migration, endocytic membrane activity and Matrigel invasion. Results Exogenous EGF enhanced the cells' in vitro metastatic behaviours (migration, endocytosis and invasion. Endogenous EGF had a similar involvement. EGF increased VGSC Nav1.7 (predominant isoform in PCa mRNA and protein expressions. Co-application of the highly specific VGSC blocker tetrodotoxin (TTX suppressed the effect of EGF on all three metastatic cell behaviours studied. Conclusion 1 EGF has a major involvement in the upregulation of functional VGSC expression in human PCa PC-3M cells. (2 VGSC activity has a significant intermediary role in potentiating effect of EGF in human PCa.

  7. Endosymbiotic theories for eukaryote origin.

    Science.gov (United States)

    Martin, William F; Garg, Sriram; Zimorski, Verena

    2015-09-26

    For over 100 years, endosymbiotic theories have figured in thoughts about the differences between prokaryotic and eukaryotic cells. More than 20 different versions of endosymbiotic theory have been presented in the literature to explain the origin of eukaryotes and their mitochondria. Very few of those models account for eukaryotic anaerobes. The role of energy and the energetic constraints that prokaryotic cell organization placed on evolutionary innovation in cell history has recently come to bear on endosymbiotic theory. Only cells that possessed mitochondria had the bioenergetic means to attain eukaryotic cell complexity, which is why there are no true intermediates in the prokaryote-to-eukaryote transition. Current versions of endosymbiotic theory have it that the host was an archaeon (an archaebacterium), not a eukaryote. Hence the evolutionary history and biology of archaea increasingly comes to bear on eukaryotic origins, more than ever before. Here, we have compiled a survey of endosymbiotic theories for the origin of eukaryotes and mitochondria, and for the origin of the eukaryotic nucleus, summarizing the essentials of each and contrasting some of their predictions to the observations. A new aspect of endosymbiosis in eukaryote evolution comes into focus from these considerations: the host for the origin of plastids was a facultative anaerobe. © 2015 The Authors.

  8. Endosymbiotic theories for eukaryote origin

    Science.gov (United States)

    Martin, William F.; Garg, Sriram; Zimorski, Verena

    2015-01-01

    For over 100 years, endosymbiotic theories have figured in thoughts about the differences between prokaryotic and eukaryotic cells. More than 20 different versions of endosymbiotic theory have been presented in the literature to explain the origin of eukaryotes and their mitochondria. Very few of those models account for eukaryotic anaerobes. The role of energy and the energetic constraints that prokaryotic cell organization placed on evolutionary innovation in cell history has recently come to bear on endosymbiotic theory. Only cells that possessed mitochondria had the bioenergetic means to attain eukaryotic cell complexity, which is why there are no true intermediates in the prokaryote-to-eukaryote transition. Current versions of endosymbiotic theory have it that the host was an archaeon (an archaebacterium), not a eukaryote. Hence the evolutionary history and biology of archaea increasingly comes to bear on eukaryotic origins, more than ever before. Here, we have compiled a survey of endosymbiotic theories for the origin of eukaryotes and mitochondria, and for the origin of the eukaryotic nucleus, summarizing the essentials of each and contrasting some of their predictions to the observations. A new aspect of endosymbiosis in eukaryote evolution comes into focus from these considerations: the host for the origin of plastids was a facultative anaerobe. PMID:26323761

  9. ProTx-II, a selective inhibitor of NaV1.7 sodium channels, blocks action potential propagation in nociceptors.

    Science.gov (United States)

    Schmalhofer, William A; Calhoun, Jeffrey; Burrows, Rachel; Bailey, Timothy; Kohler, Martin G; Weinglass, Adam B; Kaczorowski, Gregory J; Garcia, Maria L; Koltzenburg, Martin; Priest, Birgit T

    2008-11-01

    Voltage-gated sodium (Na(V)1) channels play a critical role in modulating the excitability of sensory neurons, and human genetic evidence points to Na(V)1.7 as an essential contributor to pain signaling. Human loss-of-function mutations in SCN9A, the gene encoding Na(V)1.7, cause channelopathy-associated indifference to pain (CIP), whereas gain-of-function mutations are associated with two inherited painful neuropathies. Although the human genetic data make Na(V)1.7 an attractive target for the development of analgesics, pharmacological proof-of-concept in experimental pain models requires Na(V)1.7-selective channel blockers. Here, we show that the tarantula venom peptide ProTx-II selectively interacts with Na(V)1.7 channels, inhibiting Na(V)1.7 with an IC(50) value of 0.3 nM, compared with IC(50) values of 30 to 150 nM for other heterologously expressed Na(V)1 subtypes. This subtype selectivity was abolished by a point mutation in DIIS3. It is interesting that application of ProTx-II to desheathed cutaneous nerves completely blocked the C-fiber compound action potential at concentrations that had little effect on Abeta-fiber conduction. ProTx-II application had little effect on action potential propagation of the intact nerve, which may explain why ProTx-II was not efficacious in rodent models of acute and inflammatory pain. Mono-iodo-ProTx-II ((125)I-ProTx-II) binds with high affinity (K(d) = 0.3 nM) to recombinant hNa(V)1.7 channels. Binding of (125)I-ProTx-II is insensitive to the presence of other well characterized Na(V)1 channel modulators, suggesting that ProTx-II binds to a novel site, which may be more conducive to conferring subtype selectivity than the site occupied by traditional local anesthetics and anticonvulsants. Thus, the (125)I-ProTx-II binding assay, described here, offers a new tool in the search for novel Na(V)1.7-selective blockers.

  10. Comparative study of the distribution of the alpha-subunits of voltage-gated sodium channels in normal and axotomized rat dorsal root ganglion neurons.

    Science.gov (United States)

    Fukuoka, Tetsuo; Kobayashi, Kimiko; Yamanaka, Hiroki; Obata, Koichi; Dai, Yi; Noguchi, Koichi

    2008-09-10

    We compared the distribution of the alpha-subunit mRNAs of voltage-gated sodium channels Nav1.1-1.3 and Nav1.6-1.9 and a related channel, Nax, in histochemically identified neuronal subpopulations of the rat dorsal root ganglia (DRG). In the naïve DRG, the expression of Nav1.1 and Nav1.6 was restricted to A-fiber neurons, and they were preferentially expressed by TrkC neurons, suggesting that proprioceptive neurons possess these channels. Nav1.7, -1.8, and -1.9 mRNAs were more abundant in C-fiber neurons compared with A-fiber ones. Nax was evenly expressed in both populations. Although Nav1.8 and -1.9 were preferentially expressed by TrkA neurons, other alpha-subunits were expressed independently of TrkA expression. Actually, all IB4(+) neurons expressed both Nav1.8 and -1.9, and relatively limited subpopulations of IB4(+) neurons (3% and 12%, respectively) expressed Nav1.1 and/or Nav1.6. These findings provide useful information in interpreting the electrophysiological characteristics of some neuronal subpopulations of naïve DRG. After L5 spinal nerve ligation, Nav1.3 mRNA was up-regulated mainly in A-fiber neurons in the ipsilateral L5 DRG. Although previous studies demonstrated that nerve growth factor (NGF) and glial cell-derived neurotrophic factor (GDNF) reversed this up-regulation, the Nav1.3 induction was independent of either TrkA or GFRalpha1 expression, suggesting that the induction of Nav1.3 may be one of the common responses of axotomized DRG neurons without a direct relationship to NGF/GDNF supply. (c) 2008 Wiley-Liss, Inc.

  11. Lithium-Responsive Seizure-Like Hyperexcitability Is Caused by a Mutation in the Drosophila Voltage-Gated Sodium Channel Gene paralytic

    Science.gov (United States)

    Kasuya, Junko; Ueda, Atsushi; Iyengar, Atulya; Wu, Chun-Fang

    2016-01-01

    Abstract Shudderer (Shu) is an X-linked dominant mutation in Drosophila melanogaster identified more than 40 years ago. A previous study showed that Shu caused spontaneous tremors and defects in reactive climbing behavior, and that these phenotypes were significantly suppressed when mutants were fed food containing lithium, a mood stabilizer used in the treatment of bipolar disorder (Williamson, 1982). This unique observation suggested that the Shu mutation affects genes involved in lithium-responsive neurobiological processes. In the present study, we identified Shu as a novel mutant allele of the voltage-gated sodium (Nav) channel gene paralytic (para). Given that hypomorphic para alleles and RNA interference–mediated para knockdown reduced the severity of Shu phenotypes, Shu was classified as a para hypermorphic allele. We also demonstrated that lithium could improve the behavioral abnormalities displayed by other Nav mutants, including a fly model of the human generalized epilepsy with febrile seizures plus. Our electrophysiological analysis of Shu showed that lithium treatment did not acutely suppress Nav channel activity, indicating that the rescue effect of lithium resulted from chronic physiological adjustments to this drug. Microarray analysis revealed that lithium significantly alters the expression of various genes in Shu, including those involved in innate immune responses, amino acid metabolism, and oxidation-reduction processes, raising the interesting possibility that lithium-induced modulation of these biological pathways may contribute to such adjustments. Overall, our findings demonstrate that Nav channel mutants in Drosophila are valuable genetic tools for elucidating the effects of lithium on the nervous system in the context of neurophysiology and behavior. PMID:27844061

  12. Mutually exclusive splicing regulates the Nav 1.6 sodium channel function through a combinatorial mechanism that involves three distinct splicing regulatory elements and their ligands.

    Science.gov (United States)

    Zubović, Lorena; Baralle, Marco; Baralle, Francisco E

    2012-07-01

    Mutually exclusive splicing is a form of alternative pre-mRNA processing that consists in the use of only one of a set of two or more exons. We have investigated the mechanisms involved in this process for exon 18 of the Na(v) 1.6 sodium channel transcript and its significance regarding gene-expression regulation. The 18N exon (neonatal form) has a stop codon in phase and although the mRNA can be detected by amplification methods, the truncated protein has not been observed. The switch from 18N to 18A (adult form) occurs only in a restricted set of neural tissues producing the functional channel while other tissues display the mRNA with the 18N exon also in adulthood. We demonstrate that the mRNA species carrying the stop codon is subjected to Nonsense-Mediated Decay, providing a control mechanism of channel expression. We also map a string of cis-elements within the mutually exclusive exons and in the flanking introns responsible for their strict tissue and temporal specificity. These elements bind a series of positive (RbFox-1, SRSF1, SRSF2) and negative (hnRNPA1, PTB, hnRNPA2/B1, hnRNPD-like JKTBP) splicing regulatory proteins. These splicing factors, with the exception of RbFox-1, are ubiquitous but their levels vary during development and differentiation, ensuing unique sets of tissue and temporal levels of splicing factors. The combinatorial nature of these elements is highlighted by the dominance of the elements that bind the ubiquitous factors over the tissue specific RbFox-1.

  13. Characterization of Amm VIII from Androctonus mauretanicus mauretanicus: a new scorpion toxin that discriminates between neuronal and skeletal sodium channels.

    Science.gov (United States)

    Alami, Meriem; Vacher, Hélène; Bosmans, Frank; Devaux, Christiane; Rosso, Jean-Pierre; Bougis, Pierre E; Tytgat, Jan; Darbon, Hervé; Martin-Eauclaire, Marie-France

    2003-11-01

    The venom of the scorpion Androctonus mauretanicus mauretanicus was screened by use of a specific serum directed against AaH II, the scorpion alpha-toxin of reference, with the aim of identifying new analogues. This led to the isolation of Amm VIII (7382.57 Da), which gave a highly positive response in ELISA, but was totally devoid of toxicity when injected subcutaneously into mice. In voltage-clamp experiments with rat brain type II Na+ channel rNa(v)1.2 or rat skeletal muscle Na+ channel rNa(v)1.4, expressed in Xenopus oocytes, the EC50 values of the toxin-induced slowing of inactivation were: 29+/-5 and 416+/-14 nM respectively for AmmVIII and 2.6+/-0.3 nM and 2.2+/-0.2 nM, respectively, for AaH II interactions. Accordingly, Amm VIII clearly discriminates neuronal versus muscular Na+ channel. The Amm VIII cDNA was amplified from a venom gland cDNA library and its oligonucleotide sequence determined. It shows 87% sequence homology with AaH II, but carries an unusual extension at its C-terminal end, consisting of an additional Asp due to a point mutation in the cDNA penultimate codon. We hypothesized that this extra amino acid residue could induce steric hindrance and dramatically reduce recognition of the target by Amm VIII. We constructed a model of Amm VIII based on the X-ray structure of AaH II to clarify this point. Molecular modelling showed that this C-terminal extension does not lead to an overall conformational change in Amm VIII, but drastically modifies the charge repartition and, consequently, the electrostatic dipole moment of the molecule. At last, liquid-phase radioimmunassays with poly- and monoclonal anti-(AaH II) antibodies showed the loss of conformational epitopes between AaH II and Amm VIII.

  14. Anticonvulsant mechanism of saponins fraction from adventitious roots of Ficus religiosa: possible modulation of GABAergic, calcium and sodium channel functions

    Directory of Open Access Journals (Sweden)

    Damanpreet Singh

    Full Text Available ABSTRACT In our previous studies, quantified saponins-rich fraction from adventitious root extract of Ficus religiosa L., Moraceae, showed anticonvulsant effect in acute, as well as chronic mice models of epilepsy. The present study was designed to reveal putative anticonvulsant mechanism of quantified saponins-rich fraction using target specific animal models. The anticonvulsant effect of quantified saponins-rich fraction was initially studied in maximal electroshock and pentylenetetrazol test at 1, 2 and 4 mg/kg; i.p. doses. Based on the results of initial anticonvulsant testing, different groups of mice were injected with vehicle or quantified saponins-rich fraction (4 mg/kg; i.p., 30 min prior to an injection of N-methyl-D-aspartic acid (100 mg/kg; s.c., bicuculline (5 mg/kg; i.p., strychnine hydrochloride (2 mg/kg; i.p., BAY k-8644 (37.5 µg; i.c.v., veratridine (500 µg/kg; i.p. and the convulsive episodes were studied. Treatment with the extract (1, 2 and 4 mg/kg showed significant protection in maximal electroshock and pentylenetetrazol-induced convulsion tests, in a dose-dependent manner. Moreover, quantified saponins-rich fraction at 4 mg/kg dose showed significant increase in latency to clonic convulsions, decrease in seizure severity and increase in average wave amplitude in bicuculline, BAY k-8644 and veratridine tests, respectively, as compared to vehicle control. However, SRF treatment failed to abolish N-methyl-D-aspartic acid and strychnine-induced convulsions, indicated by insignificant change in the appearance of turning behavior and onset of tonic extension, respectively, as compared to vehicle control. From the results of present study, it is concluded that quantified saponins-rich fraction suppress maximal electroshock, pentylenetetrazol, bicuculline, BAY k-8644 and veratridine-induced convulsions, indicating its GABAergic, Na+ and Ca2+ channel modulatory effects. Further it can be correlated that quantified saponins

  15. Chloride binding site of neurotransmitter sodium symporters

    DEFF Research Database (Denmark)

    Kantcheva, Adriana Krassimirova; Quick, Matthias; Shi, Lei

    2013-01-01

    Neurotransmitter:sodium symporters (NSSs) play a critical role in signaling by reuptake of neurotransmitters. Eukaryotic NSSs are chloride-dependent, whereas prokaryotic NSS homologs like LeuT are chloride-independent but contain an acidic residue (Glu290 in LeuT) at a site where eukaryotic NSSs ...

  16. Eukaryotic DNA Replication Fork.

    Science.gov (United States)

    Burgers, Peter M J; Kunkel, Thomas A

    2017-06-20

    This review focuses on the biogenesis and composition of the eukaryotic DNA replication fork, with an emphasis on the enzymes that synthesize DNA and repair discontinuities on the lagging strand of the replication fork. Physical and genetic methodologies aimed at understanding these processes are discussed. The preponderance of evidence supports a model in which DNA polymerase ε (Pol ε) carries out the bulk of leading strand DNA synthesis at an undisturbed replication fork. DNA polymerases α and δ carry out the initiation of Okazaki fragment synthesis and its elongation and maturation, respectively. This review also discusses alternative proposals, including cellular processes during which alternative forks may be utilized, and new biochemical studies with purified proteins that are aimed at reconstituting leading and lagging strand DNA synthesis separately and as an integrated replication fork.

  17. The Snake with the Scorpion's Sting: Novel Three-Finger Toxin Sodium Channel Activators from the Venom of the Long-Glanded Blue Coral Snake (Calliophis bivirgatus).

    Science.gov (United States)

    Yang, Daryl C; Deuis, Jennifer R; Dashevsky, Daniel; Dobson, James; Jackson, Timothy N W; Brust, Andreas; Xie, Bing; Koludarov, Ivan; Debono, Jordan; Hendrikx, Iwan; Hodgson, Wayne C; Josh, Peter; Nouwens, Amanda; Baillie, Gregory J; Bruxner, Timothy J C; Alewood, Paul F; Lim, Kelvin Kok Peng; Frank, Nathaniel; Vetter, Irina; Fry, Bryan G

    2016-10-18

    Millions of years of evolution have fine-tuned the ability of venom peptides to rapidly incapacitate both prey and potential predators. Toxicofera reptiles are characterized by serous-secreting mandibular or maxillary glands with heightened levels of protein expression. These glands are the core anatomical components of the toxicoferan venom system, which exists in myriad points along an evolutionary continuum. Neofunctionalisation of toxins is facilitated by positive selection at functional hotspots on the ancestral protein and venom proteins have undergone dynamic diversification in helodermatid and varanid lizards as well as advanced snakes. A spectacular point on the venom system continuum is the long-glanded blue coral snake ( Calliophis bivirgatus ), a specialist feeder that preys on fast moving, venomous snakes which have both a high likelihood of prey escape but also represent significant danger to the predator itself. The maxillary venom glands of C. bivirgatus extend one quarter of the snake's body length and nestle within the rib cavity. Despite the snake's notoriety its venom has remained largely unstudied. Here we show that the venom uniquely produces spastic paralysis, in contrast to the flaccid paralysis typically produced by neurotoxic snake venoms. The toxin responsible, which we have called calliotoxin (δ-elapitoxin-Cb1a), is a three-finger toxin (3FTx). Calliotoxin shifts the voltage-dependence of Na V 1.4 activation to more hyperpolarised potentials, inhibits inactivation, and produces large ramp currents, consistent with its profound effects on contractile force in an isolated skeletal muscle preparation. Voltage-gated sodium channels (Na V ) are a particularly attractive pharmacological target as they are involved in almost all physiological processes including action potential generation and conduction. Accordingly, venom peptides that interfere with Na V function provide a key defensive and predatory advantage to a range of invertebrate

  18. Sodium-23 MRI of whole spine at 3 Tesla using a 5-channel receive-only phased-array and a whole-body transmit resonator

    Energy Technology Data Exchange (ETDEWEB)

    Malzacher, Matthias; Kalayciyan, Raffi; Konstandin, Simon; Schad, Lothar R. [Heidelberg Univ., Mannheim (Germany). Computer Assisted Clinical Medicine; Haneder, Stefan [Heidelberg Univ., Mannheim (Germany). Clinical Radiology and Nuclear Medicine; University Hospital of Cologne, Koeln (Germany). Dept. of Radiology

    2016-05-01

    Sodium magnetic resonance imaging ({sup 23}Na MRI) is a unique and non-invasive imaging technique which provides important information on cellular level about the tissue of the human body. Several applications for {sup 23}Na MRI were investigated with regard to the examination of the tissue viability and functionality for example in the brain, the heart or the breast. The {sup 23}Na MRI technique can also be integrated as a potential monitoring instrument after radiotherapy or chemotherapy. The main contribution in this work was the adaptation of {sup 23}Na MRI for spine imaging, which can provide essential information on the integrity of the intervertebral disks with respect to the early detection of disk degeneration. In this work, a transmit-only receive-only dual resonator system was designed and developed to cover the whole human spine using {sup 23}Na MRI and increase the receive sensitivity. The resonator system consisted of an already presented {sup 23}Na whole-body resonator and a newly developed 5-channel receive-only phased-array. The resonator system was first validated using bench top and phantom measurements. A threefold SNR improvement at the depth of the spine (∝7 cm) over the whole-body resonator was achieved using the spine array. {sup 23}Na MR measurements of the human spine using the transmit-only receive-only resonator system were performed on a healthy volunteer within an acquisition time of 10 minutes. A density adapted 3D radial sequence was chosen with 6 mm isotropic resolution, 49 ms repetition time and a short echo time of 540 μs. Furthermore, it was possible to quantify the tissue sodium concentration in the intervertebral discs in the lumbar region (120 ms repetition time) using this setup.

  19. Optimal requirements for high affinity and use-dependent block of skeletal muscle sodium channel by N-benzyl analogs of tocainide-like compounds.

    Science.gov (United States)

    De Luca, Annamaria; Talon, Sophie; De Bellis, Michela; Desaphy, Jean-François; Lentini, Giovanni; Corbo, Filomena; Scilimati, Antonio; Franchini, Carlo; Tortorella, Vincenzo; Camerino, Diana Conte

    2003-10-01

    Newly synthesized tocainide analogs were tested for their state-dependent affinity and use-dependent behavior on sodium currents (INa) of adult skeletal muscle fibers by means of the Vaseline-gap voltage clamp method. The drugs had the pharmacophore amino group constrained in position alpha [N-(2,6-dimethylphenyl)pyrrolidine-2-carboxamide (To5)] or beta [N-(2,6-dimethylphenyl)pyrrolidine-3-carboxamide (To9)] in a proline-like cycle and/or linked to a lipophilic benzyl moiety as in N-benzyl-tocainide (Benzyl-Toc), 1-benzyl-To5 (Benzyl-To5), and 1-benzyl-To9 (Benzyl-To9). INa were elicited with pulses to -20 mV from different holding potentials (-140, -100, and -70 mV) and stimulation frequencies (2 and 10 Hz). All compounds were voltage-dependent and use-dependent channel blockers. The presence of a proline-like cycle increased the potency; i.e., To5 was 3- and 10-fold more effective than Toc in blocking INa at the holding potential of -140 and -70 mV, respectively. The benzyl group on the amine further enhanced drug effectiveness with the following scale: Benzyl-To9 >/= Benzyl-Toc > Benzyl-To5. At a holding potential of -100 mV and 10-Hz stimulation, Benzyl-To9 blocked INa with a half-maximal concentration of 0.5 microM, being 60 and 400 times more potent than To9 and Toc, respectively. The similar effectiveness of Benzyl-Toc and Benzyl-To9 was paralleled by a similar spatial arrangement by equilibrium geometry modeling. In addition, the latter had a higher pKa value that probably contributed to a slow kinetic during its high use-dependent behavior. Benzyl-To5 had its lowest energy level at a more folded conformation that justifies the less favorable profile among the N-benzylated analogs. The new compounds are the most potent tocainide-like sodium channel blockers so far described and have high therapeutic potentials.

  20. Coordinated role of voltage-gated sodium channels and the Na{sup +}/H{sup +} exchanger in sustaining microglial activation during inflammation

    Energy Technology Data Exchange (ETDEWEB)

    Hossain, Muhammad M. [Department of Environmental and Occupational Medicine and Environmental and Occupational Health Sciences Institute, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States); Sonsalla, Patricia K. [Department of Neurology, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States); Richardson, Jason R., E-mail: jricha3@eohsi.rutgers.edu [Department of Environmental and Occupational Medicine and Environmental and Occupational Health Sciences Institute, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States)

    2013-12-01

    Persistent neuroinflammation and microglial activation play an integral role in the pathogenesis of many neurological disorders. We investigated the role of voltage-gated sodium channels (VGSC) and Na{sup +}/H{sup +} exchangers (NHE) in the activation of immortalized microglial cells (BV-2) after lipopolysaccharide (LPS) exposure. LPS (10 and 100 ng/ml) caused a dose- and time-dependent accumulation of intracellular sodium [(Na{sup +}){sub i}] in BV-2 cells. Pre-treatment of cells with the VGSC antagonist tetrodotoxin (TTX, 1 μM) abolished short-term Na{sup +} influx, but was unable to prevent the accumulation of (Na{sup +}){sub i} observed at 6 and 24 h after LPS exposure. The NHE inhibitor cariporide (1 μM) significantly reduced accumulation of (Na{sup +}){sub i} 6 and 24 h after LPS exposure. Furthermore, LPS increased the mRNA expression and protein level of NHE-1 in a dose- and time-dependent manner, which was significantly reduced after co-treatment with TTX and/or cariporide. LPS increased production of TNF-α, ROS, and H{sub 2}O{sub 2} and expression of gp91{sup phox}, an active subunit of NADPH oxidase, in a dose- and time-dependent manner, which was significantly reduced by TTX or TTX + cariporide. Collectively, these data demonstrate a closely-linked temporal relationship between VGSC and NHE-1 in regulating function in activated microglia, which may provide avenues for therapeutic interventions aimed at reducing neuroinflammation. - Highlights: • LPS causes immediate increase in sodium through VGSC and subsequently through the NHE-1. • Inhibition of VGSC reduces increases in NHE-1 and gp91{sup phox}. • Inhibition of VGSC and NHE-1 reduces NADPH oxidase-mediated Tnf-α, ROS, and H{sub 2}O{sub 2} production. • NHE-1 and Na{sub v}1.6 may be viable targets for therapeutic interventions to reduce neuroinflammation in neurodegenerative disease.

  1. 1,25-Dihydroxyvitamin D Enhances Alveolar Fluid Clearance by Upregulating the Expression of Epithelial Sodium Channels.

    Science.gov (United States)

    Nie, Hongguang; Cui, Yong; Wu, Sihui; Ding, Yan; Li, Yanchun

    2016-01-01

    Vitamin D is implicated in the pathogenesis of asthma, acute lung injury, and other respiratory diseases. 1,25-Dihydroxyvitamin D (1,25(OH)2D3), the hormonal form of vitamin D, has been shown to reduce vascular permeability and ameliorate lung edema. Therefore, we speculate that 1,25(OH)2D3 may regulate alveolar Na(+) transport via targeting epitheli